Physiol Rev 93: 803–959, 2013
doi:10.1152/physrev.00023.2012
THE DIVERGENCE, ACTIONS, ROLES,
AND RELATIVES OF SODIUM-COUPLED
BICARBONATE TRANSPORTERS
Mark D. Parker and Walter F. Boron
Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio
L
I. INTRODUCTION
Roos and Boron (811). In 2003, Chesler (186) focused on
pH regulation in the brain. This journal reviewed vacuolar
H⫹ pumps in the contributions by Forgac in 1989 (292), by
Nelson and Harvey in 1999 (678), and by Wagner et al. in
2004 (1015). The journal considered H-K pumps in the
effort of Hersey and Sachs in 1995 (380). Na-H exchange
was the subject of the 1997 review by Wakabayashi and
co-workers (1017). Recently, Lee et al. (555) have examined HCO3⫺ secretion by the pancreas and salivary glands
(555). However, Physiological Reviews has not examined HCO3⫺ transporters per se.
A. Regulation of pH
B. Scope of This Review
pH is one of the most important parameters for life. Virtually every biological process is sensitive to changes in pH,
and some are exquisitely sensitive. Thus transporters have
evolved to regulate pH in organelles, the cytosol, and the
extracellular fluid. Not surprisingly, dysregulation of pH is
associated with a wide array of pathologies (TABLE 1), including cancer, hypertension, reperfusion injury, amyloid
deposition (e.g., in Alzheimer’s disease), and aging.
The movement of bicarbonate equivalents, HCO3⫺ itself,
CO32⫺, or the NaCO3⫺ ion pair, across the plasma membrane is an integral part of the regulation of pHi and the
transepithelial transport of solutes and fluid. Disturbances
in HCO3⫺ transporter genes are associated with a variety
of pathologies and can potentially impact any of the vast
array of pH-sensitive proteins and processes summarized
in TABLE 1.
The transporters responsible for pH regulation in various
compartments include vacuolar-type ATPases or H⫹
pumps, gastric-type H⫹-K⫹-ATPases or pumps, Na-H exchangers, and bicarbonate (HCO3⫺) transporters. Physiological Reviews last appraised the general subject of intracellular pH (pHi) regulation in 1981, with the review by
Bicarbonate transport in animals is effected by the eight
physiologically distinct mechanisms numbered 1– 8 in the
generic epithelial cell in FIGURE 1.
I.
II.
III.
IV.
V.
VI.
VII.
VIII.
INTRODUCTION
NCBT EMERGENCE AND DIVERGENCE
NCBTs AND RELATIVES IN NONMAMMALS
GENERAL FEATURES OF NCBTs
NCBTs IN MAMMALS
RELATIVES OF NCBTs IN MAMMALS
CONCLUDING REMARKS
APPENDICES
803
810
819
836
845
912
925
931
1) Conductive HCO3⫺ transport mediated by anion permeable channels such as GABA- and glycine-gated anion chan-
0031-9333/13 Copyright © 2013 the American Physiological Society
803
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Parker MD, Boron WF. The Divergence, Actions, Roles, and Relatives of SodiumCoupled Bicarbonate Transporters. Physiol Rev 93: 803–959, 2013;
doi:10.1152/physrev.00023.2012.—The mammalian Slc4 (Solute carrier 4) family
of transporters is a functionally diverse group of 10 multi-spanning membrane proteins
⫺
that includes three Cl-HCO3 exchangers (AE1–3), five Na⫹-coupled HCO3
transporters
(NCBTs), and two other unusual members (AE4, BTR1). In this review, we mainly focus on the five
mammalian NCBTs-NBCe1, NBCe2, NBCn1, NDCBE, and NBCn2. Each plays a specialized role in
⫺
maintaining intracellular pH and, by contributing to the movement of HCO3
across epithelia, in
maintaining whole-body pH and otherwise contributing to epithelial transport. Disruptions involving
NCBT genes are linked to blindness, deafness, proximal renal tubular acidosis, mental retardation, and
epilepsy. We also review AE1–3, AE4, and BTR1, addressing their relevance to the study of NCBTs.
This review draws together recent advances in our understanding of the phylogenetic origins and
physiological relevance of NCBTs and their progenitors. Underlying these advances is progress in such
diverse disciplines as physiology, molecular biology, genetics, immunocytochemistry, proteomics, and
structural biology. This review highlights the key similarities and differences between individual NCBTs
and the genes that encode them and also clarifies the sometimes confusing NCBT nomenclature.
MARK D. PARKER AND WALTER F. BORON
Table 1. The importance of pH regulation
Process
Pathological Associations
Cell survival
Acid-extruding mechanisms defend intracellular pH
from catastrophic, pro-apoptotic acidosis (e.g.,
Ref. 112). However, acidosis is anti-apoptotic
for some cells (e.g., Refs. 978, 1057).
Telomere structure is pH sensitive (417).
Tumor proliferation: In cancer cells,
enhanced acid-extrusion ability and
a lowering of local extracellular pH,
contributing to an acidic, tumorpermissive environment while
defending tumor pHi (546, 935).
Autophagy is reduced at acidic
extracellular pH (1058). Heart
failure: Hypoxia in combination
with acidosis is pro-apoptotic in
cardiac myocytes (519).
Na⫹ homeostasis
NCBTs and NHEs are secondary active
transporters that couple acid extrusion with Na⫹
influx, thereby contributing to regulation of [Na⫹]i
and plasma [Na⫹]. ENaC activity is modulated by
⫺
pH and [HCO3
] (163, 196, 730).
Cell migration and Wound
healing
Acid-extruders act as plasma membrane anchors
for cytoskeletal components (e.g., Ref. 243)
and can promote an isosmotic volume increase
at the leading edge of migrating cells (910).
Acid extrusion promotes wound healing (1062)
as well as dendritic spine growth (249).
Solute transport
Many solute carriers such as H⫹-coupled amino
acid transporters (95) influence or are
influenced by pH. Furthermore, acid-base status
influences the expression of other, nominally
pH-independent carriers (660, 688).
Protein folding/assembly
The stability and conformation of almost all
proteins is pH dependent, due to electrostatic
effects (946). Consequently, the oligomeric
state of diverse proteins (e.g., Refs. 145, 838,
1084) as well as interactions between protein
binding partners (e.g., Refs. 661, 687) can be
pH dependent.
Reperfusion injury: The influx of Na⫹
that accompanies enhanced acid
extrusion following ischemia can
tend to reverse Na⫹-Ca2⫹
exchangers, causing a pathological
increase in [Ca2⫹]i (956, 993,
997). Hypertension:
⫺
Dysregulation of H⫹ and HCO3
transporters is associated with
hypertension (89, 92, 1020).
Tumor metastasis: Acidosis, by
stimulating the acid-extruding
activity of NHE1, can promote
metastasis of tumor cells (151,
⫺
547). HCO3
, in its capacity as a
buffer, is inhibitory to metastasis
(410, 801).
Drug sensitivity: Acid-base status
can influence the efficacy and
toxicity of drugs (647, 705) and
acidosis induces drug resistance in
tumors via activation of Pglycoprotein (963).
Amyloidosis: Acidosis promotes
amyloid formation (294, 395, 784,
815, 936), potentially impacting the
severity of Alzheimer’s Disease and
scrapie. Carcinogenesis: The
stability of the tumor-suppressing
tetrameric form of a mutant p53 is
readily destabilized by mild alkalosis,
a mechanism suggested to underlie
the increased incidence of
carcinomas in individuals who carry
this mutation (250).
Protein glycosylation
An acidic environment in the Golgi is crucial for
appropriate localization of glycosyltransferases
and therefore for N-glycosylation of proteins
(799).
Some interactions between proteins and the
plasma membrane or between proteins and
cell-surface receptors are pH dependent (e.g.,
Refs. 255, 370).
Interactions at the cell surface
Amyloid deposition: Deposition of
amyloids is enhanced at acidic pH
(131, 171, 513, 785). Viral
infection: The fusion of viral
particles with the host plasma
membrane is pH dependent,
although the direction of the
dependence may vary between
viruses (e.g., Refs. 363, 548,
751, 803, 1046). Bacterial
colonization: The colonization of H.
pylori on the surface of gastric
mucosa is enhanced at acidic pH
(787). Moreover, in a porcine
model of cystic fibrosis, the acidity
of airway surface liquid diminishes
its antimicrobial properties (745).
Continued
804
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pH-Dependent Physiology
⫺
TRANSPORTERS
Na⫹-COUPLED HCO3
Table 1.—Continued
Process
pH-Dependent Physiology
⫺
Sensors for acid, alkali, and CO2/HCO3
(129,
181, 1105, 1107) are expressed in multiple
cell types, mediating the cellular effects of
acid-base status. Furthermore, numerous
receptor/ligand interactions are influenced by
pH (e.g., Refs. 227, 295, and 691).
DNA and protein synthesis
and stability
Incorporation of amino acids into polypeptides is
reduced under acidic conditions (451, 736).
pH-responsive elements in certain RNAs confer
increased lifetime to those transcripts in
acidosis (409).
Excessive neuronal firing can reduce neuronal pH
and in turn, neuronal excitability is reduced in
response to lowering extracellular and
intracellular pH (186, 187, 783). Most K⫹
channels are pH dependent (e.g., Refs. 67,
424, 1053). NCBTs play critical roles in
defending neuronal pHi and regulating the pH of
the neuronal microenvironment (via their action
in astrocytes and choroid plexus epithelia).
⫺
The fluid movement that follows HCO3
transport
maintains the clarity of the cornea (96) and lens
(65) and also maintains retinal attachment
(400, 534). In the inner ear, low endolymph pH
can reduce response of hair cells to auditory
stimuli (150).
Neuronal excitability
Special senses
Muscle contraction
Bone remodeling
Digestion
Multiple elements of excitation-contraction
coupling in cardiac, smooth, and skeletal muscle
are inhibited at low pH including
neurotransmitter release (586), gap junction
conductivity (379, 707), as well as the action of
the contractile apparatus (e.g., Refs. 286, 497,
892, 1045).
Bone remodeling requires H⫹ secretion (62) and
⫺
HCO3
resorption (797), thus bone maintenance
is exquisitely pH sensitive. Furthermore,
osteoclast survival is reduced by acidosis (e.g.,
Ref. 112).
Enamel formation (456), saliva secretion (555),
enzymatic digestion, and mucosal protection
⫺
(17) are all pH/HCO3
-dependent processes.
Type 2 diabetes mellitus: Elevated
⫺
serum HCO3
was associated with a
reduced risk of developing type 2
diabetes in a study of 650 women
(625). Tumor proliferation:
Expression of the acid sensor
TDAG8 in tumor cells enables the
cells to adapt to the extracellular
acidic environment (415). Anxiety
disorders: Acidosis and detection
of H⫹ by the acid sensor ASIC-1a
elicits acquired fear behavior.
Overexpression of ASIC-1a in mice
is a model of anxiety (204, 205,
1032, 1117).
Altered neuronal excitability:
Disruption of NCBT genes is
associated with autism, epilepsy,
mental retardation, and migraine
(360, 411, 516, 830, 930).
Loss of vision: Mutations in acidbase transporters are associated
with cataracts, glaucoma, and
retinopathy (e.g., Refs. 30, 93,
411). Acidosis induces retinopathy
in neonatal rats (391, 392). Loss
of hearing: Mutations in acid-base
transporters are associated with
hearing loss (e.g., Refs. 93, 473).
Paralysis: Lactic acidosis (e.g., Ref.
85) and renal tubular acidosis
(e.g., Ref. 119) result in muscle
weakness.
Bone remodeling defects: H⫹
secretion defects in osteoclasts
are associated with osteopetrosis
(e.g., Refs. 455, 866), whereas
whole-body acidosis can be
associated with bone dysplasia
(e.g., Refs. 313, 602).
Poor dentition: Defects in acid-base
transporters result in defective
enamel deposition (540, 617).
Ulceration: Metabolic and
respiratory acidoses increase the
incidence of gastric lesions (142,
507). Gut lumen pH is unusually
acidic in some individuals with
ulcerative colitis (690). Diarrhea:
Dysregulation of acid-base
transport can result in decreased
nutrient absorption, increased fluid
secretion, and diarrhea (388,
938, 1092).
Continued
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Cell signaling
Pathological Associations
MARK D. PARKER AND WALTER F. BORON
Table 1.—Continued
Process
pH-Dependent Physiology
Immune response (544)
Extracellular acidosis activates neutrophils (978)
but reduces TNF-␣ secretion by alveolar
macrophages (82). Superoxide production by
NADPH oxidase during the respiratory burst is
accompanied by a decrease in pHi that is
countered by the action of H⫹ channels (230).
Fertility
Multiple aspects of male and female fertility,
including sperm maturation and cervical mucus
⫺
release are influenced by pH and HCO3
(597,
665).
Pathological Associations
Tumor proliferation: The reduction
of macrophage cytotoxicity in the
acidic tumor microenvironment
would promote tumor survival (82).
Immunodeficiency: Inability to
defend macrophage pHi during
respiratory burst might reduce the
ability of macrophages to counter
bacterial infection (discussed in
Ref. 230).
Reduced fertility: Mice with
⫺
disrupted HCO3
transporters are
sub- or infertile (e.g., Refs. 165,
389, 638).
nels (98, 460), the cystic fibrosis transmembrane conductance regulator CFTR (752), ClC channels (827), and
Ca2⫹-activated chloride channels (776, 777).1
2) Apical Na⫹-independent Cl-HCO3 exchange, effected by
anion exchangers encoded by members of the solute carrier
26 (Slc26) gene family (Slc26a3, Slc26a4, Slc26a6, and
Slc26a9), reviewed in References 153, 259, and 888.2
3) A basolateral Na ⫹ -independent SO 4 -2HCO 3 exchanger, or oxalate-2HCO 3 exchange encoded by
Slc26a1 (474, 517, 525).
4) Electroneutral K/HCO3 cotransport. The molecular
identity of the responsible protein(s) has yet to be established (386, 387, 570, 1097).
5) Basolateral Na⫹-independent Cl-HCO3 exchange, mediated mainly by the electroneutral anion exchangers AE1
1
In most cases, these channels are at best poorly permeable to
⫺
HCO3
and, in most cases, the physiological significance of this permeability is not demonstrated.
2
For the purposes of this review, we depict Slc26a3, -4, -6, -7, and
-9 as electroneutral Cl-HCO3 exchangers; in fact, the molecular
action of these Slc26 transporters is controversial. Slc26a3 has
been described as being capable of electroneutral Cl-HCO3 exchange
by some (26, 908) but electrogenic 2Cl-HCO3 exchange by others
(499, 871). Slc26a4 is capable of electroneutral Cl-HCO3 exchange
(872), a description that is uncontested. Slc26a6 has been described as being capable of electroneutral Cl-HCO3 exchange by
some (185) but electrogenic Cl-2HCO3 exchange by others (499,
871, 1052). Slc26a7 has been described as an electroneutral
Cl-HCO3 exchanger (743), and also as a pH-sensitive Cl⫺ channel
with no anion exchange activity (488). Slc26a9 has been decribed
as a Cl-HCO3 exchanger of undetermined electrogenicity/electro⫺
neutrality (1055), an electrogenic nCl-HCO3 exchanger with a HCO3
⫺
independent Cl⫺ conductance (168), a HCO3
-independent Cl⫺ chan⫺
nel with no anion exchange activity (258), and also as a HCO3
stimulated Cl⫺ channel with no anion exchange activity (609). The
reasons underlying the apparent disparities among studies have not
been determined.
806
(Slc4a1),3 AE2 (Slc4a2), and AE3 (Slc4a3) and perhaps
some members of the Slc26 family (e.g., Slc26a7).
6) Electrogenic Na/HCO3 cotransport, mediated by NBCe1
(Slc4a4) and NBCe2 (Slc4a5), which are predicted to operate with varying stoichiometry in different cell-types (6a
versus 6b in FIGURE 1).
7) Electroneutral Na/HCO3 cotransport, mediated by
NBCn1 (Slc4a7) and NBCn2 (Slc4a10).
8) Na⫹-driven Cl-HCO3 exchange, mediated by NDCBE
(Slc4a8).
Groups 5– 8 include members of the Slc4 family that, in
vertebrates, are normally located in the basolateral (or
equivalent) membranes of polarized cells, in some instances
complementing the usually apical (or equivalent) distribution of certain HCO3⫺-transporting Slc26 family members.
Groups 6 – 8 are collectively referred to as Na⫹-coupled
bicarbonate transporters (NCBTs) and are the major focus
of the present review.
The general predicted topology of mammalian, and likely
all vertebrate, Slc4s is exemplified by the depiction of human NBCe1 in FIGURE 2A. Typically, each Slc4 protein has
a large NH2-terminal (Nt) cytoplasmic domain, followed
by a large multi-spanning transmembrane domain (TMD)
3
Following the recommendations of The HUGO (Human Genome
Organization) Gene Nomenclature Committee (HGNC), we use the
terms “SLC4” (gene) and “SLC4” (gene-product) only in instances
when we are specifically and exclusively referring to human genes
and products. The terms “Slc4” and “Slc4” are used in reference to
vertebrate genes/products in general (even if that grouping includes
humans), whereas “Slc4-like” and “Slc4-like” refer to related nonvertebrate genes. The common names AE, NBC, and NDCBE are
capitalized throughout, independently of parent organism.
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Proteins, processes, and pathologies in mammals that are influenced by or that influence pH. Processes and
diseases that are specifically related to NCBT function and dysfunction are discussed in detail in later sections
of the review.
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
Apical
Tight junction
Basolateral
–
HCO3–
channel
HCO3–
2 HCO3
1
3
4
Cl–
5
K+
HCO3–
HCO3–
Basolateral SO42––2 HCO3– exchanger
Electroneutral K+/HCO3– cotransporter
Basolateral Cl––HCO3– exchanger
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6a
SO42–
Na+
3 HCO3–
Electrogenic Na+/HCO3– cotransporters
Na+
Apical
Cl–-HCO3–
exchanger
HCO3–
2
Cl–
2 HCO3–
Na+
HCO3–
6b
Electroneutral Na+/HCO3– cotransporter
7
Na+
8
2 HCO3–
Cl–
Electroneutral Na+–driven
Cl–-HCO3– exchanger
FIGURE 1. Functional classifications of bicarbonate transporters. Diagram of a generic epithelial cell show⫺
ing the typical subcellular distribution of the 8 classes of HCO3
transporters. Anion channels (1) and anion
⫺
exchangers of the Slc26 family (2) perform HCO3 secretion across the apical membrane. Basolateral Slc26a1
⫺
functions as SO42--2HCO3
exchanger (3). An as yet unknown transporter (4) is presumed to be responsible for
a basolateral K/HCO3 cotransport activity in the inner medulla. Members of the Slc4 family (5–8) are usually
located in the basolateral membranes of polarized epithelia. Cl-HCO3 exchangers (5) and electrogenic Na/
⫺
HCO3 cotransporters with a calculated 1:3 stoichiometry (6a) act as acid-loaders, supporting HCO3
absorption
into the blood. Electrogenic Na/HCO3 cotransporters with a 1:2 stoichiometry (6b), electroneutral Na/HCO3
⫺
cotransporters (7), and Na⫹-driven Cl-HCO3 exchangers (8) act as acid-extruders, supporting HCO3
secretion
across the apical membrane by transporter classes 1–2.
that includes one glycosylated extracellular loop, and concludes with a shorter COOH-terminal cytoplasmic domain
(Ct). As depicted in FIGURE 2B, nonvertebrate Slc4-like (see
footnote 3) products, such as those from bacteria, fungi,
amoebas, and plants, are predicted to retain the same general topology but to have shorter Nts and to have extracellular loops of varying lengths.
The molecular identity of the Na⫹-independent Cl-HCO3
exchangers AE1-AE3 (included in group 5, above) has been
known for some time. It is more than 30 years since AE1
was first demonstrated to be the erythrocyte anion transporter (1040). The cloning of the murine Slc4a1 cDNA that
encodes AE1 was reported in 1985 (510) and was soon
followed by the discovery and cloning of Slc4a2 (23, 242)
and Slc4a3 (509) products. These three genes appeared to
be the extent of the gene family until 1997, when Romero et
al. (809) published the cDNA and the elucidated protein
sequence of an electrogenic Na/HCO3 cotransporter from
the tiger salamander, Ambystoma tigrinum. Electrogenic
Na/HCO3 cotransport had first been described in the salamander proximal tubule (PT) by Boron and Boulpaep 14
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807
MARK D. PARKER AND WALTER F. BORON
A
EL3
C
C
TMD
C
C
1
2
3
4
5
Lumen
6
7
8
9
10
11
E 13
14
Cytosol
Ct
Nt
H2N
B
TMD
Nt
1 23 4 5
EL3
6 7
9
1011 1314
E
(human NBCe1-A; 1,035 aa)
Mammalian
(bacterial ‘Nitro’; 513 aa)
Bacterial
Fungal
Plantal
8
Ct
(yeast Bor1p; 576 aa)
100 aa
(thale cress AtBOR1; 729 aa)
(slime mold Slc4-like product; 768 aa)
Amoebal
FIGURE 2. Presumed topology of NCBTs and Slc4-like transporters. Presumed topology of the electrogenic
Na/HCO3 cotransporter NBCe1 (A), representing a probable common structure for all five mammalian NCBTs
and most nonmammalian NCBTs. The model shows the extended cytosolic amino- and carboxy-terminal
domains (Nt and Ct) linked via a transmembrane domain (TMD) that includes 14 transmembrane spans (TMs),
one of which is thought to be an extended region (E) rather than an ␣-helix. In mammalian NCBTs, the third
extracellular loop (EL3) between TMs 5 and 6 usually includes multiple cysteine residues (C) and multiple
putative glycosylation sites (Y). A sequence alignment displaying these features for human NCBTs is provided
in Appendix I. Pictorial depiction of NBCe1 domain sequences aligned against homologous regions of nonvertebrate Slc4-like transporters (B). Horizontal purple bars represent protein sequence laid out from Nt to Ct.
Gaps in sequence alignment are depicted as horizontal lines. Vertical bars represent position of ␣-helical TMs.
Note the shorter Nt and EL3 in nonvertebrates. The amoebal Slc4-like transporter includes an extended Nt
(yellow region), but it shares no significant sequence identity with the extended Nt of vertebrate Slc4s. The
sequence of nonmammalian Slc4-like protein is provided in Appendix II.
years earlier (103), and the cloning of the responsible gene
product allowed sequence comparisons that importantly
demonstrated that NCBTs were members of the same Slc4
family as AE1–3. The salamander cDNA reported by Romero et al. is now recognized as the archetypal Slc4a4 gene
product. Work from several groups then revealed the existence of six further members of the vertebrate Slc4 gene
family (337, 720, 765, 767, 982, 1021), bringing the total
808
number to 10. These novel genes were designated Slc4a5
and 7–11 (Slc4a6 was rescinded, see below). The products
of 5 of these 10 Slc4 genes (NBCe1, NBCe2, NBCn1,
NBCn2, and NDCBE) have demonstrated NCBT activity.
The function of AE4, the product of Slc4a9, is controversial, but it is reported to mediate Cl-HCO3 exchange in
some heterologous systems. Bicarbonate transporter related
protein 1 (BTR1), the product of Slc4a11, likely mediates
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HOOC
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
borate transport, a function common to the Slc4-like transporters (the BORs) of some fungi and higher plants. The
values in TABLE 2 and the unrooted phylogenetic tree in
FIGURE 3 summarize the relatedness, at the level of protein
sequence, of the TMDs of human SLC4s. Note that SLC4
function follows sequence relatedness. The first major sequence classification corresponds to AEs (red group) versus
NCBTs/AE4 (gray group) versus BTR1. The second major
sequence classification corresponds to electrogenic NCBTs
(blue group) versus electroneutral NCBTs (red group).
BTR1
Putative Boron
Transporter
(SLC4A11)
AE3
(SLC4A3)
Cl-HCO3
Exchangers
AE2
(SLC4A2)
AE1
(SLC4A1)
Electroneutral Na/HCO3
Cotransporters
AE4
(SLC4A9)
NBCn2
(SLC4A10)
0.1
NDCBE
NBCe1
(SLC4A4)
(SLC4A8)
NBCn1
(SLC4A7)
NBCe2
(SLC4A5)
C. Review Outline
Electrogenic Na/HCO3
Cotransporters
In section III we review the actions and roles of NCBTs and
Slc4-like transporters in nonmammalian species. In addition to being of interest to comparative physiologists, this
discussion brings together, for the first time, data that provide insight into how the actions and roles of Slc4-like proteins have evolved to their present status in mammals.
In section IV, we look at the structural features/domains of
a typical mammalian NCBT. Here we present a second way
to consider the structural relation between NCBTs: an analysis of conserved and variable protein regions. We also
present a summary of maneuvers known to inhibit or stimulate mammalian NCBTs.
Table 2. Relatedness among human SLC4 protein sequences
FIGURE 3. Relatedness among human SLC4s. The unrooted phylogram displays the relatedness at the level of protein sequence among
the transmembrane domains of the 10 human SLC4 proteins. Note
how transporter function correlates with protein sequence similarity.
The phylogram was generated using ClustalW (183) and TreeView
(704). A sequence alignment of the 10 human SLC4s is provided in
Appendix I, and the protein sequence identity among the transmembrane domains of human SLC4s is provided in TABLE 2.
In section V we then consider, in turn, each of the 5 mammalian NCBTs and, for each, 10 categories of key characteristics. The italicized terms below correspond to the titles
of the headings in section V.
A) Summary. A précis of the key characteristics, actions,
and roles for each NCBT, serving as a quick reference guide
for the casual reader.
B) Nomenclature. A definitive guide to the naming of each
NCBT, necessary because nonstandard and redundant nomenclatures have made collation and interpretation of the
literature confusing. In each case we link the nomenclature
used in this review with a GenBank sequence accession
number.
C) Molecular action. A detailed account of the substrates
and transport modes of each NCBT.
NBCe1 NBCe2 NBCn1 NBCn2 NDCBE AE4 BTR1
AE1
AE2
AE3
NBCe1
NBCe2
NBCn1
NBCn2
NDCBE
AE4
BTR1
39
42
42
100
38
39
39
71
100
38
41
42
57
50
100
38
40
42
58
55
81
100
39
41
41
58
54
81
84
100
38
43
43
62
58
52
52
52
100
28
30
30
28
28
28
29
30
29
100
Percentage identities among the protein sequences of human
SLC4s transmembrane domains. Identities were computed by pairwise BLAST (951). AE1–AE3 share 50 – 60% identity within their
transmembrane domains. Alignments of human NCBT protein sequences are provided in Appendix I, and GenBank protein accession
numbers are provided in Appendix IV.
D) Genome. A summary of the key features of the genes
encoding each NCBT.
E) Structural features and variants. A definitive guide to the
known products created from each NCBT gene.
F) Distribution. A comprehensive detailing of the localization of NCBT transcripts and proteins from the intracellular to the whole organ level.
G) Physiological roles. A review of the known and speculated physiological roles of each NCBT in specific tissues.
H) Causes of upregulation. A consideration of the perturbations that result in upregulation of NCBT at the level of
transcript/protein abundance or activity.
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In the first major part of our review, section II, we examine
the NCBTs and Slc4-like genes from bacteria, fungi, plants,
and animals and consider how the Slc4 gene family has
diverged from a single common ancestor into the 10 members that we recognize today, including the 5 mammalian
NCBTs. In addition, we examine the genealogy of extant
vertebrate NCBT genes based on an analysis of conserved
exon boundaries. Section II should be valuable to those
interested in any Slc4 protein.
MARK D. PARKER AND WALTER F. BORON
I) Causes of downregulation. A consideration of the perturbations that result in downregulation of NCBT at the level
of transcript/protein abundance or activity.
J) Consequences of dysfunction. A review of the diverse
pathological states associated with defects and variations in
NCBT genes and products.
Characteristics G–J, taken together, provide an integrated
picture of the importance of each NCBT.
In section VII, our final section entitled “Concluding Remarks,” we draw together from Section V several recurring
themes, unresolved issues, and emerging topics in the
NCBT field.
Throughout the review we summarize critical information,
for quick reference, in the form of Tables. Here the reader
will find guides to the importance of pH regulation, the
relatedness among Slc4 and Slc4-like gene products, NCBT
inhibitors, NCBT distribution, and pathological mutations
in the SLC4A4 gene.
In our Appendices, we complement the content of the review with detailed information, such as complete sequence
alignments of the NCBT proteins and their splice variants.
We also present tables of GenBank protein accession numbers of all of the Slc4 and Slc4-like gene products and variants discussed in this review to allay confusion about nomenclature. The accession numbers are hyperlinked to the
National Center for Biotechnology Information (NCBI) database for ease of reference. We also present some additional data about NCBT distribution, namely: 1) an NCBT
expression pattern in humans and mice inferred from a
tabulation of the origins of NCBT expressed-sequence tags
deposited on a public database; 2) a discussion of “antiNBC3” immunoreactivity, which discloses a distribution
pattern for NBCn1 (Slc4a7) that is different from that suggested by other probes; and 3) a discussion of several apparently conflicting reports of AE4 (Slc4a9) localization
within the mammalian kidney. These last two appendices
will be useful for those who seek to make sense of the often
conflicting data concerning the distribution of these proteins.
This review is not intended to focus on the regulation of pHi
per se, although the NCBTs play key roles in this task.
810
II. NCBT EMERGENCE AND DIVERGENCE
A. Summary
In this section we consider how the five mammalian NCBT
genes emerged from a single primordial Slc4-like gene. As
we shall see, gene and genome duplications as well as gene
losses have resulted in the inclusion of a diverse number of
Slc4-like genes in the genomes of diverse organisms. Fungal
and plantal Slc4-like genes predominantly encode boron
transporters. In the animal lineage, distinct NCBT-like
genes appeared no later than the emergence of Eumetazoa
such as sea anemones. The most primordial Slc4-like geneproduct with NCBT activity is the Na⫹-driven anion/bicarbonate exchanger ABTS-1 from the nematode worm, Caenorhabditis elegans. The genome of the chordate sea squirt
Ciona intestinalis includes three Slc4-like genes, one of
which shares a single common ancestor with the five mammalian NCBTs and “AE4.” The emergence of individual
NCBTs was initiated by the divergence of an NBCe1/
NBCe2/“AE4” ancestor from an NBCn1/NDCBE/NBCn2
ancestor. The five mammalian NCBTs were probably distinct entities no later than the emergence of primordial vertebrates such as lampreys.
B. Emergence, From an Ancestral
Prokaryote to Early Chordates, of
AE-like, NCBT-like, and BOR-like Genes
The recent proliferation of genome sequence data, backed
up by the physiological characterization of certain products, allows us to begin to appreciate the diversity of Slc4
and Slc4-like genes and products. In FIGURE 4, which gives
examples of the major taxonomic divisions, we represent
the taxonomic relationship of diverse organisms along with
their known complement of Slc4-like genes. Some of these
products have demonstrated Cl-HCO3 exchanger (AE),
Na-coupled HCO3 transporter (NCBT), or borate trans-
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In section VI we provide a similar, though abbreviated,
consideration for the three AEs (AE1–3) and the related
products AE4 and BTR1. Section VI, A and B, with their
organization of the NCBT literature in light of the wealth of
AE data, will be of particular value to those new to the
larger Slc4 field. Our consideration of AE4 and BTR1,
which are of interest to the NCBT community, are the first
detailed reviews of these unusual family members.
Rather, the reader is referred to the review by Roos and
Boron (811), the more recent chapter by Bevensee et al.
(77), or the analysis of Boron (101). Likewise, the present
review does not focus on the kinetics or thermodynamics of
HCO3⫺ transport, for which we would recommend References 339 and 529. For a more concise overview of NCBTs,
we direct the reader to recent reviews in 2004 by Romero et
al. (807), in 2006 by Pushkin and Kurtz (772), in 2007 by
Parker and Boron (714), and in 2009 by Casey and Cordat
(153), Romero et al. (805), and Boron et al. (104). We
intend this document to provide a clear review of NCBT
genes and proteins for those new to the field, as well as an
up-to-date and comprehensive reference resource for Slc4
researchers. Note that meta-analyses and reinterpretations
of published data that do not include a link to a published
article are the opinions of the authors.
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
Prokaryota
Nitrifying bacterium
Nitrococcus mobilis
Eukaryota
ZP_01128815
Primitive
Haptophyceae
Amoebozoa
Marine Phytoplankter*
Emiliania huxleyi
Plants
Multiple fragments**
fra
ag
Primitive 7
Basidiomycota*
Inky-cap mushroom
Coprinopsis cineria
EAU85942
BOR-like 15
Porifera
Sponge*
Suberites domuncula
CAF32326 (NBCSA)
AE-like
9
BOR-like 3G
Placozoa
Tablet animal*
Trichoplax adhaerens
XP_002110840
AE-like
9
XP_002110841
AE-like
16
XP_002115332/3
XP_001765760
Primitive 13
o na
otin
na
Pezizomycotina
Bilateria
Moss*
Physcomitrella patens
Tracheophyta
Bryophyta
Saccharomycotina
Black mold
n
i err
Aspergillus nig
niger
Brewer’s Yeast
Saccharomyces cerevisiae
BOR-like 17
Cnidaria
Starlet sea anemone*
Nematostella vectensis
CAK43550
BOR-like 6G
XP_001623893
AE-like
8
XP_001766018 (PpBOR1) BOR-like 29
CAK39400
O
14
BOR-like
XP_001637907
AE-like
11
XP_001759676 (PpBOR2) BOR-like 28
CAL00656
BOR-like 23
XP_001619189**
NCBT-like 17
XP_001632673** taken
with XP_001632674**
BOR-like 24
Monocotyledons
Thale cress
Arabidopsis thaliana
Rice
Oryza sativa
75
Pseudocoelomata
Nematode worm
Caenorhabditis elegans
NP_001078071 (AtBOR1)
BOR
69
NP_001067049 (OsBOR1)
BOR
NP_191786 (AtBOR2)
BOR
59
ABD78950 (OsBOR2)
BOR-like 30
NP_001024827 (ABTS-4)
AE-like
6G
NP_187296 (AtBOR3)
BOR
45
BAG92966 (OsBOR3)
BOR
27
NP_492258 (ABTS-1)
NCBT
21
NP_172999 (AtBOR4)
BOR
27
ABD78951 (OsBOR4)
BOR-like 30
NP_509936 (ABTS-2)
BOR-like 14
NP_177619 (AtBOR5)
BOR
24
NP_001033333 (ABTS-3)
BOR-like 18
NP_197925 (AtBOR6)
BOR-like 25
NP_194977 (AtBOR7)
BOR-like 24
Mollusca
49
BOR
Coelomata
Eudicotyledons
NP_014124 (Bor1p)
Protostomia
Deuterostomia
Panarthropoda
Longfin Inshore Squid*
Echinodermata
Doryteuthis (Loligo) pealei
Chordata
Sea urchin*
Strongylocentrotus purpuratus
Fruit fly
Drosophila melanogaster
Sea squirt*
Ciona intestinalis
ABF06445
AE-like
16
NP_648357 (CG8177)
‘AE-like’ 15
XP_793649**
‘AE-like’
10
XP_002124608
ABF06444 (sqNBCe)
NCBT
18
NP_523501 (NDAE1)
NCBT
XP_791964
‘AE-like’
16
XP_002124093
NCBT-like 66
AAN75454 (sqNDCBE)
NCBT
22
NP_001073019 (Sp-NBC)
‘NCBT-like’ 21
XP_002128564
BOR-like 73
XP_786033
‘NCBT-like’ 22
XP_784629
‘BOR-like’
18
AE-like
66
15
FIGURE 4. Diversity of nonvertebrate Slc4-like transporters. The cladogram represents the evolutionary relationships of a variety of organisms, based on the taxonomy defined by The Tree of Life Web Project (http://
tolweb.org) and the taxonomy database at NCBI (839). Each organism is represented by a boxed table that
includes, in the top row, the common and scientific name of the organism. If the full genome sequence of the
organism has not been published, the organism’s name is noted with an asterisk. The remaining rows of each table
provide information about the Slc4-like proteins that are encoded by the genome of each organism. These rows are
divided into three columns: Column 1 lists the GenBank protein accession number and, where appropriate, common
name for each Slc4-like protein. Partial sequences are marked with a double asterisk. Column 2 lists the function
of each transporter (AE, NCBT, or BOR) or, if unknown, the assignment of each protein to one of the four groups
(AE-like, NCBT-like BOR-like, or Primitive) defined in text that indicates their relatedness to their reference sequences for each kingdom (red text). Column 3 shows a numerical “divergence score” (DS, see text) denoting the
extent of similarity to the reference protein. Proteins that do not bear a strong similarity to any one of the reference
proteins are marked with a “G” for “generic.” A hyperlink to the protein sequence of each transporter is provided in
Appendix II.
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Primitive 2G
XP_646790
Eumetazoa
XP_001417272
XP_001417844
3-5G
Ascidiomycota
Green alga
cim
mari
m
rinus’
Ostreococcus ‘luc
‘lucimarinus’
Embryophyta
a
a
Chlorophyta
Primitive
Social amoeba
Dictyostelium discoideum
Animals
Fungi
MARK D. PARKER AND WALTER F. BORON
1) “Primitive” (present only in bacteria and plants): most resembling the bacterial Slc4-like transporter that we have provisionally termed “Nitro” (see “Primitive” in FIGURE 4/Plants
and FIGURE 5).
2) AE-like (present only in animals): most resembling the
sea-squirt protein that shares a common ancestor with all
vertebrate Na⫹-independent Cl-HCO3 exchangers (see
Ciona “AE-like” in FIGURE 4/Sea Squirt). AE-like transporters cluster with the Ciona AE-like reference protein on
“BOR” Group II
AtBOR4*
OsBOR2
OsBOR3*
an unrooted phylogenetic tree (FIGURE 6) and exhibit a
characteristic “fingerprint” of sequence identity inasmuch
as they are more similar to NCBTs than BORs.
3) NCBT-like (present only in animals): most resembling
the sea-squirt protein that shares a common ancestor
with all vertebrate Na⫹-coupled HCO3⫺ transporters (see
“NCBT-like” in FIGURE 4/Sea Squirt). NCBT-like transporters cluster with the Ciona NCBT-like reference protein
on an unrooted phylogenetic tree (FIGURE 6) and exhibit a
characteristic “fingerprint” of sequence identity inasmuch
as they are more similar to AEs than BORs. Of course, all
invertebrate Slc4-like transporters with demonstrated Na⫹coupled HCO3⫺ transport function fall into this category.
4) BOR-like: because borate transporter proteins share little
identity across kingdoms (22–27%; see TABLE 3), we define
“BOR-like” as follows. For plants, most resembling the established borate transporter of thale cress (see “AtBOR1” in
FIGURE 4/Thale Cress) than our bacterial Slc4-like reference
protein “Nitro.” Of course, all plantal Slc4-like transporters
with demonstrated borate transport function fall into this category. For fungi, most resembling the established borate transporter of brewer’s yeast (see “Bor1p” in FIGURE 4/Brewer’s
Yeast) than “Nitro”. For animals, most resembling the seasquirt protein that shares a common ancestor with the vertebrate boron transporter BTR1 (see “BOR-like” in FIGURE
4/Sea Squirt). Thus an assignment of BOR-like character is
kingdom-specific. For example, a BOR-like transporter from
AtBOR5* AtBOR6
AtBOR7
Alga
DS:2G
OsBOR4
AtBOR2*
AtBOR1*
OsBOR1*
Alga
DS:7
AtBOR3*
Moss
Moss
PpBOR1
PpBOR2
“BOR” Group I
“Primitive”
* Demonstrated borate transporters
Moss
DS:13
FIGURE 5. Relatedness among plantal Slc4-like proteins. The unrooted phylogram displays the relatedness,
at the level of protein sequence, among the transmembrane domains of the 16 plantal Slc4-like proteins shown
in FIGURE 4. Proteins are identified by the name of parent organism and the common name or the divergence
score (DS), of each transporter, as listed in FIGURE 4. Note how the BOR and/or BOR-like transporters fall
into two groups. It is unknown if members of Group I versus Group II are functionally distinct, like members of
animal Slc4 groups in FIGURES 3 AND 6. The phylogram was generated using ClustalW (183) and TreeView
(704).
812
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porter (BOR) function and thus we have assigned them as
being AEs, NCBTs, and BORs. However, the function of
many of the products is currently unknown. The phylogenetic relationships between the proteins in these groups are
shown in FIGURE 5 (plants) and FIGURE 6 (animals, i.e.,
metazoa). We are not showing dendrograms for the other
major taxonomic divisions that contain identified Slc4-like
genes because: 1) bacteria have only two such genes, 2) the
only known Slc4 sequences from phytoplankton are fragments from an unknown number of distinct Slc4-like products that cannot be meaningfully grouped, 3) the only two
known amoebal genomes each has only one such gene, and
4) fungal Slc4-like genes are all BOR-like and differences
among them appear to reflect mainly species divergence. We
have attributed presently uncharacterized Slc4-like transporters to one of four groups, according to their relatedness
at the protein level (within their transmembrane domains)
to selected reference proteins.
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
Urchin DS:10
Placozoa
SD:9 & 16 Squid
DS:16
Anemone DS:8
Ciona “AE-like”
nc
e
Anemone DS:11
re
Fly DS:15
fe
Sponge DS:9
Re
C68177
ro
up
NBCSA
Urchin DS:16
G
Anemone DS:24
Urchin DS:16
Grou
Nematode DS:18
p Refe
rence
ABTS-3
Ciona “NCBT-like”
Urchin DS:22
Tablet DS:17
u
Gro
pR
efe
r
e
enc
Urchin DS:21
Sp-NBC
Squid NDCBE
Squid NBCe
Fly NDAE
Nematode NCBT
Nematode DS:14
ABTS-1
ABTS-2
Nematode
DS:6G
ABTS-4
Generic
0.1
FIGURE 6. Relatedness among invertebrate Slc4-like proteins. The unrooted phylogram displays the relatedness, at the level of protein sequence, among the transmembrane domains of the 23 invertebrate Slc4-like
proteins shown in FIGURE 4. Proteins are identified by the name of parent organism and the common name
or the divergence score (DS), of each transporter, as listed in FIGURE 4. Note how the transporters fall into
four groups: 1) including AEs and AE-like transporters (red), 2) including NCBTs and NCBT-like transporters
(blue), 3) including BORs and BOR-like transporters (green), and 4) a “Generic” outlier (black) that does not fall
into any of the three previous groups. The phylogram was generated using ClustalW (183) and TreeView
(704).
worms is really “Ciona-BOR-like,” and not particularly
“Bor1p-like” or “AtBOR1-like.” Within a kingdom, BORlike transporters cluster with their BOR-like reference protein
on an unrooted phylogenetic tree (FIGURES 5 AND 6) and in
the majority of cases are more similar to NCBTs than AEs.
We chose the sea squirt (Ciona intestinalis) as our animal
reference point for items 2– 4 in the list immediately above
because the sea squirt is the most primordial animal with
three genes, each of which, on the basis of deduced amino
acid sequence and conserved exon boundaries, shares a sin-
Table 3. Relatedness among representative Slc4-like transporters from bacteria and eukaryotes
Domain:
Bacteria
Kingdom:
Eukaryota
Amoebozoa
Plantae
Genus:
Nitrococcus
Dictostelium
Arabidopsis
Saccharomyces
Fungi/Metazoa
Gene product:
Nitrococcus “Nitro”
Dictyostelium
Arabidopsis AtBOR1
Saccharomyces
Bor1p
Ciona AE-like
Ciona NCBT-like
Ciona BOR-like
“Nitro”
100
“Dicty”
33
100
AtBOR1
31
29
100
Bor1p
25
26
33
AE-like
36
27
28
NCBT-like
35
28
27
BOR-like
32
36
26
100
25
100
27
41
100
22
27
28
100
Ciona
Percentage identities among the transmembrane domain sequences of Slc4-like proteins. Identities were
computed by pairwise BLAST (951). Accession numbers are provided in Figure 4. Note that gaps in protein
sequence alignments (represented in Figure 2) reduce the computed percentage identity between Slc4-like
proteins from different genera. GenBank protein accession numbers are provided in Appendix II.
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Ciona “BOR-like”
MARK D. PARKER AND WALTER F. BORON
gle common ancestor with the three mammalian AEs
(Ciona AE-like), or the five mammalian NCBTs (Ciona
NCBT-like), or the singleton mammalian BTR1 (Ciona
BOR-like).
For amoebae, we lack an amoebozoan reference protein.
Therefore, in this special case, we compared the Slc4-like
protein from social amoeba Dictyostelium to all of our
Slc4-like reference sequences. It is revealed to share most
identity with the Ciona BOR-like transporter (36%) and
“Nitro” (33%; see TABLE 3). Thus we assign it as BOR-like
with a DS of 3G compared with “Nitro” (FIGURE 4/Social
Amoeba).
1) Of the many hundreds of complete bacterial genome
sequences presently available, only two, those of the marine
bacterium Nitrococcus mobilis and the opportunistic
pathogen Segniliparus rugosus, contain an Slc4-like gene.5
2) In a sampling of 34 fungal genomes (not shown), each
includes between one and three Slc4-like genes (all BORlike). About one-third of these genomes (predominantly of
the classes Eurotiomycetes and Sordariomycetes) contain
more than one Slc4-like gene.
3) A number of overlapping and nonoverlapping Slc4-like
sequence fragments have been identified in the genome of
the phytoplankter Emilyiana huxleyi (621, 795). Analysis
of these fragments suggests that they might be derived from
more than two, and perhaps as many as four, Slc4-like
genes. Fragments of sufficient length to be reliably analyzed
appear to be technically BOR-like, although generic.
4) Only two amoebal genomes are known, each from a different species of Dictyostelium. Each genome includes one Slc4like gene, both are technically BOR-like, but generic.
5) Plant genomes, which predominantly encode BOR-like
products, include between two and seven Slc4-like genes
(FIGURE 4/Plants), the number of genes being greatest in
more recently emerged clades. This trend likely reflects a
gradual accumulation of Slc4-like paralogs, resulting from
gene/genome duplication. None of these products is more
AE-like or NCBT-like than BOR-like. Some plant strains
have multiple copies of the same BOR gene. For example,
the boron tolerant “Sahara” cultivar of barley may have
four times as many copies of the BOR1 gene as the boronsensitive cultivar “Clipper” (927).
Although our analysis is limited by the availability of complete genome sequences for key organisms, we find that the
6) Animal genomes include 2 or more Slc4-like genes, and
most vertebrate genomes include at least 10. The number of
Slc4-like genes is not always greater in more recently
emerged clades, demonstrating that some Slc4-like genes
have been lost following the emergence of certain clades
(e.g., the fruit fly has fewer Slc4-like genes than most other
animals). Animal Slc4-like products predominantly fall into
the three categories: AE-like, NCBT-like, and BOR-like
(which are underrepresented). Some fish genomes include
two similar copies of each Slc4 gene, reflecting a recent
genome duplication event.
4
The DS of an Slc4-like transporter is the difference between
1) the percent identity (computed by a pairwise BLAST alignment
at http://blast.ncbi.nlm.nih.gov; see Ref. 951) of each transporter with its most similar reference protein and 2) the averaged
percent identity of the transporter with the other reference proteins to which it has been compared.
5
Many partial, unattributed “Nitro”-like DNA sequences are
present in the environmental genome database, making it likely
that many other Slc4-like transporters are encoded in the genomes of marine bacteria. For example, nucleotide accession
numbers AACY01572225, AACY01408744, AACY01136643,
and AACY01500593.
For phytoplantkon, we also lack a reference protein. Compared against all other reference proteins, the four fragmented Slc4-like sequences appear to be primitive with DS
of 3–5G.
1. Copy number of Slc4-like genes
in diverse genomes
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Because the assignments to the AE-like, NCBT-like, and
BOR-like groups are not always clear cut, FIGURE 4 includes-for each accession number-a “divergence score”
(DS)4 that is a quantitative index of the protein’s divergence
from a hypothetical “generic state.” A perfectly generic
transporter, one that bears no greater resemblance of any
one of the reference proteins to which it is compared, has a
DS of zero. In the example case of “NCBT-like” transporters, the maximum DS is 66 because our reference NCBTlike transporter from Ciona exhibits an average 34% identity (i.e., 100% - 34% ⫽ 66%) with our AE-like and BORlike reference genes (TABLE 3). These scores provide an
index of how “AE-like,” “NCBT-like,” or “BOR-like” any
particular transporter is. We note that one nematode transporter with a DS of 6, which is more like the Ciona AE-like
gene-product than either the NCBT- or BOR-like products,
does not group with its assigned reference proteins in the
phylogenetic tree in FIGURE 6. Thus we have assigned it as
generic (noted by a “G” following the “DS” in FIGURE 4).
Based on this assessment, we have also marked with a “G”
plant and yeast transporters that have a DS of 6 or lower,
indicating their possible generic nature. Our bacterial reference protein “Nitro” is the most generic of all of the
protein considered here (DS of 3) compared with animal
reference proteins, as befits its primitive nature.
number of Slc4-like genes varies on a genome-to-genome
basis. Notable findings are as follows.
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
In the following paragraphs we discuss in further detail the
divergence of present day AE-like, NCBT-like, and BORlike genes from a single common Slc4-like progenitor. The
actions and roles of nonvertebrate Slc4-like products are
discussed in section III.
2. Archetypal and bacterial Slc4-like genes
and products
A curiosity is that “Nitro” has ⬃20% sequence identity at
the amino-acid level with certain prokaryotic sulfate permeases6 that share common ancestry with the Slc26 family
of vertebrate anion exchangers. Thus, archetypal Slc4-like
and Slc26-like genes may have been preceded by a single
common ancestral gene. Furthermore, the genome of the
archaebacterium Methanococcus maripaludis includes a sequence (YP_001548276) predicted to encode a multi-spanning membrane protein that is equally similar to “Nitro”
and the prokaryotic Slc26-like paralog BicA (p. 820).
3. Emergence of BOR-like genes and products
in fungi
The known Slc4-like products of fungi are all BOR-like. The
best known of these is Bor1p, the sole Slc4-like gene-product
from the brewer’s yeast Saccharomyces cerevisiae, encoded by
the BOR1 gene (FIGURES 2B AND 4/Brewer’s Yeast). Similar,
singleton Slc4-like genes are found in the genomes of many
other model, commercial and pathogenic species of fungi. The
genomes of yet other fungal species, representing nearly onethird of fungal species whose Slc4-like genes have been reported, contain multiple slc4-like genes. For example, Aspergillus niger (FIGURE 4/Black Mold) has three. As with the
BOR-like transporters of plants, those from fungi are more
similar to each other than to those of other kingdoms, suggesting divergence from a single fungal BOR-like ancestor, but not
necessarily indicating a common function. We discuss the action and role of Bor1p below.
6
For example, ZP_02948162 from Clostridium butyricum 5521.
The known Slc4-like products of plants are all either BORlike or “Primitive.” Slc4-like transporters in the plant kingdom are represented in FIGURE 4 by the genomes of one
species each of green alga, moss, a monocotyledonous flowering plant, and a dicotyledonous flowering plant. The relatedness at the amino acid level of these transporters is
represented in FIGURE 5.
The green alga Ostreococcus is a unicellular organism and
one of the smallest known eukaryotes, having only a single
mitochondrion and a single plastid (206). The Ostreococcus “lucimarinus” genome includes two Slc4-like genes that
appear to have diverged from a common ancestor. Both
Ostreococcus transporters retain most similarity to the bacterial ortholog7 “Nitro,” and we thus consider them “Primitive.” However, because their divergence scores are small,
they are also “Generic” or nearly “Generic.”
Representing the Slc4 complement of an early land colonizing plant, the moss Physcomitrella retains a single “Primitive” Slc4-like gene along with two BOR-like genes. The
two BOR-like genes appear to have diverged in the bryophyte lineage from a single common archetypal BOR-like
ancestor that probably also gave rise to the rice and thale
cress BORs in the “higher” plant/tracheophyte lineage.
Thus the presence of recognizable BOR-like genes in plants
appears to be contemporary with land colonization.
During the emergence of “higher plants,” the archetypal
plantal BOR-like ancestor appears to have diverged many
times. The first duplication of the BOR-like ancestral gene
appears to postdate the emergence of moss, but predate the
divergence of monocotyledons (such as rice) and eudicotyledons (such as thale cress). One copy of the archetype
retained a high degree of similarity to the original ancestral
protein and gave rise to the precursor of AtBOR1–3 as well
as OsBOR1 (BOR Group I, FIGURE 5). The second copy of
the archetype gave rise to the precursors of AtBOR4 –7 as
well as OsBOR2– 4 (BOR Group II, FIGURE 5). We discuss
the actions and roles of plantal BOR products below.
5. Emergence of AE-like, NCBT-like, and BOR-like
genes and products in animals
The emergence of animals was more or less accompanied by
the duplication of an archetypal Slc4-like gene, the inclu7
Following the recommendations of Jensen (447), we use the
term ortholog to distinguish gene/proteins with a common genetic
ancestry from different species (e.g., human SLC4A4 versus mouse
Slc4a4) and the term paralog to distinguish gene/proteins that
diverged from each other following gene duplication (e.g., human
SLC4A4 versus human SLC4A5 or human SLC4A4 versus mouse
Slc4a5). These terms are intended to refer to phylogenetic rather
than functional relatedness.
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We have tentatively dubbed “Nitro” the gene-product from
Nitrococcus (FIGURES 2B AND 4/Bacteria). A comparison of
overall protein sequence identity shared between the transmembrane domains of “Nitro” and sea squirt Slc4s shows
that “Nitro” itself is almost equally similar to AEs, NCBTs,
and BORs (TABLE 3). It is interesting to note that the cytosolic C terminus (Ct) of “Nitro” and the Ct the Slc4-like gene
product from Segniliparus both contain a sequence “LDA[D/
E]E” that is similar to the proposed binding site, in the Ct of
mammalian Slc4 proteins, for carbonic anhydrase (CA) II
(1007). Also notable, although perhaps coincidental, is that
the Ct of the Segniliparus Slc4-like transporter and the Ct of
human BTR1 both terminate with the sequence [D/E]xRP,
although the significance of that motif has not been described
for either protein.
4. Emergence of BOR-like genes and products in
true plants
MARK D. PARKER AND WALTER F. BORON
sion of sequence that encodes a substantial amino-terminal
domain, and the subsequent evolution of distinct AE-,
NCBT-, and BOR-like genes in animals. The overrepresentation of BORs and BOR-like transporters in the genomes
of plants, amoebozoa, and fungi is complemented by their
comparative underrepresentation in the genomes of animals (FIGURE 4/Animals). Most animals retain multiple AElike and NCBT-like genes but only a single BOR-like gene,
with two exceptions: the nematode worm has two BORlike paralogs and the fruit fly has none.
The most primordial, animal Slc4-like sequence that represents a complete cDNA is the AE-like transporter known as
“NBCSA” (847) from a sponge (FIGURE 4/Sponge). Notably, these placozoan and sponge Slc4-like proteins are predicted to have in place two features that are absent from
Slc4-like proteins of plants and fungi, but that are found in
vertebrate Slc4s: 1) a large cytosolic Nt and 2) an extended
third extracellular loop-between the fifth and sixth transmembrane spans (TMs), which includes cysteine residues
and multiple, putative N-glycosylation sites (FIGURE 2A).
In Slc4 evolution, the large Nt appears for the first time in
animals (e.g., tablet animal in FIGURE 4).8 The origin of the
Nt is unknown, but it is likely to be derived from a preexisting open-reading frame that became appended to the
transporter gene. An Nt-precursor gene is not identifiable as
an isolated entity in any presently available genome sequence, nor is an Slc4-independent function for the Ntprecursor protein suggested by sequence homology to other
soluble proteins. However, some mammalian Slc4 genes
express variant transcripts that encode an isolated Nt (see
below), which may be a vestige of the original genetic independence the of Nt-encoding sequence. It is noteworthy
that a region of the crystal structures of the Nt of AE1 and
NBCe1 shares substantial structural homology with some
EIIA proteins, which are components of bacterial phosphotransferase systems that can act as soluble regulators of
certain K⫹ channels and sugar transporters (245, 552).9
Clues to the divergence of AE-like, NCBT-like, and BORlike transporters in animals are provided by the visual
guides to protein identity shown in FIGURES 4 AND 6. In
tablet animals, two AE-like proteins are already distinct
8
Note that the extended Nt of the slime mold Slc4-like transporter
bears no significant sequence identity to the Nt of animal NCBTs and
likely evolved independently.
9
Precomputed structural alignments between the Nt of AE1 and
certain bacterial EIIA proteins can be accessed via http://
www.ncbi.nlm.nih.gov/Structure/vast/vastsrv.cgi?sdid⫽51159.
816
6. Emergence of AE, NCBT, and BOR activity
in animals
Borate transport is likely a primitive function of Slc4-like
transporters as evidenced by the presence of Slc4-like products with borate transport function in plants, fungi, and
animals. Bicarbonate transport function appears to be a
more recent specialization.
To date, the only nonvertebrate Slc4-like gene-product with
demonstrated Na⫹-independent Cl-HCO3 exchange activity is the AeAE from mosquitos (747), an ortholog of the
AE-like transporter from Drosophila (FIGURE 4/Fruit Fly).
Thus AE activity presumably arose prior to the divergence
of protostomes (e.g., flies) from deuterostomes (e.g., mammals).
Many nonvertebrate NCBT-like products have demonstrated NCBT function. NCBT-like proteins that perform
Na/HCO3 cotransport are common to both coelomates
(e.g., humans) and pseudocoelomates (e.g., ABTS-1 from C.
elegans, see below). Thus it seems likely that NCBT-like
products had, at the latest, acquired the ability to perform
Na⫹-coupled HCO3⫺ transport soon after the emergence of
the Bilateria, over 900 million years ago (368).
C. Divergence of Vertebrate Slc4 Genes
From an Early Chordate Slc4-like Gene
As far as we can discern from the presently available genome data, mammals, and likely most extant vertebrates,
have at least one copy of each of the five distinct, known
NCBT paralogs: Slc4a4, Slc4a5, Slc4a7, Slc4a8, and
Slc4a10 (FIGURE 3). To investigate the genetic origins of
these five genes, we look again to our reference genome
from the sea squirt Ciona intestinalis, the genome of which
includes one known NCBT-like gene. Ciona is a primordial
chordate and shares a single common ancestor with all vertebrates. Thus the singleton Ciona NCBT-like gene is likely
very similar to the archetypal vertebrate NCBT.
1. Analysis of exon-exon boundaries
We can make some inferences about the emergence of chordate and vertebrate Slc4s from their common ancestor by
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The most primordial, animal Slc4-like transporters presently identified may be in the “tablet animal” Trichoplax
adhaerens (FIGURE 4/Tablet Animal). An early draft of the
Trichoplax genome sequence indicates the presence of at
least three Slc4-like genes; two AE-like and one BOR-like.
from a BOR-like transporter. In our analysis, the most primordial organism with at least one gene each that is distinctly AE-like, NCBT-like, and BOR-like is the sea anemone (FIGURE 4/Starlet Sea Anemone). However, because the
partial sequence that we have assigned as NCBT-like is only
⬃100 amino acids long, our assignment may not accurately
reflect the nature of the full-length gene-product. Nevertheless, the divergence of NCBT-like genes must have occurred
no later than the appearance of the Bilateria because
NCBT-like genes appear in Coelomates and Pseudocoelomates (FIGURE 4).
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
analyzing the exon boundaries of Ciona and vertebrate
paralogs. FIGURE 7 shows a representative region of an Slc4
protein, from presumptive TM7 to TM14, aligned against
the mRNAs that encode this region for all ten human SLC4
genes and all three Ciona Slc4-like genes.10 In the horizontal
bars that represent mRNAs, different colored blocks represent sequences that are encoded by different exons. The
analysis in FIGURE 7 shows that human SLC4s can be
grouped into four categories by virtue of their gene structure.
Group 1 is composed of the genes that encode the human
Na⫹-independent Cl-HCO3 exchangers, AE1, AE2, and AE3.
Group 2 is composed of electroneutral NCBT genes NBCn1,
NDCBE, and NBCn2. Group 3 is composed of the electrogenic NCBT genes NBCe1, NBCe2, and the functionally controversial gene AE4. Group 4 is composed solely of the BTR1
gene that encodes the putative borate transporter.
The number of shared and unique exon boundaries among
groups provides an indication of their relatedness. For gene
regions encoding TM7 to TM14, the AEs (group 1) and
NCBTs (groups 2⫹3), which share a single exon boundary
(FIGURE 7A), are more closely related to one another than to
BTR1.
Most closely related to the Ciona AE-like gene is group 1,
which shares five exon boundaries (FIGURE 7, B, D, G, J,
AND L) with the Ciona AE-like gene, indicating that the
Ciona AE-like gene shares a single common ancestor with
all vertebrate AEs.
10
FIGURE 7 is based on the sequence alignments of Ciona and
human Slc4 genes in Appendix III.
Protein
7
8
9
10
11
13
E
14
mRNA
AE1
Group 1
AE2
AE3
Ciona AE-like
NBCn1
Group 2
NDCBE
NBCn2
NBCe1
NBCe2
Group 3
‘AE4’
Ciona NCBT-like
BTR1
Group 4
Ciona BOR-like
A
B C
D E
F G H
I
J
K
L
FIGURE 7. Analysis of common and unique exon boundaries among human SLC4 and Ciona Slc4-like genes.
The gray horizontal protein bar represents a region of a typical Slc4 protein, between putative TMs 7–14.
White numbered boxes within the protein bar mark the positions of ␣-helical TMs, similar to the representations in FIGURE 2B. Aligned below the protein bar are the corresponding regions of mRNAs that encode the
TMs 7–14 region for all 10 human SLC4 genes and all 3 Ciona Slc4-like genes. Each mRNA bar is divided into
colored boxes that represent the individual exons that comprise each sequence: thus a change in color on the
horizontal axis marks the position of an exon boundary. Exons that appear to have been derived by splitting of
a larger ancestral exon are colored green in the human lineage and purple in the Ciona lineage. Common exon
boundaries that are discussed in the text are labeled A–L. The position of the exon boundaries for each gene
are provided by NCBI Evidence Viewer (839). The sequence alignment from which this analysis is derived is
presented in Appendix III.
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Most closely related to the Ciona NCBT-like gene structure
are groups 2 and 3, which share four exon boundaries (FIGURE 7, A, C, E, AND K) with the Ciona NCBT-like indicating
that the Ciona NCBT-like shares a single common ancestor
with all vertebrate NCBTs. Exon boundaries “F,” “H,” and
“I” (FIGURE 7), none of which is found in the Ciona NCBT
gene, mark the divergence of group 2 genes (containing only
MARK D. PARKER AND WALTER F. BORON
exon boundary “H” and “I”) and group 3 genes (containing exon boundaries “F,” “H,” and “I”). It is not necessarily the case that the gain of exon boundaries “H” and “I”
predate the gain of boundary “F,” as introns may also be
lost during the course of evolution (53), but the simplest
explanation is that the group 2 (electroneutral NCBTs) archetype structure arose earlier and is the parent of the group
3 (electrogenic NCBTs plus “AE4”) archetype structure.
Finally, the most recent NCBT gene divergence created the
individual members of groups 2 and 3.
Most closely related to the Ciona BOR-like gene is group 4
(i.e., BTR1). BTR1 shares no exon boundaries with the AEs
and NCBTs in groups 2– 4, but shares six exon boundaries
with the Ciona BOR-like gene (FIGURE 7).
2. Analysis of deduced amino acid sequences
A) EMERGENCE OF THE FIVE NCBTS.
Among vertebrates, the earliest indicators of NCBT divergence are 41 NCBT-like gene
fragments in the draft genome sequence of the sea lamprey
Petromyzon marinus. By comparing fragments that have
overlapping sequence homology, we estimate that there
are at least two and perhaps as many as three NBCe1/
NBCe2-like genes (predominantly NBCe1-like) and at
least two NBCn1/NBCn2/NDCBE-like genes (predominantly NBCn1/NDCBE-like). Thus it seems that the split
between electroneutral NCBT-like and electrogenic NCBTlike genes predates the divergence of lampreys and jawed
vertebrates. Although the fragmented and incomplete nature of the sequence information makes direct correlation of
fragments to specific mammalian orthologs impossible, all
five NCBT genes may already have been distinct entities by
the time that lampreys appeared.
The most primordial vertebrate NCBT cDNA sequence described may be a fragment cloned from a cartilaginous fish,
the Atlantic stingray Dasyatis sabina (GenBank protein accession no. AAU29553). This cDNA fragment is most similar to mammalian Slc4a4 (NBCe1). The most primordial
organism with a documented set of orthologs of the five
mammalian NCBTs is the zebrafish Danio rerio, a bony
fish. The zebrafish genome contains orthologs of all Slc4
genes, with the exception of Slc4a9, indicating that the five
NCBTs were distinct entities at the point at which a common ancestral organism diverged to give rise to 1) rayfinned fishes (i.e., most modern bony fish, including zebrafish) and 2) lobe-finned fishes and tetrapods.
B) EMERGENCE OF DUPLICATE NCBT-LIKE GENES IN BONY FISHES.
All
vertebrates likely have a full complement of five NCBT
818
Let us consider the NCBT complement of zebrafish. For
clarity we will provisionally refer to duplicate Slc4 genes as
Slc4aX.1 and Slc4aX.2. In the case of Slc4a4, the sole reported Slc4a4.1 product (aka zNBCe1a aka zNBCe1-B aka
NBCe1.1) most resembles the mammalian NBCe1-B variant (see below for a discussion of NBCe1 variants) inasmuch as it includes Nt sequence similar to the auto inhibitory and IRBIT (IP3 receptor binding protein released with
inositol 1,4,5 trisphosphate)-binding determinants of
NBCe1-B/C and terminates with an NBCe1-A/B-like Ct. An
analysis of the Slc4a4.1 gene suggests that it would be unable to produce an NBCe1-A-like or an NBCe1-D-like transcript (926): specifically, 5= extension of exon 4a (see FIGURE 17) would not append a sequence to zebrafish NBCe1
that has obvious sequence similarity with the autostimulatory domain of mammalian NBCe1-A and NBCe1-D. Although Slc4a4.1 does have the capacity to encode a
NBCe1-C like variant, the corresponding transcript has not
been isolated. However, the presence of Slc4a4.1 variant
products that lacks splice cassette I sequence (599), i.e., an
NBCe1-E-like sequence indicates that posttranscriptional
processing of NBCe1 does occur in these fish. Thus Slc4a4.1
is demonstrated to encode NBCe1-B and NBCe1-E-like sequences, but also has the potential to encode an NBCe1-Clike sequence.
The sole reported Slc4a4.2 product (aka zNBCe1b aka
NBCe1.2) is a partial clone that includes an NBCe1-B/Clike Nt, but lacks Ct sequence. A predicted complete open
reading frame terminates with an NBCe1-C-like Ct that
includes a PDZ-domain binding sequence. If each of the
duplicate Slc4a4 genes has permanently taken on the character of a specific mammalian-like Slc4a4 splice variant, the
distribution of “NBCe1-B versus NBCe1-C” in zebrafish
could be controlled at the transcriptional level rather than,
as in mammals, at the posttranscriptional level. Presumably
these duplicate NCBT-like genes could serve as genetic back
up for each other, although preliminary studies that reveal
distinct distribution patterns (561) suggest that each may
have carved out its own physiological niche.
Slc4a9. The unusual Slc4a9 gene-product
was initially described as NBC5 due to its relatedness to
NCBT sequences, but was subsequently redesignated as
AE4 following reports that the rabbit and rat orthologs are
capable of Cl-HCO3 exchange activity. Slc4a9 clearly
shares a common genetic origin with electrogenic NCBTs
(FIGURES 3 AND 7), yet an Slc4a9 gene is notably absent
C) EMERGENCE OF
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To trace the divergence of individual genes within these
groups, we must rely on assessments of overall protein sequence relatedness, such as those presented in TABLE 3,
depicted in FIGURE 3, and discussed in the following section.
genes, as evidenced by the presence of Slc4a4, Slc4a5,
Slc4a7, Slc4a8, and Slc4a10 in the genomes of zebrafish and
African clawed frogs (i.e., Xenopus). However, the complement of Slc4 genes may vary between genera. For example,
due to a whole-genome duplication, zebrafish and many
other fishes have two copies of at least Slc4a1 (561), Slc4a2
(882), Slc4a4 (167, 561), Slc4a5, and Slc4a10 (1101).
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
from the draft genome of Danio rerio. Indeed, no Slc4a9
genes or products have been reported from any non-tetrapodan species. These observations suggest that Slc4a9 is a
tetrapod-specific gene.
tetrapodan Slc4a9. It is more likely that a later Slc4a4 gene
duplication in the tetrapod lineage gave rise to the precursors
of mammalian Slc4a4 and slc4a9. Such interspecific divergence between Slc4a9 sequences could result in Slc4a9 products from different animals having different function and distribution, a subject that we discuss later in this review. Therefore, it may be helpful to think of Slc4a9 products not as a
singular entity but as a group of related proteins, the genes for
which diverged following a “recent” Slc4a4 duplication.
The deduced amino-acid sequence of Slc4a9 orthologs is
not as well conserved as those of its closest paralog, NBCe1.
Human, rabbit, rat, and mouse orthologs of NBCe1 are
96 –99% identical to each other, whereas Slc4a9 orthologs
only share 79 –91% identity among those same species. The
Nt sequence of Slc4a9 orthologs are more divergent (70 –
89% identity) than their TMD sequence (85–93% identity).
The greater divergence of Slc4a9 compared with NBCe1
may reflect a reduced selective pressure to retain electrogenic NCBT function.
III. NCBTs AND RELATIVES IN
NONMAMMALS
A comparison of vertebrate NBCe1, NBCe2, and Slc4a9
deduced protein sequences (FIGURE 8) provides important
information about the origin of Slc4a9. The fish Danio rerio
has no Slc4a9 gene, but two copies of the Slc4a4 gene
(Slc4a4.1 and Slc4a4.2). The amphibian Xenopus tropicalis
has an AE4-like gene, but Xenopus “AE4” is actually more
identical at the amino acid level to human NBCe1 (66%
identity between TMDs) than to human “AE4” (63% in the
TMD). In the fowl Gallus gallus, “AE4” shares an equal
degree of identity with human NBCe1 and human “AE4”
(66% between the TMDs). Only in the mammalian lineage
has the Slc4a9 gene diverged sufficiently to be clearly distinguishable from Slc4a4. The divergence of Danio
Slc4a4.1 and Slc4a4.2, likely contemporary with a wholegenome duplication event (40), postdates the divergence of
ray-finned fishes and lobe-finned fishes/tetrapods. Thus it is
unlikely that either Slc4a4.1 or Slc4a4.2 is an ortholog of
human
SLC4A4
fowl
zebrafish Slc4a4
Slc4a4.2
NBCe1
1. Cation-coupled bicarbonate transport in bacteria
CO2 sequestration by photosynthetic cyanobacteria makes
a significant contribution to the global carbon cycle (284).
The efficiency of carbon fixing by cyanobacteria is enhanced by a CO2-concentrating mechanism, of which cation-coupled HCO3⫺ transport is a vital component (recently
reviewed by Price in Ref. 759). In animals, all Na⫹-coupled
HCO3⫺ transport performed by Slc4 proteins. However,
none of the prokaryotic cation-coupled HCO3⫺ transporters
identified to date are Slc4-like.
In the freshwater cyanobacterium Synechococcus sp. strain
PCC 7942, HCO3⫺ transport is a high-affinity, primary active process that is induced under CO2-limiting conditions
(701). HCO3⫺ transport is effected by an ABC (ATP binding
mouse
Slc4a4
frog
Slc4a4
zebrafish
Slc4a4.1
frog
“Slc4a9”
fowl
“Slc4a9”
zebrafish
Slc4a5.2
zebrafish
Slc4a5.1
mouse
Slc4a5
NBCe2
frog
Slc4a5
human
SLC4A9
human
SLC4A5
mouse
Slc4a9
“AE4”
0.1
fowl
Slc4a5
FIGURE 8. Divergence of NBCe1, NBCe2, and “AE4” in vertebrates. The unrooted phylogram displays the
relatedness, at the level of protein sequence, among the transmembrane domains of NBCe1, NBCe2, and “AE4”
for zebrafish (Danio rerio), frogs (Xenopus tropicalis), fowl (Gallus gallus), mice (Mus musculus), and humans. The
phylogram was generated using ClustalW (183) and TreeView (704). The GenBank protein accession numbers for
each transporter are provided in Appendix IV.
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A. Bacteria
MARK D. PARKER AND WALTER F. BORON
In another freshwater photosynthetic cyanobacterium, Synechococcus sp. strain PCC 6803, deletion of the cmp gene
cluster has little effect on HCO3⫺ transport (877). Another
transporter called BicA, a paralog of the vertebrate Slc26
family of anion exchangers, has been suggested to be responsible for HCO3⫺ uptake by Synechocystis PCC6803
under normal conditions (760). Furthermore, in this strain,
CO2 limitation induces expression of a Na⫹-dependent
HCO3⫺ transporter, called SbtA, that appears to have no
eukaryotic equivalent. It is not known whether SbtA
cotransports Na⫹ with HCO3⫺ and if so, in what ratio.
However, it has been suggested that the process is driven by
an inwardly directed Na⫹ gradient established by an active
Na⫹-extrusion pump (877).
2. Bacterial Slc4-like transporters
To date, the only reported occurrences of Slc4 orthologs in
identifiable prokaryotic genomes are singular examples
from the marine nitrifying bacterium Nitrococcus mobilis
(FIGURES 2B AND 4/Bacteria) and the pathogenic bacterium
Segniliparus rugosus. At 513 amino acids in length, the
Nitrococcus clone, that we have provisionally termed “Nitro,” is the most compact of all known Slc4-like transporters. “Nitro” lacks many of the extended extramembranous
regions of its vertebrate SLC4 counterparts, but is predicted
to retain their topology in the transmembrane domain (FIGURE 2B). The codon usage pattern of the “Nitro” gene is
more similar to that of eukaryotes than of bacteria (e.g., E.
coli). The function of “Nitro” has yet to be fully characterized, but when heterologously expressed in Xenopus
oocytes, Nitro does not mediate detectable HCO3⫺ transport but does permit the electroneutral and Na⫹-independent influx of 36Cl (713). It is intriguing to speculate that its
retention in a nitrifying bacterium, which imports toxic
NO2⫺ and exports NO3⫺, may indicate a role for “Nitro” in
NO2-NO3 exchange. This hypothesis is especially tempting
820
in light of the homology between NO3⫺ and HCO3⫺ transporters in cyanobacteria (701), the role of an Arabidopsis
ClC ortholog as a H/NO3 cotransporter (222), and the penchant of mammalian AE2 for NO3⫺ as a nonphysiological
substrate (401). A possible BOR-like action of the archetypal Slc4-like gene-product is suggested by a consideration
of Slc4-like transporters encoded in the eukaryotic domain.
Only Slc4-like products with borate transport function are
present across the kingdoms of plants (e.g., AtBOR1), fungi
(e.g., Bor1), and animals (e.g., BTR1). Moreover, among
plants, amoebozoa, and fungi, no HCO3⫺ transporters are
known.
B. Fungi
In eukaryotic organisms of primordial origin, such as yeast,
no Slc4-like proteins with NCBT function have yet been
identified. The best characterized example of a fungal Slc4like gene is BOR1 (aka YNL275W), the singular example
from the baker’s yeast Saccharomyces cerevisiae. Its product Bor1p is a 576-amino acid nonglycosylated transporter
that is similar in predicted secondary structure to its bacterial ortholog “Nitro,” except that the predicted Nt and Ct
are slightly longer (FIGURE 2B).
In Saccharomyces, Bor1p is localized to the plasma membrane (941, 1098), where it functions as a boron, or borate,
efflux pathway, allowing cells to survive in media containing high levels of boric acid (689, 941, 943). A phosphate
transporter, Pho88p, has been identified as a partner of
Bor1p in a split-ubiquitin-based yeast two-hybrid screen
(646), suggesting that Bor1p may be part of a larger integral
membrane protein complex. In light of the observations
that Bor1p is 1) not downregulated under boron-limiting
conditions (442), 2) not strongly upregulated by high borate levels (442), 3) not the sole candidate borate transporter in this organism (116, 476, 689), and 4) does not
compensate for boron efflux defects in an ATR1-deletion
strain (476),11 it has been suggested that Bor1p may have
another as yet uncharacterized function (442).
A report that an overexpressed Bor1p-GFP fusion protein is
enriched in vacuolar preparations compared with total cell
homogenate (229) has been cited as evidence that the transporter is primarily localized to an internal compartment.
However, this report should be interpreted carefully for
three reasons. First, it conflicts with the earlier work on
endogenously expressed Bor1p, noted above, that supports
the plasma membrane localization of Bor1p protein and
action (689, 941, 943, 1098). Second, overexpression of
GFP-tagged Bor1p could swamp the trafficking machinery
and lead to aberrant protein localization. Third, this report
11
Atr1p is a member of the multidrug-resistant transporter protein family originally noted for its ability to confer aminotriazole tolerance (464).
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cassette) protein assembly, called BCT1, encoded by the
products of cmpABCD gene cluster (701). Together these
four components create a complex equivalent to a mammalian ABC-type transporter that, in mammals, would be encoded by a single gene. The components of this transport
complex are highly similar to those of a nitrate/nitrite transporter assembly from the same species, encoded by the nitrate assimilation (nirA) operon (701). Within BCT1,
CmpA is an extracellular membrane-anchored HCO3⫺
binding lipoprotein that confers high affinity to the transport process. CmpB is the membrane-multispanning HCO3⫺
permease. CmpC and CmpD are intracellular ATPase subunits (511). CmpD is also predicted to have a solute-binding/transport modulatory role, based on its homology to the
nitrite/nitrate transporter component NrtD (501). Structural data indicate that HCO3⫺ binding to the extracellular
subunit CmpA is strongly Ca2⫹, but not Na⫹ dependent,
although it is presently unclear whether Ca2⫹ is cotransported with HCO3⫺ (511).
⫺
TRANSPORTERS
Na⫹-COUPLED HCO3
also describes increased vacuolar fragmentation in a Bor1p
deletant strain (229), which conflicts with the findings of a
later study (442), thereby weakening the association between Bor1p and the vacuole.
C. Phytoplankton
The phytoplankter Emilyiana huxleyi is surrounded by a
shell (a coccosphere) composed of CaCO3 plates (coccoliths). Coccoliths are formed from Ca2⫹ and CO32⫺/HCO3⫺
in internal compartments (coccolith vesicles) and are exocytosed onto the cell surface. Coccoliths are an important
sink of carbon in the global carbon cycle (e.g., the White
Cliffs of Dover are composed of coccoliths), but the physiological role of coccoliths is unknown. One possibility is
that coccoliths are a store of carbon for photosynthesis
(860). Alternatively, coccoliths may be a sink for excess
Ca2⫹, a desalting mechanism that would parallel the deposition of CaCO3 in the intestines of marine fishes. The molecular action of Slc4-like transporters in phytoplankton is
unknown, but the abundance of one of these Slc4-like products in Emilyiana huxleyi increases in the presence of extracellular Ca2⫹. One possibility is that Slc4-like products
might be responsible for HCO3⫺ influx across the plasma
membrane (621). Another is that cytoplasmic carbonic anhydrases produce HCO3⫺ from CO2 when CO2 is abundant.
In either situation, a transporter, conceivably an Slc4-like
protein, would move HCO3⫺ from the cytoplasm, across the
vesicle membrane, and into coccolith vesicles. However, the
molecular actions and the subcellular locations of any Slc4like gene-product from phytoplankton are presently unknown.
Considering the primordial boron transport function of
Slc4-like proteins, it is also possible that some phytoplank-
D. Amoebae
Valproic acid (VPA; 2-propylpentanoic acid) is a commonly
prescribed anticonvulsant that acts upon ion channels as
well as intracellular targets such as histone deacetylases
(173). At doses above 1 mM, VPA is toxic to the model
unicellular slime mold Dictyostelium discoideum; VPA-resistant strains have their singleton Slc4-like gene disrupted
(960). The link between the Slc4-like gene and VPA transport in slime mold is strengthened by the inhibition of VPA
uptake by the Slc4-blockers DIDS and tenidap and by inhibition of VPA uptake by extracellular HCO3⫺ (960). VPA
uptake is independent of extracellular Na⫹ but stimulated
by acidic extracellular pH (960). Thus it is possible that the
protonated form of VPA moves into the slime mold, perhaps via an Slc4-like protein. Heterologous expression
studies would be helpful to determine whether the Slc4-like
protein is capable of such activity. It is unlikely that VPA is
the physiological substrate of this transporter, and it is unknown if HCO3⫺ is carried by the Slc4-like transporter. An
intriguing possibility is that VPA is a substrate or inhibitor
of mammalian Slc4s. If VPA is a substrate of mammalian
Slc4s, these transporters could promote VPA action upon
intracellular targets. On the other hand, if VPA blocks neuronal NCBTs, the resulting fall in pHi could dampen neuronal excitability, contributing to the anticonvulsive properties of the drug.
E. Plants
Algae, moss, and both mono- and dicotyledonous flowering
plants each have their own unique complement of Slc4-like
genes (FIGURE 4/Plants) that bear more sequence similarity
among themselves than to any of their animal orthologs.
These plantal Slc4-like genes are structurally similar to their
yeast homologs, having short Nt and Ct cytosolic domains,
but appear to always include an extended extracellular loop
between TMs 9 and 10 (FIGURE 2B). The only plantal Slc4like transporters characterized to date are those from flowering plants. The founder member BOR1 (see next section)
is a boron-efflux transporter from the thale cress Arabidopsis thaliana (943) and shares many properties with the yeast
Slc4-like protein Bor1p. Boron is a highly significant element for plants: boron cross-linked rhamnogalacturonan II
dimers are important cell-wall components (reviewed in
Ref. 692). Too little boron can cause reproductive and
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Bor1p binds to stilbene derivatives such as DIDS and SITS
(inhibitors of mammalian HCO3⫺ transporter), but there is
no indication that Bor1p can transport HCO3⫺. Moreover,
reports differ as to whether Bor1p-mediated boron efflux is
inhibited by the presence of NaHCO3 in the growth medium (442, 943). Furthermore, as Na⫹ and Cl⫺ accumulation in yeast is unaffected by genetic ablation or overexpression of Bor1p (441, 442), it seems unlikely that Bor1p
shares any common substrates with mammalian NCBTs.
However, other anions may at least interact with Bor1p, as
evidenced by the displacement of Bor1p from a SITS-affinity column by high concentrations of Br⫺, Cl⫺, HCO3⫺, I⫺,
or NO3⫺ (1098). Borate efflux by yeast is faster at more
acidic extracellular pH, which is consistent with the hypothesis that uphill borate efflux is driven by an inward H⫹
gradient (442), i.e., H/borate exchange. Inasmuch as the
genetic diversity among Slc4-like products in fungi is at least
as great as among their animal paralogs (e.g., human AE1
versus BTR1), it is possible that not all fungal Slc4-like
transporters share the same molecular action as Saccharomyces Bor1p.
ton Slc4-like transporters might transport boron. Indeed,
coccoliths do contain boron. However, the influx of uncharged H2BO3 across phytoplankton membranes is
thought sufficient to account for the observed coccolith boron content (911). Even if this hypothesis were true, it
would not preclude a role of an Slc4-like transporter as a
borate efflux pathway in the plasma membrane, analogous
to the role of Bor1p in yeast.
MARK D. PARKER AND WALTER F. BORON
growth problems in plants, whereas excessive boron can be
toxic (reviewed in Ref. 86). Boron transport is likely a complex process, aside from Slc4-like transporters, other plant
proteins such as the aquaporin-like NIP5 (945, 1089) and
NIP6 (947) are necessary for efficient boron transport
throughout the plant (FIGURE 9).
and to seven BOR genes in thale cress (dicotyledonous),
most paralogs within each group arose independently and
the numbering is arbitrary. For example, although OsBOR1 in rice and AtBOR1 in thale cress are truly orthologous, OsBOR2 in rice is not the direct ortholog of AtBOR2
in thale cress. The relatedness of some plantal Slc4 products
is as shown in the dendrogram in FIGURE 5.
For at least two reasons, the nomenclature for plantal Slc4like transporters requires careful interpretation: 1) Not all
products named “BOR” have demonstrated boron transport function. 2) BOR genes from dicotyledonous genomes
do not have exact orthologs in monocotyledonous genomes. Thus, although a common ancestor presumably
gave rise to four BOR genes in rice (monocotyledonous)
B
OsBOR1
AtBOR1/2
B (out)
NIP?
AtBOR4
NIP5
B (in)
Stele
EN
(CS)
Cortex
EX
(CS)
B (in)
EP
Stele
EN
(CS)
Cortex
Rice root
Cross section
(elongation zone)
Arabidopsis root
Cross section
(elongation zone)
FIGURE 9. Roles of Slc4-like boron transporters in boron uptake and boron tolerance in the roots of plants.
Radial cross sections through the roots of rice (A) and thale cress (B). Boron can enter the roots from the soil
via the intercellular apoplast (blue shaded area), but is blocked from the root vasculature (in the stele) by
casparian strips (CS). NIP boron channels, BOR1, and BOR2 transporters in root cells, provide a transcellular
pathway through the epidermis (EP), exodermis (EX), cortex, and endodermis (EN) directing boron towards the
stele and past the CS allowing boron to access the xylem. In thale cress, AtBOR4 directs excess boron into the
soil.
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A
It is interesting to note that the plantal BOR-like transporters can be separated, according to protein sequence relatedness, into three distinct groups. The first group, “Primitive,” includes plantal Slc4-like products of unknown function. The second and third groups, “BOR group 1” and
“BOR group II,” both include demonstrated borate trans-
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
As boron availability and toxicity are critical determinants
of crop growth, and boron in plants acts as an antimicrobial
agent, the function of BOR transporters and the linkage of
BOR gene variation to enhanced boron tolerance is currently of considerable interest in plant physiology. Of special importance to animal physiologists is the growing number of reports of BOR-gene mutations, which may reveal
much about the structure/function relationships of Slc4s.
Notable is the coincidence, discussed below, that a mutation identified in the Arabidopsis AtBOR1 gene also occurs
in human AE1, where the mutation is associated with hereditary spherocytosis. In the following two sections, we
summarize the current knowledge concerning the physiological roles of BOR transporters in monocotyledonous and
dicotyledonous plants.
1. Boron transport in monocotyledonous plants
A) RICE.
The genome of rice (Oryza sativa) contains four
Slc4-like genes named OsBOR1– 4 (FIGURE 4/Rice). OsBOR1 and OsBOR3 both mediate boron efflux. At present
very little is known about the physiological roles of OsBOR2 and OsBOR4.
I) OsBOR1. The heterologously expressed OsBOR1-GFP
fusion protein localizes at/near the plasma membrane of
onion epidermal cells (674). In terms of the amino acid
sequence, of the seven Arabidopsis AtBORs, OsBOR1 is
most, and equally, similar to AtBOR1 and AtBOR2 (FIGURE 5). In terms of function, OsBOR1 is also similar to
AtBOR1 and -2, mediating boron efflux at the level of individual cells, and mediating boron uptake (i.e., root to
shoot) at the level of the whole plant (FIGURE 9A). Solutes
and water can travel freely throughout the root apoplast (an
extracellular space that includes cell walls) but are barred
from the xylem-surrounding apoplast by two corky casparian strips. By analogy with boron uptake pathways in Arabidopsis, it is likely that boron crosses the basal membrane
of rice root cells via aquaporin-like NIP5 transporters and
exits root cells across the apical membrane via BORs (256,
674, 944).12 The presence of OsBOR1 in root cells that
span the exodermal casparian strip likely provides a transcellular efflux pathway by which boron is directed towards
the endodermis where OsBOR1 in endodermal root cells
would finally transport boron into the xylem-surrounding
apoplast of the stele on the other side of the endodermal
strip (674).
In the root cells surrounding the xylem, the expression of
OsBOR1 is constitutive under normal-boron conditions
and is only modestly increased by boron starvation (674).
On the other hand, in the exodermis, prolonged boron deficiency massively increases the expression of OsBOR1
(674), thereby enhancing boron extraction from the soil.
II) OsBOR3. OsBOR3 functions as a boron-efflux transporter necessary for normal growth under boron-limited
conditions (675) and probably plays a similar role to OsBOR1. The expression of OsBOR3 is regulated such that
the OsBOR3 promoter drives exodermal expression in root
tips, but endodermal expression in the root elongation zone
(675).
B) BARLEY. Variations in the sensitivity of barley cultivars
to high boron levels have been linked to the Slc4-like
HvBOR2 (aka bot1) gene locus (788, 927). Of the transporters displayed in FIGURE 5, HvBOR2 is most like OsBOR2. HvBOR2 transcripts are expressed in roots and leaf
blade tips. In the latter, the transporter may contribute to
the excretion of boron in guttation fluid (927), the liquid
that some vascular plants secrete onto the leaf surface.
Four observations underlie the boron-tolerance of the
hardy “Sahara” cultivar, which can withstand high boron
levels, compared with boron-sensitive cultivars such as
“Clipper” and “Schooner”: 1) Southern blotting suggests
that the genome of “Sahara” may have four times as many
copies of the HvBOR2 gene than “Clipper” (927). 2) Realtime quantitative PCR (qPCR) suggests that HvBOR2 transcripts may be many hundred-fold more plentiful in “Sahara” than in “Clipper” and “Schooner” (788, 927). 3) In
conditions of elevated boron levels, “Sahara” maintains an
abundance of HvBOR2 transcripts, whereas “Schooner” is
unable to substantially increase HvBOR2 transcript abundance from its constitutively low level (788). 4) Heterologously expressed “Sahara” HvBOR2 is superior to “Clipper” HvBOR2 at enhancing the boron tolerance of yeast
(927). Of 11 differences in nucleotide sequence between the
2 cDNAs, 2 are predicted to change the protein sequence.
The first is the “Clipper” L305S polymorphism, which
would disrupt a conserved Leu residue in putative TM8.
12
According to the terminology recommended in Reference 296,
apical membranes face the shoot apex and basal membranes face
the root apex.
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porters (marked with an asterisk in FIGURE 5, and discussed
in the following sections). By analogy to similar groupings
observed for mammalian Slc4s (e.g., FIGURE 3), it is possible
that the molecular action of borate transport is different
between groups I and II. However, nothing is presently
known about the borate transport mode of any plantal
transporter. The sole feature by which BOR-like transporters currently can be categorized is the ability to confer tolerance to low-boron stress versus high-boron stress. This
distinction may follow the polarity of BOR expression
within plants cells: a boron-efflux transporter in the apical
membrane of plant cells will move boron in the direction of
the shoots, whereas a boron-efflux transporter in the basal
membrane will move boron in the direction of the soil (FIGURE 9). Note that both groups I and II contain members that
confer tolerance to low-boron stress (e.g., AtBOR1 and
OsBOR3).
MARK D. PARKER AND WALTER F. BORON
The orthologous mutant L750C in rat NBCe1 causes a 50%
loss of wild-type activity and is predicted to be located in a
very conformationally sensitive part of the ion-translocation pathway (633). Thus, if borate transport via HvBOR2
and bicarbonate transport via NBCe1 share commonality
in their translocation pathways, one explanation for the
relatively poor ability of the “Clipper” versus “Sahara”
gene to confer boron tolerance to yeast is a disrupted boronefflux pathway. The second polymorphism is D592G, in a
poorly conserved region of the cytosolic COOH terminus,
close to the last putative transmembrane segment. What
effect, if any, this amino-acid substitution would have on
the HvBOR2 transporter has yet to be elucidated.
C) WHEAT.
Arabidopsis thaliana is a popular model
organism used for the study of flowering plants. The Arabidopsis genome includes seven Slc4-like genes (FIGURE
4/Thale cress and FIGURE 5).
With regard to AtBOR1 localization at the tissue level, AtBOR1-GFP is expressed throughout the roots, but only under boron-limiting conditions, and mainly in the apical
membranes of cells (see footnote 12), opposing the basal
distribution of the boron-uptake transporter NIP5 (944).
AtBOR1 expression in the roots is also enriched in the
membranes of endodermal (EN) cells surrounding the stele
that contains xylem vessels (650), which transport water
and solutes from the roots to the rest of the plant (FIGURE
9B).
I) AtBOR1. In 1997, Noguchi and co-workers (686) reported the creation, by tilling, of a mutant Arabidopsis
strain called bor1–1. Unlike wild-type plants, bor1–1 failed
to thrive in boron-limiting conditions and produced fewer
seeds. Linkage analysis showed that the reduced boron content of this and a similar mutant cultivar is due to mutation
of the AtBOR1 gene (943). This study provided the first
evidence, in any species, of an Slc4-like gene-product being
involved in boron transport. One of the mutations in AtBOR1, G86E, is located in the short intracelullar sequence
linking putative TM segments 2 and 3 (943) (see FIGURE 2B
for putative BOR1 topology) and is orthologous to the naturally occurring human AE1 mutation G455E, which is
associated with hereditary spherocytosis (433). It is not yet
clear for either AE1 or AtBOR1 whether the G to E mutation causes a functional or trafficking defect in the transporter. A second mutant Arabidopsis strain bor1–2 is associated with an S74P mutation in AtBOR1, at a position
midway through putative TM2 (943). The inappropriate
positioning of a Pro residue in a helical region is likely to be
very disruptive. For example, the L522P mutation in TM4
of human NBCe1-A leads to rapid protein degradation
(241).
The cartoon in FIGURE 9B outlines a proposal for how the
thale cress root transports boron from the soil to the xylem
(568, 789). Solutes and water can travel freely throughout
the root apoplast (extracellular space) but are barred by a
single casparian strip from the apoplast surrounding the
xylem. Boron enters root cells via aquaporin-like NIP5
transporters on the basal membrane (256, 944). The presence of AtBOR1 in the apical membrane of root cells provides a transcellular efflux pathway that directs boron in
stepwise fashion through the cortex towards the endodermis, where AtBOR1 would finally transport boron across
the casparian strip into the xylem-surrounding apoplast of
the stele. Indeed, bor1–1 mutants have reduced boron content in the xylem exudate (943) as well as in shoots, shoot
apices, and rosette leaves (650), but not the roots (943).
Thus the critical AtBOR1-dependent step in the “root to
shoot” boron-transport pathway is presumably the endodermal step responsible for xylem loading (943). This hypothesis is consistent with the observation that AtBOR1
enhances plant tolerance to boron-limiting conditions, and
also explains why, when AtBOR1-GFP is overexpressed in
a transgenic Arabidopsis strain, the protein does not afford
increased protection from toxic levels of boron (650). How-
2. Boron transport in dicotyledonous plants
A) THALE CRESS.
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In wheat (Triticum aestivum), TaBOR2 is an
OsBOR2- and bot1/HvBOR2-like gene-product associated with increased high-boron tolerance in certain cultivars (788), consistent with a role in root to soil boron
efflux. The TaBOR2 gene is expressed at a higher level in
the boron-tolerant “India” cultivar over the boron-sensitive
“WIMMC*10” cultivar (788). Furthermore, in response to
boron-excess, boron-tolerant strains maintain substantial TaBOR2 transcript levels, whereas boron-sensitive strains are
unable to substantially upregulate TaBOR2 transcript abundance from its constitutively low level (788).
The evidence that AtBOR1 is a plasma-membrane protein is
the localization of heterologously expressed AtBOR1-GFP
fusion protein at/near the plasma membrane of tobacco leaf
cells (943). Five lines of evidence indicate that AtBOR1
mediates boron uptake at the organismal level (i.e., boron
efflux from root cell into the xylem; see FIGURE 9B) and
makes an important contribution towards “root-to-shoot”
boron transport. 1) The AtBOR1 gene locus is associated
with tolerance to boron-limiting conditions (1089). 2) In a
transgenic Arabidopsis strain, boron-limiting conditions
upregulate the expression of AtBOR1-GFP in roots,
whereas the protein is mainly expressed in shoots when
boron is in ready supply (942). 3) The restoration of high
levels of boron after a period of boron limitation triggers
the endocytosis and degradation of AtBOR1-GFP (942).
4) bor1–1 mutant plants, with mutant AtBOR1 genes, have
a reduced boron content (650, 943) and exhibit reduced
dimerization of rhamnogalacturonan II (see above and Ref.
685). Conversely, 5) the overexpression of AtBOR1-GFP in
transgenic plants enhances boron accumulation in the shoot
and shoot apices by about five times (650).
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
dopsis has no reported boron-related phenotype (653). AtBOR5 transcripts are enriched in guard cells and sepals
(1038) and are upregulated approximately eightfold in response to nitrate starvation of seedlings (1118).
II) AtBOR2. This transporter shares many properties with
AtBOR1, to which it is 90% identical. Like AtBOR1, AtBOR2 mediates boron efflux when heterologously expressed in yeast (652). Disruption of the AtBOR2 gene in
Arabidopsis is associated, under boron-limiting conditions,
with retardation of both overall plant growth and elongation of cells at the root tip. Indeed, the AtBOR2 promoter is
active in root tips (652). In seeds, a 24-h imbibing period
induces AtBOR2 gene expression (1038). AtBOR2 likely
contributes, in parallel with AtBOR1, to the directed “rootto-shoot” movement of boron depicted in FIGURE 9B.
VI) AtBOR6 and -7. Very little information is available on
the expression and function of AtBOR6 and AtBOR7. AtBOR6 transcripts are enriched in mature versus immature
pollen (1118).
III) AtBOR3. Transcripts have been detected in shoot
guard cells, trichomes, and root cortex (653) as well as
stigma and ovaries (1038). Although AtBOR3 mediates boron efflux when expressed in yeast, disruption of the AtBOR3 gene in Arabidopsis has no obvious phenotype.
However, a bor1/bor2/bor3 triple mutant suffers more
root-growth retardation than a bor1/bor2 double mutant,
consistent with the hypothesis that AtBOR3 activity can
compensate for defects in AtBOR1 and AtBOR2 expression
(653). Thus AtBOR3 likely contributes to the directed
“root-to-shoot” movement of boron depicted in FIGURE 9B.
IV) AtBOR4. The distribution of a GFP-tagged AtBOR4
construct in Arabidopsis plants indicates that this transporter is normally expressed in the basal (soil facing) membranes of root epidermal cells (651) and transcripts are
additionally detected in stamens (1038). In the root cells of
transgenic plants, elevated boron levels result in increased
AtBOR4-GFP expression (651). Furthermore, transgenic
Arabidopsis and Sativa (rice) that are overexpressing AtBOR4-GFP, from a non-native promoter, exhibit enhanced
tolerance to boric acid (461, 651). These observations are
consistent with the idea that AtBOR4 normally exports
excess boron from root to soil (651), as depicted in FIGURE
9B. An unexpected observation is that transgenic rice plants
that heterologously express exceptionally large quantities
of AtBOR4-GFP RNA (⬃100-fold more than the aforementioned transgenic rice plants tolerant to boric acid) exhibit a paradoxical diminution in tolerance to boric acid
(461), as if excessive expression of AtBOR4 creates a rootto-shoot boron transport pathway. In principle, this could
result from errant accumulation of AtBOR4 in the apical
membranes of root epidermal cells, where AtBOR4 activity
would parallel that of AtBOR1 and AtBOR2.
V) AtBOR5. AtBOR5 mediates boron efflux when expressed in yeast (653). The AtBOR5 gene is situated in a
genetic locus associated with tolerance to low-boron stress
(1089), although disruption of the AtBOR5 gene in Arabi-
B) GRAPEVINE. Analysis of the Vitis vinifera genome indicates
that grapevine plants have six Slc4-like genes, all of which
are BOR-like (735).13 Three are most similar to AtBOR1
and -2 and are members of group 1 in FIGURE 5. Three are
most similar to AtBOR4 and -5 and are members of group
II in FIGURE 5. Interestingly, none of the six Vitis BOR-like
genes appear to be direct orthologs of any of the seven
Arabidopsis BORs (735). The lack of orthology between
Arabidopsis and Vitis BORs is likely a complication of independent gene-duplication and gene-loss events following
the divergence of the two organisms from a common ancestor.
I) VvBOR1. Although not a direct ortholog of AtBOR1,
VvBOR1 is at least the most closely related paralog of AtBOR1 in the grapevine genome. Heterologous expression
of VvBOR1 compensates for loss of boron efflux pathways
in Bor1p-deficient Saccharomyces as well as in AtBOR1deficient Arabidopsis (735), indicating that VvBOR1 is a
boron efflux transporter and normally plays a role in “rootto-shoot” boron transport (mimicking the action of AtBOR1 in FIGURE 9B). It has been suggested, in light of the
reduced fertility of bor1–1 mutant plants, that VvBOR1
action promotes the fertility of individual Vitis flowers and
thereby reduces the incidence of formation of small, seedless “shot” berries that can be formed parthenocarpically
from unfertilized flowers (735). Consistent with this hypothesis, VvBOR1 transcript abundance and boron content
are both lower in shot versus seeded grapes, although a
causal link has yet to be established (735).
F. Invertebrate Animals
The first NCBT to be described, the Na⫹-driven Cl-HCO3
exchanger, was originally detected in squid axons and snail
neurons (105, 106, 111, 826). Moreover, experiments on
these preparations, as well as barnacle muscle fibers (110)
and crayfish neurons (658), first elucidated the importance
of these transporter activities for pHi regulation. In the
post-genomic era, many invertebrate HCO3⫺ transport activities have been specifically attributed to Slc4-like products. It is possible to see structural features, actions, and
physiological roles of these transporters that are shared
13
The GenBank accession numbers are provided in Appendix II.
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ever, in the context of a unicellular organism, heterologous
expression of AtBOR1-GFP in a Bor1p-deficient yeast
strain promotes boron efflux and increases tolerance to boric acid (650, 943).
MARK D. PARKER AND WALTER F. BORON
with some mammalian Slc4s. The invertebrate Slc4-like
proteins are structurally diverse, but have a similar inferred
topology to their vertebrate counterparts, including extended Nt and Ct (e.g., FIGURE 2A).
trans-side requirement for Cl⫺ to mediate Na⫹ and HCO3⫺
efflux) have not been formally demonstrated. At least the
molecular action of ABTS-1 is qualitatively indistinguishable from that of the Na⫹-driven anion exchanger NDAE1
from Drosophila (see Ref. 71).
1. Sponge
2. Nematode worms
The C. elegans genome contains four Slc4-like genes, abts-1
(anion bicarbonate transporter-1 aka CeNBC) through
abts-4 (FIGURE 4/Pseuodocoelomata). The abts-1 gene has
two alternative promoters that are active in different cell
types (71), and abts-4 has at least two splice variants (876).
Yet more predicted transcript variants are represented on
Wormbase.14
A) DISTRIBUTION. In transgenic worms, the promoters of these
four abts genes drive GFP expression in neurons (abt-1– 4),
hypodermal cells (abts-1 and -3), body wall, pharynx, and
vulval muscle cells (abts-1) as well as the intestine (abts-1
and -4) (71, 584, 876). Additional sites of expression for
ABTS-2 and ABTS-4 protein are revealed in transgenic
worms in which the natural termination codons of the genes
are replaced by an in-frame GFP open-reading frame. In
these animals, ABTS-2-GFP is expressed in the excretory
cell of larvae and the ovaries of adults, whereas ABTS-4GFP is expressed in gut cells (876). Both transporter fusions
exhibited a basolateral distribution.
B) MOLECULAR ACTION.
To date, only the function of ABTS-1
(aka ceNBC), which, of the four ABTS proteins bears most
sequence similarity to vertebrate Slc4s, has been characterized in detail. When heterologously expressed in Xenopus
oocytes, ABTS-1 mediates a robust 36Cl influx and also
mediates a detectable Cl-HCO3 exchange activity (71, 876).
Furthermore ABTS-1 mediates electroneutral Na/HCO3
cotransport (71, 804). Taken together, these actions could
be consistent with Na⫹-driven Cl-HCO3 exchange, although the hallmarks of classical NDCBE activity (i.e.,
Na⫹- and HCO3⫺-dependent Cl⫺ efflux and an absolute
14
http://wormbase.sanger.ac.uk.
826
ABTS-1 also transports iodide (71). Of additional interest
are the observations that abts-1 transcript and protein
abundance doubled during arsenite exposure and that abts1-null worms are hypersensitive to arsenite toxicity (584).
Arsenite causes intracellular acidification in a human cell
line (425), leading Liao and co-workers to suggest that, if
arsenite also lowers pHi in worms, abts-1-null worms may
be unable to adequately counter the drop in pHi, leading to
apoptosis (584). Although untested, another intriguing possibility is that ABTS-1 itself might counter arsenite toxicity by
providing an arsenite-efflux pathway, paralleling the borate
tolerance conferred by yeast and plantal Slc4-like products.
The substrates of ABTS-2, -3, and -4 are unknown. ABTS-2
does not mediate substantial Cl⫺ or oxalate2⫺ uptake when
expressed in Xenopus oocytes (876).
C) PHYSIOLOGICAL ROLE OF ABTS-1.
In mammals, the concerted
efforts of a Na⫹ driven Cl-HCO3 exchanger (NDCBE) and
a K/Cl cotransporter (KCC-2) are hypothesized to play a
role in nervous system maturation by lowering intracellular
[Cl⫺] and potentiating the inhibitory effect of GABAergic
and glycinergic signaling. In the case of C. elegans, ABTS-1
(together with KCC-2) is thought to play a similar role in
the maturation of GABAergic signaling (see Refs. 71 and
948 as well as FIGURE 10) because of the following observations of neuronal hyperexcitability in abts-1-null worms.
1) abts-1-null worms are hypersensitive to the postsynaptic
acetylcholinesterase inhibitor aldicarb as well as to the nicotinic acetylcholine receptor agonist levamisole (361, 584),
indicating excessive ACh release.
2) Worms with a defective 5-HT reuptake transporter are
typically hypersensitive to the inhibitory (i.e., hyperpolarizing) effects of 5-HT, causing the worms to slow down more
than normal. Worms with mutations in the abts-1 gene
(which would lead instead to depolarization) lose this hypersensitivity (361).
3) Hermaphrodite specific neurons (HSNs), which innervate the vulval muscles, express ABTS-1. In wild-type
worms, the GABA receptor agonist muscimol inhibits egg
laying via an inhibitory effect on the GABAergic HSNs. In
worms carrying a mutation in abts-1, muscimol has no
effect on egg laying, that is, GABA no longer elicits an
inhibitory response.
4) Worms with a mutation in the G protein-coupled receptor EGL-47 (659) typically exhibit a reduction in egg laying.
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In the demosponge Suberites domuncula, cells associated
with the siliceous endoskeleton contain transcripts encoding an Slc4-like transporter “NBCSA” (847). Three lines of
evidence suggest that NBCSA may be a silicate transporter. 1) Many Slc4 transporters are stilbene-sensitive,
and sponge cells have a DIDS-inhibitable silicate uptake
activity; 2) NBCSA is the only Slc4-like transporter identified so far in this organism; and 3) NBCSA transcripts are
upregulated in sponge cells by the presence of silicic acid in
the bathing medium (847). No data address the issue of
whether the transporter is Na⫹-coupled or has the ability to
transport HCO3⫺.
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
A
B
Neurons of wild-type worm
Neurons of abts-1 null worm
Agonist
(e.g., GABA)
K+
Agonist
(e.g., GABA)
Cl–
Cl–
KCC-2
K+
ABTS-1
Na+
Cl–
KCC-2
2 HCO3–
Cl–
ABTS-1
BTS
Cl–
–
KCC-2 and ABTS-1 lower [Cl–]i
Vm more positive than ECl
Stimulus is hyperpolarizing
Neurotransmitter release inhibited
1)
2)
3)
4)
Reduced ability to lower [Cl–]i
Vm more negative than ECl
Stimulus is depolarizing
Neurotransmitter release stimulated
FIGURE 10. Role of the Na⫹-driven anion exchanger ABTS-1 in the neurons of nematode worms. In wild-type
C. elegans worms, ABTS-1 and the K/Cl cotransporter KCC-2 lower intracellular [Cl⫺] rendering GABAergic,
glutamatergic, and 5-HT-ergic signals inhibitory to neurotransmitter release (A). In abts-1—null worms, the
reduced ability to lower intracellular [Cl⫺] reduces the potency of inhibitory signals (B).
Egg laying is restored in worms with an additional mutation
in abts-1 (71).
5) Muscimol causes body-wall muscles to hyperpolarize,
resulting in an increase in body length. However, in worms
with a disrupted abts-1 allele, muscimol causes a decrease in
body length (71).
3. Annelid worms
Although the molecular identity of an annelid NCBT has
yet to be elucidated, multiple studies have demonstrated the
presence of NCBT activity in these organisms. NCBT activity in annelidan cells was first demonstrated in 1985 by
Schlue and Thomas, who studied the medicinal leech
Hirudo medicinalis (841). In leech Retzius neurons, Schlue
and Thomas showed that pHi recovery from an acid-load in
the presence of CO2/HCO3⫺ is mediated by the dual action
of an amiloride-sensitive Na-H exchanger (NHE) activity as
well as a SITS-sensitive electroneutral Na/HCO3 cotransport activity that was proposed, although not demonstrated, to be due to a Na⫹-driven Cl-HCO3 exchanger
(841). A third pHi regulatory mechanism was described in
leech neuropile glial cells, a SITS-insensitive NCBT, the activity of which was accompanied by a small membrane
hyperpolarization (235). This electrogenic glial transporter,
suggested to be an electrogenic NBC operating with a Na⫹:
HCO3⫺ stoichiometry of 1:2, was later demonstrated to be
blocked by DIDS (236) and to readily perform electrogenic
Li/HCO3 cotransport (671). The DIDS-sensitive current
carried by the transporter has a reversal potential close to
the resting potential of the glial membrane (671) and thus
the net direction of HCO3⫺ transport mediated by the leech
glial NCBT may either be inwards or outwards, depending
on the membrane potential (Vm) of the cell (239) and the
extracellular pH (233). In this way, HCO3⫺ transport across
the glial membrane can substantially modulate pHo to
counter changes induced by neuronal activity (234, 812).
The high-affinity of the transporter (Km ⬍1 mM) for HCO3⫺
means that the system contributes to pHi regulation in these
cells even in the nominal absence of HCO3⫺ (237). Further
experiments on leech giant glia indicate that the action of
electrogenic NCBT in these cells could influence the rate of
glutamate uptake through excitatory amino acid transporters, contributing towards termination of synaptic transmission (238). In summary, functional data would suggest that
the genome of the leech includes at least two NCBT-like
genes.
4. Mollusks
Snail neurons and squid giant axons are classic systems
for the study of intracellular pH regulatory mechanisms,
and it was in these cell types that the first Na⫹-driven
Cl-HCO3 exchanger (105, 106, 111, 826, 965, 966) and
K/HCO3 cotransporter (220, 386, 387, 1097) activities
were identified. To date, three Slc4-like genes have been
cloned from squid (i.e., Loligo pealei) giant fiber lobe:
sqNBCe, sqNDCBE, and the AE-like “SF4” (FIGURE
4/Mollusca). It is unknown whether squid genomes include a BOR-like gene.
Characterized as an electroneutral Na⫹-driven ClHCO3 exchanger, this gene-product is also known as “SF1”
(1008). This is not an Slc4a8 gene product, but sqNDCBE at
least shares a common ancestor with mammalian electroA) sqNDCBe.
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1)
2)
3)
4)
+
MARK D. PARKER AND WALTER F. BORON
neutral NCBTs. Transcripts of sqNDCBE are detected by
northern blot in the giant fiber lobe, optic lobe, heart,
and stellate ganglion (1008). The physiological characteristics of sqNDCBE expressed in Xenopus oocytes differs in two respects from the Na⫹-driven Cl-HCO3 exchange activity reported from squid axons. 1) In oocytes
but not axons, Li⫹ can support sqNDCBE-mediated
HCO3⫺ transport ⬃75% as well as Na⫹. 2) In oocytes but
not axons, sqNDCBE-mediated HCO3⫺ transport can be
readily driven in the efflux direction by removal of bath
Na⫹. The precise reasons for these discrepancies have yet
to be resolved (1008).
B) sqNBCe.
The predicted amino acid sequence of sqNBCe (aka
“SF3”) has a higher overall identity to the electroneutral rather
than electrogenic vertebrate Slc4s. Thus it was surprising that
SF3, when heterologously expressed in Xenopus oocytes,
proved to be an electrogenic Na/HCO3 cotransporter, named
sqNBCe, the first electrogenic NCBT to be cloned from an
invertebrate (746). This observation highlights a potential
problem with making sequence-based predictions of transporter function. sqNBCe transcripts have a very different distribution to those of sqNDCBE and are predominantly detected by Northern blot in the gill and heart with additional
expression in the giant fiber lobe (746). As expressed in
oocytes, sqNBCe is unable to support Li⫹-stimulated HCO3⫺
transport, unlike the situation for the electrogenic NBC from
leech glia, studied in situ. An intriguing observation, again for
sqNBCe expressed in oocytes, is that removal of extracellular
Na⫹ causes a prolonged inhibition of the transporter that is
not reversed by restoring Na⫹ to the bath (746).
Its deduced amino acid sequence suggests that SF4,
the third Slc4-like transporter to be cloned from giant fiber
lobe, is an AE-like gene-product. The function of SF4 has
yet to be reported.
Clues as to the role of NCBTs in
mollusks come from a recent study of the cuttlefish Sepia
officinalis. Adult cuttlefish counter acidosis under conditions of chronic hypercapnia by elevating plasma [HCO3⫺]
(396). Cuttlefish express two NCBTs in their gill epithelia:
“soNBC” and “soNDCBE” (362), orthologs of sqNBCe
and sqNDCBE. If soNBC is an electrogenic NCBT, it would
be positioned to mediate an increased branchial HCO3⫺
reabsorption in these animals under hypercapnic conditions, per the role of branchial NBCe1 in fish. However,
soNBC transcript abundance is not specifically altered by
chronic elevations of PCO2 in juveniles and is paradoxically
decreased in embryos and hatchlings (362). It is possible in
juveniles that upregulation of soNBCe occurs at the post
transcriptional level, or that pH regulation is effected via an
alternative mechanism.
In snail neurons, an NDCBE-like activity contributes to pHi
regulation (966) and therefore likely maintains neuronal
excitability, per the action of NCBTs in mammalian neurons. However, it has not been formally established
whether the activity described in snail neurons is mediated
by a Na⫹-driven Cl-HCO3 exchanger or the tightly-coupled
action of an AE and an NHE (966).
5. Insects
No insect genome reported to date appears to contain more
than three Slc4-like genes, and none is known to include a
BOR-like gene. The best-studied insect genome, that of the
fruit fly Drosophila melanogaster, encodes two Slc4-like
proteins (FIGURE 4/Panarthropoda): NDAE1 and CG8177.
Multiple splice variants have been reported for each geneproduct.
Drosophila NDAE1 (“Na⫹-driven anion exchanger”) has been characterized as a Na⫹-driven ClHCO3 (or Na⫹-driven Cl-OH) exchanger with a small associated anion leak (71, 810). Drosophila NDAE1 is also
capable of mediating a substantial DIDS-sensitive NO3⫺ influx when heterologously expressed in Xenopus oocytes
(852). An ortholog of NDAE1, AgNDAE1, from the mosquito Anopheles gambiae, mediates a similar Na⫹-driven
anion exchange activity and is also capable of mediating
some I⫺ influx (589).
A) NDAE1.
In Drosophila, NDAE1 is widely expressed throughout the
gut, Malpighian tubules, nervous system, and sensilla, with
the majority of protein being basolaterally distributed (589,
854). NDAE1 transcripts have also been demonstrated in a
specific subset of myocytes15 in the heart region of the cardiac tube of Drosophila (737). In the mosquito Aedes ae-
C) SF4.
828
15
Those that coexpress the cardiac homeotic products Tin and
Abd-A. NDAE1 expression is dependent on the expression of Abd-A
(737).
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In situ, squid-axon NDCBE activity requires ATP, possibly for the phosphorylation of the transporter or an essential activator (107, 221). In addition, three lines of
kinetic evidence are consistent with the hypothesis that
the squid-axon transporter, in situ, actually transports
the NaCO3⫺ ion pair. 1) Reciprocal changes in [Na⫹]o
and [HCO3⫺]o have no effect on the flux as long as the
product [Na⫹]o ⫻ [HCO3⫺]o is maintained fixed at constant extracellular pH (pHo) (111). Indeed, at a fixed
pHo, this product is proportional to [NaCO3⫺]o. 2)
Changes in pHo have no effect on the flux as long as
[NaCO3⫺]o is fixed (109). 3) The reversible stilbene derivative DNDS (a divalent anion) appears to be a competitive inhibitor not only with extracellular HCO3⫺ but also
with extracellular Na⫹. A kinetic analysis is consistent
with the hypothesis that DNDS in fact competes with the
NaCO3⫺ ion pair (108). Studies in frogs (534) and rabbits
(see p. 847) indicate that vertebrate NCBTs do not transport the NaCO3⫺ ion pair.
D) PHYSIOLOGICAL ROLE.
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
gypti, multiple NDAE1 transcript splice variants have been
detected in the Malpighian tubules of adults (1070), in
which NDAE1 protein is localized to the basolateral membranes of principal cells (589). A preliminary immunohistochemical study also localized NDAE1 to the basolateral
membrane of the anterior stomach epithelia of Aedes aegypti larvae (654). An ortholog of NDAE1 likely mediates
the stilbene-sensitive Na⫹-driven Cl-HCO3 exchange activity that has been detected in locust neurons (851). Also like
NDAE1, this locust transporter is active in the absence of
HCO3⫺. The importance of NDAE1 for insect health is underscored by the lethal nature of a P-element insertion in the
Drosophila ndae1 5= untranslated region (UTR) (810).
6. Echinoderms
A single Slc4-like gene has been cloned from the testes of the
sea urchin Strongylocentrotus purpuratus (358), although
genome analysis suggests that sea urchins may have four
other Slc4-like genes (FIGURE 4/Echinoderms). The NCBTlike protein product of the cloned gene-“Sp-NBC”-is concentrated in the flagellar membrane of sea urchin sperm,
where, as suggested by the authors of that study, it may play
16
This terminology is not intended to infer that AaAE1 is a direct
ortholog of mammalian Slc4a1, but instead refers to AaAE1 being
the first of two Na⫹-independent AEs cloned from insects.
7. Urochordates
The draft genome of the sea squirt Ciona intestinalis predicts
the existence of three Slc4-like genes, one each that can be
described as AE-like, NCBT-like, and BOR-like (FIGURE 4/
Chordata). An in situ hybridization study shows that the
NCBT-like gene is transcribed in the brain and visceral
ganglion of tailbud embryos (837). These data are reinforced by the distribution of Ciona expressed sequence tags
(ESTs), which suggests an exclusively neuronal expression
of the NBC-like gene (837). EST data further indicate that
the AE-like gene is expressed in the digestive gland, heart,
and hemocytes, whereas the BOR-like gene is expressed in
the heart, hemocytes, and neural complex (837). At present,
nothing is known of the molecular action or physiological
role of the Ciona Slc4-like transporters.
G. Nonmammalian Vertebrates
It is likely that most vertebrate genomes encode orthologs of
the three AEs, five NCBTs, and the singular BOR encoded by
the human genome. However, not all vertebrate genomes include an Slc4a9 ortholog. The following sections summarize
our current knowledge of the five NCBTs, as well as the BOR,
in nonmammalian vertebrates. Note that the overwhelming
majority of the published work in this area concerns electrogenic NCBTs of fishes and amphibians.
1. Cartilaginous fishes
As far as we are aware, only one study has addressed the role
of NCBTs in cartilaginous fishes. From the spiny dogfish
(Squalus acanthias), Bleich et al. (84) studied isolated perfused
rectal gland tubules, which contribute to whole animal osmoregulation by secreting a hyperosmotic NaCl solution (137).
The molecular mechanism of NaCl secretion by tubule cells is
represented in FIGURE 11. In cells from these tubules, pHi
recovery from an acid load (imposed by an NH4⫹ prepulse; Ref. 106) requires basolateral Na⫹, is slowed by
removing CO2/HCO3⫺, and is inhibited by DIDS. Thus
these cells probably have an NCBT at the basolateral
membrane. In separate experiments, the authors also observed that either reducing basolateral [Cl⫺] or depolarizing the cell causes a slow rise in pHi. They proposed a
basolateral Na⫹-driven Cl-HCO3 exchanger with an unexpected voltage dependence, which they explained by
suggesting that depolarization leads to a rise in [Cl⫺]i,
which in turn enhances Na⫹-driven Cl-HCO3 exchange.
Other possibilities include the following: 1) basolateral
Na⫹-driven Cl-HCO3 exchanger in parallel with a depolarization-induced alkalinization (DIA; see Refs. 884 and
885) that is totally independent of the HCO3⫺ transporter; 2) basolateral electroneutral NBC in parallel with
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B) CG8177 AND AeAE. CG8177 is an AE-like gene-product, the
distribution and molecular action of which has yet to be
fully elucidated in Drosophila. The name CG8177 refers to
“computed gene.” However, a preliminary study suggests
that the protein mediates a 36Cl influx when expressed in
Xenopus oocytes (454). In Drosophila larvae, CG8177,
also termed DAE (Drosophila anion exchanger), is located
in the basal membranes of interstitial cells of the midgut
(263). Although RNAi knockdown of CG8177 in the
midgut did not result in a phenotypic change in one study,
global knockdown of CG8177 is lethal (263). A study of
one of the two CG8177 orthologs from the mosquito Aedes
aegypti (termed AeAE or sometimes AaAE1),16 localized
the transporter to the basal membrane of stellate cells in the
Malpighian tubules (589, 747). The same distribution is
also observed for the Anopheles gambiae ortholog AgAE1
(589). Besides the Malpighian tubules, “AE1” is abundant
in the gastric cecae and anterior midgut of larval Aedes and
Anopheles, the lumen of which maintains an extremely alkaline pH to aid digestion (589, 590). When heterologously
expressed in Xenopus oocytes, AeAE mediates stilbene-sensitive, Na⫹-independent Cl-HCO3 exchange (747). AeAE
could be responsible for the DIDS-sensitive basolateral ClHCO3 exchange activity detected in the anterior segment of
mosquito rectal saltglands (912). Little is known about the
second AE-like gene-product from mosquitos (AgAE2) except for a report that it is widely expressed thought the gut
of mosquito larvae (589).
a role in sperm capacitation and regulation of sperm motility (358).
MARK D. PARKER AND WALTER F. BORON
Duct lumen
Tight junction
Interstitial fluid
Na+
K+
Na-K pump
CFTR
Na+
–
Cl
Na+
++
cAMP
HCO3–
?
H+
NHE
Cl–
H+
Na+
NKCC
–
2 Cl
HCO3–
CA
K+
KCNQ1
Cl–?
Slc4?
Na+
CO2
H2O
–
Rectal Gland
2 HCO3
Rectal gland epithelium
Intestine
Rectum
FIGURE 11. Role of an NCBT in the rectal gland of cartilaginous fish. The rectal gland duct joins the intestine
at a point upstream of the rectum (see cartoon dogfish). In rectal gland epithelia, the Na-K pump maintains a
low intracellular [Na⫹] driving basolateral Na/K/Cl cotransporter (NKCC) activity that supplies Cl⫺ for secretion across the apical membrane by CFTR. Anion secretion draws Na⫹ and H2O through a paracellular
pathway, resulting in the secretion of a NaCl-rich solution from the interstitial fluid/blood. CO2 accumulation is
dissipated by intracellular carbonic anhydrase (CA). Respiratory acidosis is prevented by NHE and NCBT action.
⫺
NHE and NCBT could also support HCO3
secretion via the unidentified apical anion exchanger, that is likely a
member of the Slc26 family (84). KCNQ1 is a voltage-sensitive K⫹ channel (1018), also known as Kv7.1.
a Cl-HCO3 exchanger, plus an independent DIA; and 3)
basolateral electrogenic NBC in parallel with a basolateral Cl-HCO3 exchanger or Cl⫺ channel. The presence of
a basolateral NCBT is consistent with the stimulation of
rectal-gland NaCl secretion by the infusion of NaHCO3
into the blood, a maneuver that mimics the post-prandial
metabolic alkalosis known as the “alkaline tide” (1042).
Note that, in mammals, it is typically basolateral NBCe1
and/or NBCn1 that supports fluid and salt secretion
across epithelia by maintaining pHi and, under stimulated conditions, supplying HCO3⫺ for secretion (e.g., see
below).
mitochondrion-rich (MR) pavement cells17 of the gills as
well as other cells in the intestines.
A) MOLECULAR ACTION OF BONY FISH NBCe1. Currently, of the
NCBT orthologs expressed by bony fishes, only NBCe1 has
been cloned and functionally characterized. NBCe1 clones
from Osorezan dace (Tribolodon hakonensis; Ref. 382),
pufferfish (Takifugu obscurus; Ref. 526), and zebrafish
(Danio rerio; Ref. 926) all mediate electrogenic Na/HCO3
cotransport activity when expressed in Xenopus oocytes. In
addition, pufferfish NBCe1 is capable of electrogenic Li/
HCO3 cotransport (167). The electrogenicity of gulf toadfish (Opsanus beta) NBCe1 has not been demonstrated, but
2. Bony fishes
In bony fishes, NCBTs are vitally important to pHi and salt
homeostasis, processes that are mainly associated with the
830
17
According to terminology of Perry et al. (741), these cells are
referred to as MR cells in freshwater fish and chloride cells in
saltwater fish.
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K+
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
the clone mediates a HCO3⫺-dependent Na-influx into Xenopus oocytes with a Km for HCO3⫺ of ⬃8.5 mM (952).
Many of these observations could be explained if the
HCO3⫺-independent conductance associated with pufferfish
NBCe1 (observation 2 above) persists in the presence of
CO2/HCO3⫺ and makes a substantial contribution towards
Vm. Such a conductance could interfere with measurements
of true Na/HCO3 cotransport activity. A similar phenomenon has been described in the case of the HCO3⫺-independent conductance associated with the human NBCe1 mutant A799V (721). The HCO3⫺-independent conductance
associated with pufferfish NBCe1 is also reminiscent of the
conductive features of trout AE1 (663) and mammalian
NBCn1 (189).
B) DISTRIBUTION OF NCBTs IN BONY FISHES.
In the gill lamellae of
Osorezan dace, trout (Oncorhynchus mykiss), and zebrafish, immunocytochemistry confirms the basolateral distribution of NBCe1 protein in a subpopulation of MR cells
(382, 726, 925). Here NBCe1 is located in a “cytoplasmic”
compartment, which the authors of the dace study attribute
to an extensive basolateral system of infoldings (382), similar to those of salamander and mammalian PTs (632). Indeed, these MR cells express many transporters common to
mammalian renal epithelia (reviewed in Refs. 278, 406). In
zebrafish gills, NBCe1 colocalizes with NCC in a subpopulation of MR cells that do not express AE1 or the H pump
(561).
Outside the gill, NBCe1 mRNAs are detected in trout heart,
liver, stomach, white muscle (741, 742), toadfish brain
(952) and zebrafish brain, intestine, skin, and eye (561),
specifically in the corneal endothelium,18 ganglion cell
layer, rods, and cones (925, 926). In zebrafish embryos,
NBCe1 is localized to the pronephros, specifically the anterior tubules and ducts, optic cup, and the ependymal cells
that line the brain ventricles (926). NBCn1 and BTR1 expression appears to be widespread in zebrafish, whereas
transcripts of NBCe2, NDCBE, and NBCn2 appear partic-
18
aka the corneal posterior epithelium.
C) ROLE OF NCBTs IN BONY FISHES.
I) pH homeostasis. In primary cultures of gill epithelia from the freshwater rainbow
trout, a stilbene-sensitive Na⫹-dependent HCO3⫺ transport
process is a major contributor to pHi homeostasis (1043). A
striking example of how NCBTs can contribute to the pHi
homeostasis of teleost fish is provided by the Osorezan
dace, which, aside from spawning season, lives in a lake that
has a pH of ⬃3.5. However, the MR cells in the gills of
Osorezan dace are uniquely able to adapt to the acidic environment by the coordinated transcriptional upregulation
of NHE3, CA II, the Na-K pump, and NBCe1 (382), a
complete branchial Na/HCO3 uptake system (FIGURE 12A) that
closely resembles that responsible for HCO3⫺ reabsorption in the
mammalian proximal tubule (PT). Other, unadaptable fish species cannot survive in these acidic conditions due to a fatal combination of acidosis and an inability to accumulate salts against
the osmotic gradient. Indeed, in the gills of zebrafish, NBCe1
transcript abundance is reduced in response to water acidification
(561).
The molecular response of fishes to hypercapnia appears
to vary among species, but generally hypercapnia results
in a compensatory increase in HCO3⫺ reabsorption to
counter acidosis. In trout, a hypercapnic challenge leads
to a transient upregulation of branchial NBCe1 mRNA at
1– 4 h, and a delayed rise in renal NBCe1 mRNA levels
from 6 to 24 h (741). In the marine eelpout (Zoarces
viviparous), a 24-h exposure to hypercapnia causes a
paradoxical decrease in NBCe1 mRNA abundance in the
gills, whereas NBCe1 transcript abundance gradually increases during a 6-wk period of chronic hypercapnia
(232). In eels, which reportedly have no branchial
NBCe1, hypercapnia elicits a rapid upregulation of renal
NBCe1 mRNA that can reach levels 300-fold greater
than basal after 12 h (741). If these changes correlate
with increased HCO3⫺ reabsorption by the gills and kidney, these would tend to protect the fish from acidosis. In
the African lungfish (Protopterus annectens), on the
other hand, the abundance of branchial and renal
NBCe1, at least at the level of mRNA, are unaltered by
acid-base disturbances. Instead, these animals use ventilatory control to blow off excess CO2 and thereby
achieve whole body pH homeostasis (322).
Regarding the liver, evidence suggests that an electrogenic
Na/HCO3 cotransport activity is important for pHi homeostasis in trout hepatocytes (305), although, as in mammals
(8), this activity may be attributable to NBCe2.
Regarding the stomach, trout NBCe1 transcripts are detected in both the antrum and the corpus, where the trans-
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Pufferfish NBCe1, as expressed in Xenopus oocytes, exhibits a number of unique features not described for other
NBCe1 orthologs: 1) oocytes expressing pufferfish NBCe1
are unusually loaded with Na⫹; 2) pufferfish NBCe1 exhibits an inwardly rectifying, HCO3⫺-independent, ion conductance; 3) pufferfish NBCe1 exhibits an inwardly rectifying
CO2/HCO3⫺-dependent conductance; 4) the apparent Km of
the cotransporter for extracellular Na⫹ is voltage dependent (the apparent affinity being lower in the negative voltage range); and 5) the reversal potential (Erev) for the
cotransporter is not substantially altered by a reduction in
[Na⫹]o, as if the stoichiometry of the transporter is variable
(167).
ularly abundant in zebrafish brain and eye (561). Furthermore, in zebrafish, an abundance of NDCBE transcripts is
notable in the heart and NBCn2 expression is notable in the
spleen (561).
MARK D. PARKER AND WALTER F. BORON
A
B
Water
Tight junction
Interstitial
fluid
Intestinal
lumen
Interstitial
fluid
3 Na+
ENaC
H+
HCO3
2 K+
Na+
Na-K pump
HCO3–
H+
Na-K pump
HCO3–
CA
CO2
NBCe1
+
Na+
cAMP
3 HCO3–
H+
Gill MR Cell (Trout)
NBCe1
H2O
Na+
2 HCO3–
Intestinal Enterocyte (Pufferfish)
FIGURE 12. Role of NCBTs in the gills and intestines of bony fishes. In the gill epithelia of freshwater fish such
⫺
absorption from the water. In the gut epithelia of marine
as trout (A), NBCe1 contributes to Na⫹ and HCO3
⫺
fishes such as pufferfish (B), NBCe1 contributes to HCO3
secretion into the gut lumen. ENaC is an epithelial
Na⫹ channel. The identities of the non-Slc4 transporters involved in these pathways have not been determined
for all species in which these systems have been identified.
porter is hypothesized to support a protective secretion of
HCO3⫺ onto the apical surface of the cells. However, contrary to those authors expectations, NBCe1 mRNA levels
fell with dietary acidification (918).
II) Salt homeostasis. Killifish (Fundus heteroclitis) are vulnerable to Na⫹ loss by fluctuation in salinity in their environment, as reviewed briefly by Scott et al. (856). In these
fish (856) and in Japanese eels (981), NBCe1 is a constitutive player in a freshwater inducible branchial Na⫹ uptake
system, similar to that shown in FIGURE 12A. Following the
same theme, the MR cells of Mozambique tilapia (Oreochromis mossambicus) tend to exhibit reduced expression
of NHE3 and NBCe1 when the salinity of their environment is increased, although the data do not achieve statistical significance (306).
Marine fish desalt seawater as it passes along the gut.
HCO3⫺ secretion into the gut lumen plays a key role in
one aspect of this desalting, the removal of Ca2⫹ and
Mg2⫹ from the gut lumen as the secreted HCO3⫺ precipitates concentrated divalent cations as an excretable carbonate deposit (reviewed in Ref. 1037). A role for NBCe1
in this process is suggested by the presence of NBCe1
transcripts in the intestines of the gulf toadfish (952),
trout (343, 741), and pufferfish (526). Moreover, deposit
832
formation by isolated mucosal layers from the intestines
of the sea bass (Cicentrarchus labrax) depends on basolateral Na⫹ and HCO3⫺ (281). Immunocytochemistry
demonstrates a basolateral distribution for NBCe1 in
intestinal epithelium (526), where the cotransporter
would presumably operate with a 2:1 stoichiometry (FIG⫺
URE 12B). The apical step of HCO3 secretion is likely
effected by Slc26a6 (526). Transferring these fishes from
freshwater into seawater, or in the case of the toadfish
from less saline to more saline seawater, causes the upregulation of NBCe1 transcripts in their intestine (343,
526, 952). This would presumably support the increased
base excretion observed in these animals during seawater
acclimatization (741).
III) Development. Inhibition of NBCe1 translation in
zebrafish embryos produces developmental defects, including hydrocephalus, retinal distention, and the presence of unidentified particulate matter in the ventricular
spaces (926).
3. Amphibians
The first description of a Na-coupled HCO3⫺ transporter
that is independent of Cl, the electrogenic Na/HCO3
cotransporter, came from Boron and Boulpaep’s 1983
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CO2
H2O
Na
2 K+
Slc26a6
CA
NHE3
3 Na+
Cl–
–
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
study of pHi regulation in the proximal tubule of the tiger
salamander Ambystoma tigrinum (103). They demonstrated NCBT activity at the basolateral membrane of the
PT epithelia (103). A similar activity is present in the PT of
the mudpuppy Necturus maculosus (606). The injection of
poly(A) mRNA from salamander PT into Xenopus oocytes
led to the cloning of the first NCBT cDNA, which encodes
a protein termed aNBC (Ambystoma Na bicarbonate
cotransporter; Ref. 809). We now recognize aNBC19 as
Ambystoma NBCe1-A; a product of the Ambystoma Slc4a4
gene.20
A) MOLECULAR ACTION OF AMPHIBIAN NBCe1.
B) DISTRIBUTION OF NCBTs IN AMPHIBIANS. I) NBCe1. The majority of renal Ambystoma NBCe1 protein is localized to the
basal folds of the late distal tubule of this mesonephric
kidney, with a smaller basolateral presence in the PT (632,
844). A developmental expression pattern of NBCe1 in Xenopus laevis pronephroi is revealed by in situ hybridization
experiments (1102). Low abundance of Xenopus NBCe1
(“XNBC1”) mRNA occurs in the early and late PTs at
developmental stage NF29,21 but a greater abundance of
XNBC1 is present in the late distal segment by stage NF33
(1102), where its distribution overlaps with that of Ca2
(1103). A more recent model of the Xenopus pronephros,
based on a large-scale in situ hybridization mapping of transcripts, localizes a significant population of NBCe1 transcripts to an early distal tubule region “DT(1)” that is homologous to the mammalian thick ascending limb (581,
779). Aside from the pronephric presence, NBCe1 transcripts are detected in the cranial ganglia, nasal pit, otic
vesicle, somites, hatching gland, and cement gland of Xenopus embryos as well as in the bladder, brain, and small
intestine of Ambystoma (809).
19
GenBank protein accession O13134.
Nomenclature guidelines for Xenopus genes are provided at
http://www.xenbase.org/gene/static/geneNomenclature.jsp.
21
About 1.5 days post-fertilization. “Nieuwkoop Faber” developmental stages are defined in Ref. 1, and the images that accompany
the defintions are reproduced online at the Xenbase: Xenopus laevis
and Xenopus tropicalis biology and genomics resource (http://
www.xenbase.org/anatomy/static/NF/NF-all.jsp).
20
III) NBCn1. Slc4a7 transcripts that encode the electroneutral Na/HCO3 cotransporter NBCn1 are detected by in situ
hybridization mainly in the central nervous system of Xenopus embyros, with an additional presence in the PT, epidermis and external gills. Slc4a7 is expressed at an earlier
developmental stage than slc4a10.
IV) NDCBE. We are unaware of any reports concerning the
distribution of Slc4a8 products in amphibians, although if it
exhibits a similar expression pattern to its mammalian ortholog, we might expect Slc4a8 to be abundantly expressed in
neurons.
V) NBCn2. Slc4a10 transcripts are detected by in situ hybridization mainly in the central nervous system (brain,
retina, and spinal cord) and the pineal gland of Xenopus
embryos and at a later developmental stage than Slc4a7.
VI) Slc4a9. We are unaware of any reports concerning the
distribution of Slc4a9 products in amphibians.
VII) BTR1. Transcripts for another Slc4 family member
Slc4a11 (referred to as “XNBC2”) are evident in the early
PT at developmental stage NF26. Some transient expression also occurs in the early distal segment, but is much
diminished by stage NF38 (1102).
C) ROLE OF NCBTs IN AMPHIBIANS. I) Vision. In the amphibian eye,
electrogenic Na/HCO3 cotransport activity, presently unattributed to a specific NCBT, has been detected in 1) lens epithelia of the cane toad Bufo marinus (1039), 2) optic nerve
glial cells of Necturus (50, 51), and 3) retinal glial cells of both
Necturus (51) and Ambystoma (680). In retinal glial cells from
Ambystoma, electrogenic NCBT activity is calculated to operate with a Na⫹:HCO3⫺ stoichiometry of 1:3 and is preferentially localized at the glial end-feet (681, 682). In glial cells
from both the retina and optic nerve, the electrogenic NCBT
activity contributes towards maintaining a slightly alkaline
resting pHi (49, 681), implying that the electrogenic NBC mediates a net HCO3⫺ uptake even when operating with a stoichiometry of 1:3 (see below). When retinal glial cells from
Ambystoma are depolarized, the resulting HCO3⫺ influx mediated by the electrogenic NCBT causes a drop in pHo that
could serve to balance the extracellular alkalinizations resulting from neuronal activity or light stimulation of the retina
(681). Because the NCBT-mediated drop in pHo is localized at
22
Data from The Xenopus Gene Expression Database (http://
www.euregene.org/xgebase/pages/entry_page.html).
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Currently, of the
NCBT orthologs expressed by amphibians, only an
NBCe1-A ortholog from the salamander Ambystoma has
been cloned and functionally characterized. This protein is
an electrogenic Na/HCO3 cotransporter with kinetic properties that are very similar to mammalian (i.e., rat)
NBCe1-A (339). Like mammalian NBCe1, salamander
NBCe1 is blocked by DIDS (339). Kinetic data suggest that
an NBCe1/NBCe2-like activity in frog retinal pigment epithelium does not, unlike the NCBT activity in squid giant
axons, transport the ion pair NaCO3⫺ inasmuch as the Km
for Na⫹ of this activity appears to be independent of
[CO32⫺] (534).
II) NBCe2. Slc4a5 transcripts that encode the second electrogenic NCBT, NBCe2, are detected by in situ hybridization of
Xenopus oocytes and appear to persist in most tissues
throughout embryonic development.22 Note that isolated, defollicated oocytes exhibit no detectable electrogenic NCBT
activity (1009).
MARK D. PARKER AND WALTER F. BORON
the glial endfeet that contact blood vessels, and because blood
vessels dilate in response to a fall in pHo, it has been suggested
that NCBT activity may also contribute to a mechanism that
increases blood flow during neuronal activity (681).
A
II) Mucosal protection. In the stomach of the edible frog
Rana esculenta, NCBT activity is present at the basolateral
membranes of the oxynt(ic)opeptic cells that alternately secrete both HCl and HCO3⫺ at the surface of the gastric
fundus (Ref. 209 and FIGURE 13B). In the edible frog, the
NCBT activity is clearly mediated by an electrogenic Na/
HCO3 cotransporter, whereas in the in the North American
bullfrog Rana castesbiana, it is not clear whether the transporter is electrogenic or electroneutral (1074). In both
cases, NCBT activity, the basolateral step in the secretion of
HCO3⫺ into the mucus that covers the stomach surface,
would play a protective role by helping to counter the acidifying effect of HCl back-diffusion from the gastric lumen,
and in the process would keep pHi relatively high (208,
1074). Electrogenic NCBT activity in the oxynt(ic)opeptic
cells of Rana esculenta is stimulated by carbachol but inhibited by histamine (228).23
23
Although carbachol and histamine both stimulate HCl secretion.
B
Subretinal
space
Tight junction
Choroid
Stomach
lumen
Interstitial
fluid
H2O
Na-K pump
H-K pump
2 K+
K+
3 Na+
H+
3 Na+
2 K+
Na-K pump
NKCC
Cl–
HCO3–
Na+
AE
K+
2 Cl–
Cl–
HCO3–
NCBT
NCBT
Cl–
Na+
HCO3–
2 HCO3–
2 HCO3–
Slc26?
Retinal Pigmented Epithelium
(Frog)
Na+
Oxyntopeptic Cell
(Frog)
FIGURE 13. Role of NCBTs in the retinas and stomachs of amphibia. In the retinal pigment epithelia of frogs
(A), an electrogenic NCBT exhibits an unusual apical distribution and contributes towards fluid absorption from
the subretinal space, promoting retinal attachment. Although undemonstrated in frogs, study of mammalian
RPE indicates that the apical NCBT could be either NBCe1 or NBCe2 and the basolateral AE could be AE2 (11).
⫺
In gastric epithelia of frogs (B), an electrogenic NCBT contributes towards HCO3
secretion onto the cell surface
⫺
that protects cells from acid attack. The transporter responsible for moving HCO3
across the luminal
membrane of these cells has not been identified, although both Slc26a6 and Slc26a9 have been suggested to
perform this function in mammalian gastric mucosa (744, 1055).
834
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The apical membrane of bullfrog retinal pigment epithelia
(RPE) has stilbene-sensitive electrogenic Na/HCO3 cotransport activity (400, 532) that is a major contributor to the
transepithelial HCO3⫺ absorption from retina to blood. This
HCO3⫺ absorption helps to drive fluid absorption across the
RPE, preventing subretinal edema and promoting retinal attachment (533, 536). The absorption of HCO3⫺ per se lowers
subretinal pHo (535). It has been suggested that the NBC activity may be responsible for the lowering of subretinal pHo
that occurs in response to a light-induced reduction in [K⫹]o
(585). FIGURE 13A shows a model of the role of electrogenic
NCBT activity in the transepithelial ion transport across the
amphibian RPE. In RPE, the apical polarity of normally basolaterally distributed transporters such as NCBTs and the Na
pump is related to an unusual, partial reversal of polarized
protein distribution in these cells (reviewed in Ref. 627). By
analogy to the distribution of NCBTs in rats and humans, the
apical NCBT in the RPE of bullfrogs is probably an Slc4a4
product (11, 94), although Slc4a5 transcripts are expressed in
human RPE and, in mice, retinal detachment is a phenotype of
Slc4a5 gene disruption (see p. 880).
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
In the Necturus PT, the movement of Cl⫺ across the basolateral membrane has a strong trans-side dependence on
Na⫹ and HCO3⫺, consistent with the presence of a Na⫹driven Cl-HCO3 exchanger (356). On the other hand, the
data are also consistent with the presence of a basolateral
Cl⫺ channel in parallel with the subsequently identified
electrogenic NBC activity (606), with voltage changes providing indirect coupling of Cl⫺ to Na⫹ and HCO3⫺. Viewed
somewhat differently, if a cell has a Cl⫺ conductance and an
A
Lumen
Tight junction
Interstitial
fluid
NHE
Na-K pump
3 Na+
Na+
H+
++
cAMP
2 K+
electrogenic NBC in the same membrane, it would be very
difficult, using only classical electrophysiological approaches, to resolve the presence of a Na⫹-driven Cl-HCO3
exchanger (see also our discussion of the dogfish NDCBE
with unusual voltage dependence on p. 829).
4. Reptiles
Orthologs of all mammalian Slc4s, with the exception of
NBCe2, are identifiable in the draft genome of the green
anole lizard Anolis carolinensis. However, we know of no
definitive demonstration of NCBT activity in any reptilian
cell or tissue. The lack of reports concerning reptilian
NCBT activity is likely related to 1) the underrepresentation
of reptiles among physiological model organisms and 2) the
unusual acid-base physiology of the reptiles that have been
studied. For example, Alligator mississippiensis excretes an
unusually alkaline urine and has a low plasma [HCO3⫺]
(566). The PT epithelia of these animals apparently do not
express NHE or CAII (1000), proteins that are considered
necessary for substantial HCO3⫺ reabsorption in mammals.
Furthermore, the distal renal epithelia of alligators actually
mediate a net secretion of HCO3⫺ under normal conditions
(565, 1000), reminiscent of collecting ducts of mammals fed
an alkaline diet (e.g., see Refs. 271 and 319). However, the
working of an as-yet unidentified HCO3⫺ reabsorbtive
mechanism is disclosed in alligator distal tubules when tubular HCO3⫺ secretion is blocked by acetazolamide (565).
In alligators, HCO3⫺ secretion may serve to balance the
renal excretion of NH4⫹ (due to the high pH of the urine,
pNH3 in alligator urine is ⬃0.1 mmHg; Ref. 566) that is
necessary due to their inability to synthesize urea (565).
Even from studies of snakes, which are capable of acidifying
their urine, there are no reports of NCBT activity in isolated
proximal or distal renal tubules (217, 494).
5. Birds
NHE
Na+
H+
HCO3–
cAMP
CA
++
H+
NBCe1-A
Na+
CO2
3 HCO3–
H2O
Proximal or Distal Tubule Cell
(Salamander)
FIGURE 14. Role of NCBTs in the renal tubules of amphibia. In
proximal and distal renal tubule epithelia of amphibia, NBCe1 con⫺
reabsorption pathway,
tributes towards a H⫹ secretion/HCO3
which maintains whole body pH within a narrow physiological range.
Orthologs of all 10 mammalian Slc4s are identifiable in the
draft genome of the fowl Gallus gallus. However, published
studies of avian NCBT activity are few in number.
Nephrons in avian kidneys are graded into three categories,
according to differences in the length of their loops of Henle
(briefly reviewed in Ref. 122): 1) long-looped “mammalianlike” nephrons; 2) short-looped “reptilian-like” nephrons;
and, falling between the two extreme forms, 3) a population
of “transitional” nephrons. A stilbene-sensitive NCBT activity, most consistent with the presence of NDCBE, is detected in isolated nonperfused PT from chicken transitional
(493) and long-looped nephrons (122, 123), but not in
short-looped nephrons (122, 629).
NCBT activity has also been detected in studies of nonrenal
avian cells. The steady-state pHi of cultured chick embryonic heart cells is maintained at a level higher than electrochemical equilibrium by a combination of NHE and SITS-
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III) Renal HCO3⫺ reabsorption. aNBC has a similar molecular physiology to mammalian NBCe1-A (338) and likely
plays the same important role in HCO3⫺ reabsorption from
the glomerular filtrate. However, the acidification of amphibian tubule fluid is predominantly achieved in the late
distal tubule, by an electrogenic Na⫹-dependent process
(749), as demonstrated by early experiments on Necturus
and leopard frog (Rana pipiens) renal tubules (657) and
later measurements of bicarbonate reabsorption in Ambystoma maculatum renal tubules (1088). FIGURE 14 shows
models of the role of NBCe1 in bicarbonate reabsorption by
amphibian pronephric epithelia in the proximal and distal
tubules. Norepinephrine has an inhibitory effect upon
NBCe1-mediated HCO3⫺ reabsorption in the Ambystoma
PT, perhaps by elevating cAMP levels (2), a factor that is
also inhibitory to electrogenic NCBT activity in rabbit renal
tubule preparations (821).
MARK D. PARKER AND WALTER F. BORON
sensitive NDCBE-like activities, both of which play a role in
recovery from an acid load imposed by an NH4⫹ prepulse
(594). In chicken chondrocytes, an NDCBE-like activity is
expected to contribute to the recovery from intracellular
acidosis that would accompany a mechanical load (218).
Stilbene-sensitive NCBT activity has also been reported in
chicken enterocytes (734) and colonocytes (146).
IV. GENERAL FEATURES OF NCBTs
A. General Structural Features of
Mammalian NCBTs
Because the Na⫹-independent Cl-HCO3 exchangers (i.e.,
AE1–3) in the Slc4 family are 28 –34% identical to NCBTs
at the amino acid level (807), it is likely that NCBTs share
many common structural features with AEs. Studies concerning the structure of NCBTs are therefore heavily supplemented by reference to the wealth of data produced by
ongoing studies into the structure of AE1. However, because of their differing functions, crucial structural differences are likely to exist between the AEs and NCBTs
(1112), and perhaps even among NCBTs. Here, using as
our template a model of AE1 structure, refined with new
data from recent studies of NBCe1 topology (see Refs.
1112, 1113 as well as FIGURE 2A), we consider the common
structural features of mammalian NCBTs and note some
key differences 1) between NCBTs and AE1 and 2) among
individual NCBTs. Although this section is intended to refer
specifically to mammalian NCBTs, many conclusions likely
hold true for most vertebrate NCBT-like transporters, and
even some invertebrate NCBTs, which are predicted to have
a similar topology to mammalian NCBTs.
The NCBTs are glycosylated membrane proteins with predicted nonglycosylated molecular weights of between 116
and 140 kDa. As shown in FIGURES 2A AND 15, each transporter has three major domains: a large 46 – 66 kDa cytosolic Nt, a ⬃60 kDa transmembrane domain (TMD) encompassing 12–14 TMs, and a smaller ⬃10 –14 kDa cytosolic Ct.
FIGURE 15 shows the TMD with 13 ␣-helical spans plus an
“extended structure” linking TM11 and TM13, as well as
836
1. Oligomerization
The detection of NBCe1 dimers in rat kidney sections (865)
and NBCe1, as well as NBCe2, tetramers in the human
embryonic kidney cell line HEK-293 (773) indicates that
NCBTs, like AE1, form higher oligomers. Like AE1,
NBCe1 molecules, and likely all NCBTs, form oligomers
stabilized at multiple contact points. Oligomerization is
presumed to be a prerequisite for functional expression of
the transporter.24 In the case of AE1, homodimers are stabilized by Nt-Nt interactions (1091) as well as TMD-TMD
interactions (791, 1023). AE1 tetramers are dimers of homodimers that are linked, at contact points in their Nt, by
cytoskeletal proteins such as ankyrin (156). It is unknown
whether NBCe1 tetramerization requires an accessory protein.
Size-exclusion chromatography indicates that the isolated
Nt of human NBCe1, human NBCe2, and rat NBCn1 all
form homodimers (320), and preliminary X-ray diffraction
data demonstrates that the NBCe1-Nt dimer is stabilized by
interlocking arms (321), homologous to those that stabilize
AE1-Nt dimers (1091). Evidence of TMD-TMD interactions (or perhaps even Nt-Ct interactions) within an NBCe1
dimer is provided by experiments in which an NBCe1 construct that lacks a Ct coimmunoprecipitates with an NBCe1
molecule that lacks an Nt (276). Unlike AE1 dimers,
NBCe1 dimers are further stabilized by disulfide bridges
between cysteine residues in the third extracellular loops
(EL3 in FIGURE 15) of opposing monomers (471, 632, 834).
Some evidence suggests that NBCe1 monomers within a
dimer are capable of functioning independently (471). A
concatameric NBCe1 molecule was created in which a mutant NBCe1 monomer [T442C, which can be selectively
blocked with (2-sulfonatoethyl) methanethiosulfonate, also
known as MTSES, a cysteine-reactive reagent] was joined to
a wild-type monomer (WT, which is unaffected by MTSES).
Unlike a WT-WT concatamer that is not inhibited by
MTSES and a T442C-T442C concatamer that is 100%
blocked by MTSES, hybrid concatamers are only 50%
blocked, as if the WT monomer within the dimer operates
independently of the blocked T442C monomer (471). Another possibility is that MTSES binding to the WT-T442C
heterodimer produces a 50% blockade of each monomer
within the dimer.
2. NCBT domain structure
Based on the presence of alternating variable and conserved
regions of protein sequence, we can consider each of the
24
We define functional expression as the product of surface expression and the intrinsic transporter activity of individual molecules.
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The human genome, and likely every mammalian genome,
includes 10 SLC4 genes. Five of these have been unequivocally characterized as encoding NCBTs: Slc4a4 (NBCe1),
Slc4a5 (NBCe2), Slc4a7 (NBCn1), Slc4a8 (NDCBE), and
Slc4a10 (NBCn2) as displayed in FIGURE 3. Each has a
distinct molecular action, distribution, and role, considered
in section V. In the present section, we consider features
that are common among NCBTs, including oligomeric
state, domain structure (e.g., predicted topology, conserved
sequence motifs), and maneuvers that inhibit or stimulate
transport.
an extended, glycosylated extracellular loop (EL3) between
TMs 5 and 6.
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
7
EL3
C
C
C
C
Lumen
1
Cytosol
5
Nt-TMD
linker
2
Nt loop
5
6
7
8
9
10
11
E 13
14
Ct core 9
TMs6–14 8
Ct
4
Nt
core 1
Ct appendage
10
HOOC
2
Nt appendage 1
NH2
FIGURE 15. Domains and subdomains of NCBTs. Representation of a typical NCBT showing the three
domains (Nt, TMD, and Ct) together with the 10 numbered subdomains that comprise them. The common and
unique features among NCBTs in each subdomain are discussed in the text. An annotated alignment of human
NCBTs showing the division of subdomains is provided in Appendix I.
three major NCBT domains as being divided into a total of
10 subdomains (see diagram in FIGURE 15 and sequence
alignments in Appendix I). The first five subdomains are all
part of the Nt. The TMD includes the sixth (transmembrane
spans 1–5, TMs1–5), seventh (third extracellular loop,
EL3), and eighth (TMs 6 –14) subdomains. Finally, the Ct
consists of two subdomains (a conserved core and a variable
region). We now will discuss each domain and subdomain
individually. Unless stated otherwise, the amino acid residue numbers, provided as a guide, refer to the human renal
variant of NBCe1 (NBCe1-A; GenBank protein accession
no. NP_003750; see guide to NBCe1 nomenclature below).
A) THE CYTOSOLIC Nt. The Nt can be divided into five subdomains (Nt appendage, Nt core 1, Nt loop, Nt core 2, and
Nt-TMD linker; FIGURE 15). The protein sequences of the
appendage, loop, and linker subdomains of the Nt differ
greatly among NCBTs, and often include splice cassettes
(reviewed in Ref. 104). On the other hand, the protein sequences of the two core subdomains are well conserved
among NCBTs and, according to the preliminary crystalstructure data (321) and by comparison to the crystal structure of the AE1 Nt (1091), form the structural core of the
Nt. The core of the Nt exhibits considerable structural homology to certain bacterial EIIA proteins, a class that function as cytosolic regulators of membrane proteins.
In the case of the AEs, the Nt is not vital for either cellsurface presentation or transporter activity, but rather in-
cludes binding sites for protein partners and determinants
that direct the trafficking of the transporter. Studies of
NBCe1 suggest that the Nt of NCBTs is not vital for cell
surface presentation of the rest of the molecule (276, 575,
634) but that it is required for NCBT activity (276, 634).
Preliminary studies show that coexpression of an isolated
NBCe1-A Nt enables the otherwise inactive NBCe1 TMD
to perform electrogenic Na/HCO3 cotransport in Xenopus
oocytes, indicating that the Nt is an activating binding partner of the TMD (724). Similarly the TMD of the human
chloride channel ClC-1 is activated by its cytosolic domain
(842, 1050). The mode of action by which the NBCe1 Nt
activates the TMD is unknown. In the case of ClCs, the
cytosolic domain is thought to act as a scaffold that influences the alignment of transmembrane spans within the
TMD (283) as well as sensor that conveys information to
the TMD (reviewed in Ref. 66). Structural studies of a ClC
homolog from a red alga reveal an extensive interface between the transmembrane and cytosolic domains (283).
I) Nt appendage (subdomain 1). The protein sequence of
the Nt appendage is poorly conserved among NCBTs.
Transcription from alternative promoters leads to great divergence in sequence and size of this subdomain (41–92
amino acids). The result may be protein variants with little
or no homology in the affected region (e.g., NBCe1-A versus NBCe1-B) or variants with an effectively truncated Nt
in which translation initiates at an otherwise “internal”
Met residue (e.g., NDCBE-A versus NDCBE-C). Inasmuch
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3
4
TMs1–5 6
Nt
core 2
Nt
3
TMD
MARK D. PARKER AND WALTER F. BORON
as the electron density corresponding to sequence encoded
by residues 1– 62 is sparse in X-ray diffraction data gathered from crystals of NBCe1-A Nt, it is likely that the Nt
appendage is either loosely structured or is structured but
tethered to the Nt core 1 subdomain by a flexible linker.
It is unknown how the ASD and AID exert their effects
upon the NCBT TMD. The Nt appendage of NBCe2 is
different from that of other NCBTs both in primary sequence and charge distribution, suggesting that NBCe2
may not contain a typical Nt AID or ASD.
The Nt appendage also includes a number of potential
phosphorylation sites, four of which–Ser89, Ser91, and
Tyr92 of NBCn1-B (210, 385, 700) and Thr49 of NBCe1-B
(345)– have been demonstrated to be phosphorylated in
vivo. Thr49 is required for cAMP-induced activation of
NBCe1-B, although phosphorylation of Thr49 is not (345).
II) Nt core 1 (subdomain 2). This ⬃65-amino-acid-long
region is intertwined in three dimensions with Nt core 2 and
together the two subdomains form the core structure of the
Nt. Over a quarter of the sequence in Nt core 1 consists of
Glu/Asp residues. In AE2, the Nt residues that confer pH
sensitivity to Cl⫺ transport are particularly concentrated in
Nt core 1 (summarized in Ref. 907).
Centrally positioned in Nt core 1 is the well-conserved
“ETARWIKFEE” signature sequence, more precisely for
NCBTs “W87[K/R]E[S/T]ARW[I/L]KFEE92”, that marks
the start of conservation between NCBTs and AEs. By
structural homology with the AE1 Nt, residues within this
motif are predicted to form charge interactions with each
other (Arg86 interacts with Glu92) and residues in Nt core
2 (Arg86 interacts with Lys227; Glu91 interacts with
Arg298; see Refs. 166 and 577). Together, these residues
are situated at one end of a “tunnel” of polar residues
within the Nt that has been proposed to be part of an
ion-permeation pathway (166). Speaking to the proposed
importance of and interaction between Glu91 and Arg298
is the severe phenotype (discussed below) of the naturally
occurring human mutation R298S (411). Mutation of ei-
838
One preliminary report suggests that a conserved Cys120
towards the end of Nt core 1 is important for the functional
expression and oligomerization of NBCe1 (52). In all
NCBTs, this cysteine falls within a region homologous to
the first ␣-helix in the AE1 Nt, which in the three-dimensional structure is near but not at the Nt dimer interface
(1091). In NBCe2 and AE2, this region contains a leucinezipper motif (542).
III) Nt loop (subdomain 3). In the AE1 Nt, this variable
region corresponds to a flexible “hinge,” including 10 residues that are not defined in the crystal structure, that is
likely to be a loop that extends from the core structure of the
Nt, linking the two core subdomains. In AE1, the Nt loop
contains determinants of protein 4.2 binding (469) and
ankyrin binding (169, 252). The Nt loop exhibits strong
sequence conservation between NBCe1 and NBCe2 and
among NBCn1, NBCn2, and NDCBE, but not between
these two sets.
In NCBTs, the Nt loop can vary in length due to the inclusion/exclusion of splice cassettes (e.g., cassette I of NBCe1,
cassette II of NBCn1, and cassette A of NBCn2). As with the
variable sequence in the Nt appendage, the splice cassettes
within the Nt loop are nonessential to transporter function
(201, 317), suggestive of a regulatory or protein-binding
role for the Nt loop. Concordantly, a preliminary report
suggests that cassette II of NBCn1 interacts with calcineurin
A (715).
IV) Nt core 2 (subdomain 4). This region, the second and
longer conserved region in the Nt (encompassing ⬃132
amino acids), includes the interlocking arms that are critical
for dimerization of the Nt (320, 1091). An AE1-Nt based
homology model of the NBCe1 Nt predicts that Arg298 can
form charge interactions with either of two residues that are
adjacent in the three-dimensional structure, Glu91 in Nt
core 1 and Glu295 in Nt core 2 (166). Finally, at least in the
case of NBCe1, Lys227 is predicted to interact with residue
Glu92 in Nt core 1 (166). As mentioned earlier, these polar
residues line a tunnel within the Nt that is important for
normal functioning of NBCe1.
V) Nt-TMD linker (subdomain 5). A flexible linker joins the
core structure of the Nt and the TMD. The majority of this
region has a disordered structure in AE1 (1091) and is
poorly conserved among Slc4s. In NBCe1 and NBCe2, this
region contains an additional glycine-rich sequence that
25
Defects that reduce the intrinsic transporter activity of individual
molecules.
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Reflecting its variable nature, most of the sequence within
the Nt appendage is nonessential for NCBT activity (634,
718). However, this region can include such elements as:
1) the autostimulatory domain (ASD) of NBCe1-A, the inclusion of which stimulates NBCe1 activity; 2) the autoinhibitory domain (AID) of at least NBCe1-B and NBCn2, the
inclusion of which inhibits NCBT activity; and 3) the IRBIT
binding determinants (IBD) of at least NBCe1-B and
NBCn2-B, and presumably also the IBD of NBCn1-B and
NDCBE-B, the inclusion of which confers sensitivity of the
NCBT to stimulation by the cytosolic protein IRBIT. Sequestration of the Nt AID may be one of the mechanisms by
which IRBIT activates NCBTs (559, 718, 859).
ther Glu91 or Arg298 can cause trafficking and per-molecule transport defects25 in NBCe1 (166, 411, 577), whereas
the complementary compound mutant E91R/R298E has
near-normal activity (166).
⫺
TRANSPORTERS
Na⫹-COUPLED HCO3
B) THE TMD.
This region comprises three subdomains: transmembrane spans (TMs) 1–5, the large extracellular loop
(EL3), and TMs6 –14 (FIGURE 15). Although neither TMs
1–5 nor TMs 6 –14 of AE1 or NBCe1 are capable of HCO3⫺
transport by themselves, when coexpressed as two separate
fragments in Xenopus oocytes, TMs 1–5 and TMs 6 –14 are
capable of self-associating to recreate the transport activity
of the full-length protein (AE1 or NBCe1, see Refs. 353 and
723). The TMDs of NCBTs have a high degree of sequence
identity. A high-resolution crystal structure has yet to be
reported for the transmembrane domain of any Slc4 family
member, but topology models predict a 10 –14 TMs together with the hydrophilic loops that link them (48, 302,
1116). Although one group had proposed 10-TM model
(950), new preliminary data generated by probing the
chemical accessibility of introduced cysteine residues
(1114, 1115) are consistent, between TM1–TM8, with the
model in FIGURE 2. For the results of extensive mutagenesis
studies that highlight residues within this domain important
to NBCe1 folding and function, we refer the reader to studies from the Kurtz laboratory (e.g., Refs. 5 and 1112).
As noted above, the TMD of NBCe1, plus its Ct, is capable
of trafficking to the cell membrane without the Nt, yet it is
nonfunctional (276, 634). The TMD also includes determinants for the electrogenicity/electroneutrality of transport
cycles (178, 179, 193).
I) TMs 1–5 (subdomain 6). Sequence conservation between
NCBTs and AE1 extends throughout the first five putative
TM spans, which are linked by short, hydrophilic loops.
TM1 contains residues that appear to lie in the ion-translocation pathway. Indeed, in a study employing cysteinescanning mutagenesis, residues predicted to map along one
edge of a TM1 helix were targets of Cys-reactive agents that
blocked transport activity (1110).
By homology to AE1, TM2 and TM3 may form a re-entrant
loop that is stabilized in the membrane more by interactions
with surrounding TMs than by protein-lipid interactions
(188). Thus TM2 and TM3 may not be topogenic without
TM1 and TM4 in place (188, 703). The observation that
two neighboring mutations in TM3–G485S and G486R,
both associated with proximal renal tubular acidosis
(pRTA)– cause per-molecule defects in NBCe1 without apparently affecting protein delivery to the plasma membrane
(393, 576, 929, 930) suggests that TM3 residues are important for ion translocation.
The extracellular end of TM5 leading into the third extracellular loop contains a conserved lysine (807), Lys559 in
NBCe1. In NBCe1, this Lys residue is the second K in the
motif “KKMIK,” which plays a critical role in both the
reversible and irreversible interaction of disulfonic stilbenes
that inhibit anion transport (611). The determinants of stilbene inhibition are discussed in greater detail below; other
compounds known to inhibit NCBT activity are considered. Although all five NCBTs retain this Lys, the Na/HCO3
cotransport activity of NBCn1 is relatively insensitive to
blockade by DIDS.
II) EL3 (subdomain 7). The third extracellular loop of
NCBTs is an extended region (⬃86 amino acids) that links
TMs 5 and 6. The integrity of this loop is not vital for
NCBT activity (Boron lab, unpublished data; see Ref. 723).
Moreover, a 9-amino acid hemagglutinin epitope-tag can
be introduced into EL3 of NBCe1 without disruption of
NBCe1 activity (634). Despite a high degree of sequence
conservation between NBCe1 and NBCe2 and among
NBCn1, NBCn2, and NDCBE in this region, the only globally conserved motifs are a series of consensus N-linked
glycosylation sites and four cysteine residues. We will consider these two features in the following paragraphs.
NBCe1 (190, 515), NBCn1 (174), NBCn2 (177, 755), and
NDCBE (176) are all glycosylated in vivo, as evidenced by
an increase in gel mobility upon PNGase F treatment. Human NBCe1 and NBCn2 have three sites, human NBCn1
and NBCe2 have four, and human NDCBE has only two.
All are within EL3; however, not all of the N-glycosylation
sites in an NCBT may actually be glycosylated. In the case
of NBCe1, which has three putative sites, only the distal
two sites of the form “Asn-Xaa-Thr,” but not the proximal
“Asn-Xaa-Ser” site, are normally glycosylated in Xenopus
oocytes (190) (Xaa is a 3-letter placeholder for any amino
acid). Glycosylation does not appear to be vital for the
NCBT function of NBCe1 (190), but a mutant NBCn2 in
which all three glycosylatable Asn residues are replaced by
Gln, exhibits poor protein expression compared with the
wild-type transporter (177).
Within an NBCe1 dimer, the four conserved cysteine residues (Cys583, Cys585, Cys630, and Cys642) form disulfide
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may confer some extra flexibility between the Nt and TMD,
although a precise role for this region has not yet been
described. The length of the Gly-rich region in NBCe2 (23
Gly in a stretch of 30 residues in human NBCe2) varies
among mammalian species, is reduced to three or four Gly
residues in zebrafish NBCe2 isoforms, and is absent altogether in the predicted protein sequence of Xenopus tropicalis NBCe2. Conservation among NCBTs returns close to
the start of TM1 at the D405IKRK409 motif, which is homologous to the protein-4.1–interaction motif in AE1
(452). Indeed, 4.1B and NBCe1 are colocalized in, and can
be coimmunoprecipitated from, murine PT epithelia (957,
958). The NBCe1 protein complex also includes the membrane-associated guanylate kinase homolog p55 (957),
which can act as cytoskeletal anchor (reviewed in Ref. 41).
Asp405 as well as Asp416 in the conserved portion of the
linker are critical for plasma membrane targeting of NBCe1
(574).
MARK D. PARKER AND WALTER F. BORON
bonds. Cys583 and Cys585 form an intramolecular disulfide bond with each other, and Cys630 and Cys642 intermolecularly bond to their counterparts within an NBCe1
dimer (1111).
C) THE CYTOSOLIC Ct. The Ct (90 –105 amino acids) consists of
two subdomains: a conserved region (Ct core) and a variable region (Ct appendage). The variable region is the site of
extensive variation in splicing, which potentially enables
each transporter to interact with a variety of protein binding partners. As evidenced by studies on NBCe1-A and
NBCn1, determinants within the Ct are vital for the stable
plasma membrane expression of NCBTs (e.g., Refs. 276,
578, 603, and 930). A study of the isolated Ct domain of
NBCn1 (603) indicates that this domain is relatively unstructured, although a subsequent study of a smaller peptide corresponding to sequence within the NBCe1 Ct reveals some ␣-helical content (573).
I) Ct core (subdomain 9). The protein sequence of the first
subdomain of the Ct is well conserved among NCBTs and
includes three notable motifs that are discussed below: 1) a
dihydrophobic trafficking signal, 2) aspartate clusters, and
3) lysine clusters.
II) FL targeting motif. An “FL” sequence in the Ct of
NBCe1 is necessary for the basolateral presentation of the
transporter, and is located in a region determined by CD
spectroscopy to have some ␣-helical content (573). Deletion
of the last 92 amino acids (thereby deleting the “FL” motif)
of NBCe1-A causes the protein to be destabilized in the
840
III) Asp clusters. A motif similar to the “LDADD” sequence
in AE1 has been reported to be important for CA II binding
and consequently NBCe1 activation (68, 350, 604, 764),
the CAII metabolon hypothesis. Furthermore, expression of
NBCn1 is reported to cause a redistribution of CAII to the
plasma membrane of HEK cells (604). However, in subsequent studies by others, peptides corresponding to human AE1, NBCe1, or NDCBE Ct do not bind CA II in
vitro (748) and a CA II-dependent activation of NBCe1
cannot be demonstrated by co-expressing NBCe1 and CA
II in Xenopus oocytes (1063), by coinjecting purified CA
II into NBCe1-expressing oocytes (613), or by expression
of an NBCe1-CA II fusion protein (613). The evidence
presented in favor and against a physiologically relevant
interaction between NCBTs and CA II was recently evaluated in Reference 102.
IV) Lys clusters. Following the Asp cluster is a long stretch
of charged residues that are characteristic of the NCBTs but
not the AEs. The most striking example is in NBCe1, which
has a string of 17 consecutive charged residues, 12 of which
are lysines.
V) Ct appendage (subdomain 10). The terminal subdomain
of the Ct can vary greatly among NCBT isoforms. Some of
the features that can be included or excluded by alternative
splicing in this region are listed below.
A) Arg-based ER localization signals. The Ct of both
NBCe1-A and NBCe2c contain “R-X-R” sequences that, in
many other transporters, prevents forward trafficking of
transporter molecules until the signal is masked by oligomer
formation, or interaction with a binding partner (recently
reviewed in Ref. 644). The relevance of this motif in NCBTs
has yet to be tested, but it is notable that a truncated
NBCe1-A that lacks the last 65 amino acids of the Ct (including an “RER” sequence) has a dominant-negative effect
on the forward trafficking of full-length NBCe1-A molecules (930).
B) Motifs for binding PDZ domains. A class I PDZ-domain– binding sequence, conforming to an “-ET[T/S/C]L”
consensus (874, 992), is common to NBCe1-C (79), NBCn1
(769), and NBCn2-C/D (317) and mediates interactions
between the transporter and cytoskeletal scaffolding proteins such as NHERF1 (562, 711, 769), harmonin (790),
and PSD-95 (780). These binding partners serve as foci for
clustering of membrane proteins, such that NCBTs may
associate with the vacuolar-type H⫹-pump (769), the N-
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III) TMs 6 –14 (subdomain 8). Global conservation of protein sequence among Slc4s continues throughout the remainder of the TMD. Gly723 in the fourth extracellular
loop (EL4) of NBCe1 is reported to be necessary for interaction with CA IV (32). This loop also contains determinants of transporter action, inasmuch as the electrogenicity
versus electroneutrality of chimeric NBCe1/NBCn1 transporters depends on the origin (NBCe1 versus NBCn1) of
EL4 (178). A cysteine residue (Cys916) at the putative intracellular end of TM12 is palmitoylated in the related
transporter AE1 (698). However, this Cys residue is not
necessary for the function or surface expression in heterologous systems of either AE1 (154, 465) or NBCe1 (471).
Cysteine-scanning mutagenesis studies suggest structural
differences between AE1 and NBCe1 in this region (1112)
and that residues in TM8 of NBCe1 lie along the ion-translocation pathway (633). Of particular interest in this region
are Leu750 in TM8 that appears to lie in a conformationally sensitive part of the transporter (633) and the pRTAassociated residue Ala799 in the vicinity of TM9/TM10,
mutation of which causes per-molecule transport defects
and elicits an unusual DIDS-stimulated, HCO3⫺-independent conductance in NBCe1 (721) that is similar to that
observed for NBCn1.
basolateral membrane and to be mistargeted to the apical
membrane of an opossum kidney cell line (276). A study in
Xenopus oocytes demonstrates that deletion of the last 41
amino acids (deleting the “FL” motif) from NBCe1-A is
sufficient to cause near-total intracellular retention of the
transporter (634).
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
methyl-D-aspartic acid (NMDA) receptor (780), and pertinent to the infrequent examples of apical NCBT localization, CFTR (711). The extreme Ct sequence of NBCe2c
“-SYSL” has been suggested to resemble a class II PDZdomain ligand (768), conforming to a “-X-␸-X-␸” consensus (874). No currently identified NDCBE variant terminates with a consensus PDZ-binding domain. A truncated
NBCn1 that lacks only the PDZ-domain binding sequence
traffics to the plasma membrane of HEK cells and mediates
a similar transport activity to full-length NBCn1 (711).
Thus, at least in heterologous systems, the PDZ-binding
motif is not critical for functional expression of NCBTs.
B. Inhibition and Stimulation of NCBTs
1. NCBT inhibition
NCBTs are amenable to blockade although chemical inhibitors specific to any one native NCBT has yet to be reported.
Thus the pharmacological tools for distinguishing NCBTs
from each other are currently lacking. On the other side of
the coin, an NCBT activity that is not stilbene sensitive
typically correlates with the presence of a single NCBT,
namely NBCn1. Furthermore, blockade of selected NCBT
molecules can be achieved by mutagenic introduction of
inhibitor binding sites (e.g., Ref. 471) and the use of specific
antibodies or antisense probes appear to be promising
methods for effective knockdown of specific NCBTs (see
below).
Demonstrated NCBT inhibitors and methods of NCBT inhibition are listed below and the chemical structures of pharmacological agents mentioned are presented in TABLE 4.
Interventions that downregulate NCBT transcription,
translation, and activity in vivo are discussed for each
NCBT in section V.
A) STILBENE DISULFONATES.
All NCBTs, with the exception of
NBCn1, are inhibited by stilbene derivatives such as DIDS
As is the case for AE1 (144, 569), inhibition of NBCe1 by
DIDS is temporally biphasic (611). The first phase is a
rapid, reversible component of inhibition that presumably
reflects an ionic interaction with the protein. In the case of
NBCe1-A, this inhibition depends to a large extent on three
lysine residues, in the motif KKMIK—located at the putative extracellular end of TM5. Replacing all three Lys residues with either Asn or Asp results in a 10-fold or more
increase in Ki, and replacing with three Glu residues results
in a 20-fold increase (611). However, it is the second lysine
(K559 for human NBCe1-A) that is the most important of
the three for DIDS inhibition and, indeed, only K559 that is
conserved among all five human NCBTs. However, the influence of this lysine must be context dependent, inasmuch
as the poorly DIDS-sensitive NBCn1 includes the TM5 motif “EKLFD”.28 A preliminary report suggests that mutating this motif to “EKLFK” renders the Na/HCO3 cotransport activity of NBCn1 readily inhibitable by 500 ␮M DIDS
(191).
The second, slower phase of inhibition by extracellular
DIDS is irreversible and presumably reflects the covalent
reaction of the bifunctional DIDS molecule with the –NH2
moiety of one or more Lys residues, although in principle
the electrophilic isothiocyanate groups of DIDS could derivatize the nucleophilic side chains of other amino acid
residues, such as Cys, His, Ser, or Tyr (622). Because
irreversible DIDS inhibition still occurs with the mutants
NNMIN and RRMIR, the covalent reaction requires additional determinants elsewhere in the molecule that have
yet to be identified (611).
DIDS is also capable of inhibiting NBCe1 when applied to
the intracellular side of the protein in cell-detached plasma
membrane patches (264, 381, 634).
26
Substituted stilbenes undergo cis-trans photoisomerization, the
cis isoforms being less potent than the trans isoforms as Slc4
inhibitors (848, 880, 1001).
27
In fact, the Na⫹ conductance mediated by NBCn1 is stimulated
after prolonged exposure to 500 ␮M DIDS (189, 201).
28
This motif is “KLFH” in mouse and rat orthologs of NBCn1.
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C) Autoinhibitory sequence. Alternative splicing of NBCe1 at
its extreme Ct can result in the inclusion of a 46-amino-acid
appendage (in NBCe1-A/B) or a 61-amino-acid appendage
(in NBCe1-C). Although NBCe1-B and NBCe1-C have similar intrinsic activities (634), NBCe1-C has a greater activity
than NBCe1-B when the Nt auto inhibitory domain of both
is neutralized by Nt truncation (634) or by IRBIT coexpression (967). It is unknown to what extent the 61-amino acid
appendage exerts a stimulatory effect or the 43-amino acid
appendage exerts an inhibitory effect. Similarly, alternative
splicing of NDCBE at its extreme Ct can result in the inclusion of a 17-amino acid Ct sequence (see NDCBE-B/D below) that is inhibitory to the functional expression of the
transporter (717).
(337, 835, 1009, 1021) and DNDS (634).26 DIDS is a disulfonic stilbene (i.e., it has 2 negative charges). DIDS blocks
the HCO3⫺-dependent conductance mediated by NBCe1
(611) in Xenopus oocytes with an apparent Ki of ⬃40 ␮M
(596, 611), which is less potent than its blockade of AE1 in
oocytes (Ki ⬃6 ␮M; Ref. 1044). In an exploratory setting,
we consider 200 ␮M DIDS to be an appropriate experimental concentration to achieve a substantial block of the activities of NBCe1, NBCe2, NDCBE, and NBCn2. NBCn1 is
unique inasmuch as it is poorly inhibited by even 500␮M
DIDS.27
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596, 611
264, 611
596
Ki ⫽ 36-40 ␮M when applied to oocytes
expressing NBCe1. Also blocks
NBCe2, NDCBE, and NBCn2 (typically
used at 200 ␮M). Blockade of NBCn1
is poor even at 500 ␮M.
Ki ⫽ 13-25 ␮M when applied to oocytes
expressing NBCe1. Also blocks
NBCe2. Untested on electroneutral
NCBTs.
Ki ⫽ 100 ␮M when applied to oocytes
expressing NBCe1. Untested on
other NCBTs.
Stilbene disulfonate (e.g., DIDS;
trans isomer depicted)
Tenidap
Niflumic Acid
Continued
Reference Nos.
Chemical Structure
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842
Application
Name
Table 4. NCBT inhibitors
MARK D. PARKER AND WALTER F. BORON
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Detailed information concerning the use of the NCBT inhibitors listed in this table, together with a list of drugs that have been suggested but not demonstrated to act on NCBTs, is
provided in the text. Chemical structures were drawn using ChemBioDraw Ultra version 12.0 (Perkin Elmer, Akron, OH).
247
50 ␮M is sufficient to block NCBT-like
activity in cholangiocarcinoma cells.
Untested on heterologously expressed
NCBTs.
S3705
Not disclosed
160, 545
Ki ⫽ 1.7 ␮M when applied to ventricular
myocytes, which express at least
NBCe1 and NBCn1. 10-30 ␮M
⫺
blocks Na⫹-dependent HCO3
transporters in tumor cells which
express at least NBCn1. Untested on
heterologously expressed NCBTs.
S0859
596
Reference Nos.
264
Ki ⫽ 10 ␮M when applied to oocytes
expressing NBCe1. Untested on
other NCBTs.
Application
500 ␮M effects a total block of NBCe1
currents when applied to the cytosolic
face of oocyte membrane patches.
Untested on other NCBTs and on the
extracellular face of NBCe1.
Chemical Structure
Benzamil
diBAC oxono(e.g., diBA(3)C4)
Name
Table 4.—Continued
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
843
MARK D. PARKER AND WALTER F. BORON
Tenidap29 blocks,
but with only partial reversibility, at least NBCe1 (Ki ⬃15–25
␮M; Refs. 264, 611) and NBCe2 (869). Niflumic acid, an
NSAID often used to inhibit anion channels, blocks at least
NBCe1 (Ki ⬃100 ␮M; Ref. 596).
B) NONSTEROIDAL ANTI-INFLAMMATORY DRUGS.
porter antibody (74). The antibody reacted on western blots
with a 56-kDa protein and apparently blocked NBCe1 activity (74). However, after the cloning of NBCe1, we now
appreciate that the immunoreactive protein is too small to
have been NBCe1-A. Thus the blockade must have been
indirect. The identity of the 56-kDa protein is unknown.
C) DIBAC OXONOL DYES. These fluorescent, voltage-sensitive
dyes block at least NBCe1 (Ki ⬃10 ␮M; Ref. 596).
H) ANTISENSE PROBES.
D) BENZAMIL. This analog of amiloride effects a complete, yet
reversible block of rat NBCe1 when applied to the cytosolic
surface of oocyte membrane patches at 500 ␮M (264).
I) PHOSPHATASES.
F) S3705. This agent, when applied at a concentration of 40 ␮M,
blocks at least the NCBT activity present in breast carcinoma
(1041) and cholangiocarcinoma (247) cell lines, slowing tumor growth and, in the latter case, promoting apoptosis. Although both studies report blockade of NDCBE-like activity,
the molecular identities of the NCBT responsible are not demonstrated. These cell lines likely express at least NBCn1 (546)
as well as a stilbene-sensitive NCBT.
G) ANTIBODIES.
An alternative approach to target specific
NCBTs involves the use of inhibitory antibodies directed
against extracellular epitopes, or to reduce NCBT transcript, and consequently protein, abundance via antisense
technology. In two studies, the action of NBCe1 was inhibited using antibodies raised against an epitope in the third
(i.e., longest) extracellular loop of NBCe1 (225, 481). In a
third study, conducted prior to the cloning of NBCe1, rabbit proximal tubule vesicles enriched for Na/HCO3 cotransporter activity were used to raise an anti-Na/HCO3 cotrans29
Developed by Pfizer Inc (New York, NY).
Developed by Sanofi-Aventis U.S. LLC (Bridgewater, NJ). A synthesis protocol based on commercially available compounds has
been developed by Larsen and co-workers and is provided in Ref.
545.
31
Bachmann et al. cite unpublished observations from Aventis
Laboratories that S0859 blocks NBCe1 with a Ki ⬃6 ␮M, but does
not block NBC2/3 (i.e., NDCBE) as expressed in CHO cells.
32
The use of S0859 as an “NBC1 blocker” (849) or an “NBCn1
inhibitor” (546) in cells in which these transporters are the dominant
NCBT paralog does not constitute a demonstration of paralog specificity of the compound.
30
844
The time-dependent rundown of NBCe1-A
activity in excised Xenopus oocyte macropatches can be
slowed by maneuvers that inhibit protein phosphatase activity, indicating that at least NBCe1-A can be inhibited by
dephosphorylation (1049).
J) INTRACELLULAR MAGNESIUM.
In bovine parotic acinar cells,
as well as in mammalian cell lines overexpressing NBCe1-B,
NBCe1 activity is inhibited by intracellular Mg2⫹ (1066).
Although this effect has not been demonstrated to be direct,
a mutant NBCe1 construct that lacks the Nt sequence specific to NBCe1-B (a region that includes the AID as well as
IRBIT binding determinants) exhibits a substantially reduced sensitivity to Mg2⫹ (1066). In HEK cells, the coexpression of IRBIT also reduces the Mg2⫹ sensitivity of
NBCe1-B (1067).
K) MOLECULAR BIOLOGICAL APPROACHES. The per-molecule activity of NBCe1 is reduced by the removal of the Nt ASD, or
the inclusion of the Nt AID (634). The per-molecule activity
of NDCBE is reduced by the inclusion of the Ct AID (717).
L) AGENTS SUGGESTED, BUT NOT PROVEN, TO BLOCK NCBTs.
The
anticonvulsant levetiracetam (aka Keppra) and the diuretic
hydrochlorothiazide (HCTZ) have both been reported to
inhibit NCBT activities in isolated tissues (567, 571), but
evidence of direct interaction of these drugs with NCBTs is
presently lacking. Although direct blockade of NCBTs
would indeed be anticonvulsive, the properties of levetiracetam instead appear to be a consequence of its interaction
with synaptic vesicle protein 2 (SV2A; see Ref. 618). The
possibility that levetiracetam exerts an indirect effect on the
functional expression of NCBTs cannot be excluded.
The psychoactive alkaloid harmaline blocks Na⫹-coupled
transporters, perhaps by interaction with Na⫹-binding determinants (47, 864). When applied at 200 ␮M, harmaline
is reported to effect a near-total, yet reversible block of at
least 1) the Na⫹ and HCO3⫺ dependent pHi recovery in
HEK cells expressing human NBCe1 (39), 2) the electrogenic NCBT activity in salamander Müller cells (680), and
3) the HCO3⫺-dependent Na⫹ flux in basolateral membrane
vesicle preparations from rat (332) and rabbit (893, 897)
renal cortex. However, 200 ␮M harmaline does not substantially inhibit human NBCe1-A expressed in Xenopus
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This drug30 blocks at least the NCBT activity present in 1) colonic crypt cells (likely a combination of NBCe1
and NBCn1 action, see Ref. 55),31 2) ventricular myocytes
(likely a combination of NBCe1, NBCe2, and NBCn1 action, see Ref. 160), 3) coronary endothelial cells (likely a
combination of NBCe1 and NBCn1 action, see Ref. 523),
and 4) mammalian tumor cell lines (likely NBCn1, see Ref.
545). Thus S0859 may be the only reported potent inhibitor
of NBCn1. S0859 may be more specific than stilbene derivatives, inasmuch as it is reported to be ineffective at blocking Na⫹-independent Cl-HCO3 exchanger activity (160).
There are presently no reports of S0859 action on any of the
five NCBTs expressed in isolation.32
E) S0859.
shRNAs, siRNAs, and hammerhead
ribozymes have been used to reduce the abundance of specific NCBTs (90, 546, 593, 642, 989).
⫺
TRANSPORTERS
Na⫹-COUPLED HCO3
oocytes (Lee, Boron, and Parker, unpublished data), and
thus blockade of NCBT activity by harmaline in renal membranes may be an indirect effect of blockade of other Na⫹dependent transporters.
2. NCBT stimulation
Physiological stimuli that enhance transcription, translation, and activity of individual NCBTs in vivo are discussed,
for each NCBT, in section V. Other maneuvers that enhance
the activity of NCBTs are listed below.
Injection of PIP2 doubles the functional expression of
NBCe1-B and NBCe1-C in intact oocytes, via a pathway
that can be mimicked by IP3 injection and/or elevation of
cytoplasmic [Ca2⫹] (968). The stimulation of NBCe1-B/C
by IP3 injection is blocked by the kinase inhibitor staurosporine, consistent with the involvement of endogenous kinase activity (968).
B) G PROTEIN–COUPLED RECEPTOR AGONISTS. Exposing oocytes to
lysophosphatidic acid (LPA), which binds to endogenous LPA
receptors and presumably acts via a pathway mimicked by
PIP2/IP3 injection (462), increases the per-molecule activity of
exogenously expressed NBCe1-C (968). A nuance is that LPA
application, but not PIP2/IP3 injection, also increases the
plasma membrane abundance of NBCe1-B (but not
NBCe1-C) via a Ca2⫹-independent mechanism (968).
C) ANTI-NBCe1 ANTIBODIES.
Application of an antibody raised
against EL4 of NBCe1 stimulates NBCe1-like activity in
myocytes (225).
D) INCLUSION OF AUTOSTIMULATORY SEQUENCE.
Full-length
NBCe1-A exhibits a twofold greater activity than an
NBCe1 construct that lacks the Nt ASD (559, 634). It is
possible, although untested, that all NCBTs would also be
stimulated by the replacement of their Nt appendages with
NBCe1 autostimulatory sequence. Note that autostimulatory sequences specific to NBCe2, NBCn1, NDCBE, or
NBCn2 have yet to be reported.
E) REMOVAL OF AUTOINHIBITORY SEQUENCE.
Truncation of the
Nt AID from NBCe1-B/C and NBCn2 (634, 718) or truncation of the Ct AID from NDCBE-B/D (717) increases
NCBT activity.
F) IRBIT. This soluble, 60-kDa protein is an important activator of certain NCBTs. Coexpression of IRBIT with
NBCe1-B, NBCn1-B, NBCn2-B, or NDCBE-B (722, 881,
In mammalian cells, the effect of IRBIT upon NBCe1-B is
twofold. In addition to stimulating the per-molecule activity of NBCe1-B, IRBIT, by antagonizing the WNK/SPAK
signaling pathway, also causes an increase in plasma membrane abundance of NBCe1-B (1075).
Other IRBIT binding partners include CFTR (1076), the IP3
receptor (43), NHE3 (371), and the cleavage and polyadenylation specificity factor CPSF (483).
V. NCBTs IN MAMMALS
Each of the five mammalian NCBTs–Slc4a4 (NBCe1),
Slc4a5 (NBCe2), Slc4a7 (NBCn1), Slc4a8 (NDCBE), and
Slc4a10 (NBCn2)–plays a vital and unique role in acid-base
homeostasis, and each has been the subject of much investigation. The two major roles played by NCBTs, reviewed
here in section V, are 1) maintenance of pHi, local extracellular pH, interstitial pH, and plasma pH within a normal
range (all NCBTs); and 2) support of transepithelial anion
and fluid movement (e.g., NBCe1 and NBCn1 in salivary
gland epithelia).
We will detail crucial similarities and differences in the action, distribution, and role of each transporter, with the
caveat that not all information on a particular NCBT may
be transferrable among all mammalian species. For example, in one recent comparative analysis, NBCe1 transcripts
were noticeably more abundant in preparations from human duodenum, than in equivalent samples isolated from
mice and rats (485).
In this section, we consider first the two electrogenic NCBTs
(NBCe1 and NBCe2), and then the three electroneutral
NCBTs (NBCn1, NDCBE, and NBCn2), these groupings
reflecting the relatedness of the two major groups of NCBTs
in FIGURE 3 AND TABLE 2. In section VI, we discuss the AEs
(encoded by Slc4a1–3) and two other related Slc4-family
members, Slc4a9 and Slc4a11. Also noteworthy, although
not discussed further in this review, is the presence, in rat
medullary thick ascending limb (mTAL) cells, of a stilbenesensitive, electroneutral K/HCO3 cotransport mechanism
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A) PIP2. Application of PIP2 to the intracellular face of excised Xenopus oocytes patches containing NBCe1-A stimulates transport (1049). However, PIP2 is rapidly hydrolyzed when injected into intact oocytes, an observation that
may explain why injection of PIP2 does not result in the
stimulation of NBCe1-A in whole cells (968).
1067), nNCBTs with defined autoinhibitory domains in the
Nt, stimulates NCBT activity, in part by binding to the Nt
and relieving transporter autoinhibition (559, 881). IRBIT
must undergo a series of phosphorylations to become active, although the optimal phosphorylation state remains
undefined (246, 881). Maximal stimulation of NBCe1-B
activity by IRBIT, greater than that achieved by removal of
the Nt AID, can be accomplished using a potent mutant
IRBIT that lacks a PP-1 docking site and thus presumably
becomes suitably phosphorylated (559). IRBIT has no effect on NBCe1-A because this variant has an autostimulatory domain but neither an IRBIT binding site or autoinhibitory domain (559, 634, 881).
MARK D. PARKER AND WALTER F. BORON
(570), which has yet to be attributed to the activity of a
specific transporter.
A. Mammalian Electrogenic NCBTs: NBCe1
and NBCe2
Of the five mammalian NCBTs, only NBCe1 and NBCe2
perform electrogenic Na⫹/HCO3⫺ cotransport. The molecular actions of NBCe1 and NBCe2 appear to be virtually
indistinguishable; the major differences between NBCe1
and NBCe2 reside in their distribution and perhaps in their
means of regulation.
1. NBCe1 (Slc4a4)
In keeping with these roles, NBCe1 dysfunction is associated with alterations in neuronal excitability (e.g., epilepsy), fluid-movement defects (e.g., corneal edema), and
acid-base disturbances (e.g., proximal renal tubular acidosis or pRTA).
NBCe1 has five known variants (-A through -E). NBCe1-A
is the constitutively active renal variant. NBCe1-B, -C, and
probably also -E are stimulated by the soluble protein
IRBIT. NBCe1 is upregulated in acidosis and hypercapnia,
conditions in which the action of NBCe1 would raise
[HCO3⫺] in the blood plasma and/or intracellular fluid,
thereby stabilizing pH. However, in some instances, the
obligatory influx of Na⫹ that is coupled to the movement of
HCO3⫺ into cells can contribute towards ischemic damage.
NBCe1 is downregulated in conditions such as alkalosis
and Na⫹ loading, when the requirement for NBCe1 action
is reduced.
B) NOMENCLATURE OF Slc4a4 PRODUCTS.
The nomenclature of
Slc4a4 products has gradually evolved over the last 15
years. The original Slc4a4 gene-product, cloned from salamander kidney, was termed simply NBC for Na bicarbonate cotransporter (809). Prefixes have been variously added
to this acronym to reflect either the genus of animal from
which the NBC was cloned (e.g., rNBC was used to refer to
rat Slc4a4 products), the organ from which the NBC was
846
Despite the multiplicity of acronyms, only five mammalian
NBCe1 variants have been described to date. The current nomenclature defines Slc4a4 products as follows: NBCe1-A (previously known as rNBC, aNBC, kNBC, kNBC1, hkNBCe1),
NBCe1-B (previously known as pNBC, pNBC1, rpNBC, hhNBC, hcNBC, rb1NBC), NBCe1-C (previously known as
bNBC1, rb2NBC), NBCe1-D, or NBCe1-E.
The unique and common features of each of these variants
are discussed below.
C) MOLECULAR ACTION OF NBCe.
I) Physiological substrates.
NBCe1 was the first NCBT to be cloned from mammals
(138, 140, 806, 808) and, like its amphibian ortholog, catalyzes the cotransport of 1 Na⫹ with 2 or 3 HCO3⫺ equivalents (FIGURE 16), resulting in the net movement of negative
charge in the direction of net transport. There are a number of
potential mechanisms by which electrogenic Na/HCO3
cotransport could occur. Preliminary reports suggests that it is
CO32⫺, rather than HCO3⫺, that is the transported anion when
the transporter is working with a 1:2 stoichiometry (336,
560), as shown in FIGURE 16B. It cannot be ruled out that the
NaCO3⫺ ion pair is the transported anion (FIGURE 16C as well
as Refs. 16 and 448), although potential evidence against this
possibility are as follows: 1) the poor Li/HCO3 cotransport
activity of NBCe1 that indicates a stronger cation selectivity
than might be achieved in the case of NaCO3– versus LiCO3⫺
(39) and 2) the reported inhibition of NBCe1 by harmaline
and inhibition by benzamil, drugs that interact with Na⫹ binding sites. Kinetic studies in other animals indicate that squid
NDCBE, but not frog NBCe1, can transport the NaCO3⫺ ion
pair.
II) Apparent stoichiometry shift. NBCe1 certainly can function with a Na⫹:HCO3⫺ stoichiometry of 1:2 and appears
capable of operating with a stoichiometry of 1:3. Stoichiometry plays a pivotal role in determining the direction of
net transport. With an NBCe1 stoichiometry of 1:2, and
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A) SUMMARY. The electrogenic Na/HCO3 cotransporter
NBCe1 (encoded by the Slc4a4 gene) is present in many
organ systems throughout the body but is notably abundant
in the following: 1) plasma membranes of neurons and glia
in the central nervous system, where changes in pHi and
pHo modulate neuronal excitability; 2) basolateral membranes of secretory epithelia, where NBCe1 mediates a
HCO3⫺ influx that supports luminal HCO3⫺ secretion; and
3) basolateral membranes of renal PT cells, where NBCe1
mediates a HCO3⫺ efflux that is critical for the secretion of
H⫹ into the tubule lumen, one consequence of which is
HCO3⫺ reabsorption. This activity helps maintain a normal
plasma [HCO3⫺].
cloned (e.g., kNBC was used to refer to kidney Slc4a4 products), or both (e.g., rkNBC was used to refer to rat kidney
Slc4a4 products). In a case where more than one Slc4a4
splice variant was identified in an organ from a particular
species, some authors added a number to the prefix (e.g.,
rb1NBC and rb2NBC were used to refer to two distinct
products of the Slc4a4 gene from rat brain). Following the
cloning of a cDNA from a second NBC-encoding gene, the
original NBC was referred to as NBC1 (e.g., kNBC1 and
pNBC1 distinguished kidney and pancreas Slc4a4 products) and the new ones given higher numbers (and not always different ones). Finally, with the cloning of electroneutral NBCs, a lowercase “e” for electrogenic was inserted
into the acronym (e.g., NBCe1 and NBCn1 distinguish the
electrogenic Slc4a4 gene-product from the electroneutral
Slc4a7 gene-product) and thus the original, electrogenic
Na/HCO3 cotransporter was finally renamed NBCe1 (100).
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
NBCe1 or NBCe2 in 1:2 mode
Na+
HCO3–
HCO3–
A
Na+
CO3–
B
NaCO3–
C
Na+
HCO3–
D
H+
NBCe1 or NBCe2 in 1:3 mode
H+
E
HCO3–
HCO3–
HCO3–
Na+
G
CO32–
NaCO3–
H
HCO3–
HCO3–
Na+
I
HCO3–
HCO3–
Na+
H+
J
CO32–
NaCO3–
FIGURE 16. Molecular action of electrogenic NCBTs. Possible molecular mechanisms by which an electro⫺
genic NCBT could operate with an apparent Na⫹:HCO3
stoichiometry of 1:2 (A–D in top panel) and 1:3 (E–J in
2⫺
⫺
bottom panel). Note that we have not considered any models that are based on CO3
-HCO3
exchange
⫺
(considered for NBCn1 in FIGURE 30), or models of HCO3
-stimulated electrogenic Na-2H exchange. We have
also omitted mechanisms of 1:3 stoichiometry that could result from modification of model D.
typical ion concentration and voltage profiles across the
membrane, Vm is more positive than the reversal potential
and thus NBCe1 mediates a net influx of HCO3⫺ equivalents
(for thermodynamic calculations, see examples in Refs.
103, 339, and 349). With a stoichiometry of 1:3, however,
Vm would be more negative than Erev so that NBCe1 would
mediate a net efflux of HCO3⫺.
In astrocytes (75), parotid acinar cells (1065), corneal endothelial vesicles (543), pancreatic duct cells (344), ventricular myocytes (14, 1006) or when overexpressed in Xenopus oocytes (381, 853) and HEK cells (870), NBCe1 operates with a 1:2 stoichiometry, mediating net Na⫹ and
HCO3⫺ influx (FIGURE 16, A–D, most likely FIGURE 16B).
One study reports that NBCe1 also operates with a 1:2
stoichiometry in rabbit PTs (858) (where NBCe1 mediates
net Na⫹ and HCO3⫺ efflux) but in other studies, renal
NBCe1, including that of rabbit, is calculated to operate
with an apparent 1:3 Na:HCO3 stoichiometry (348, 896,
1085). Indeed, some studies suggest that NBCe1 could fulfill its physiological mission of HCO3⫺ reabsorption only if
it operated with a stoichiometry of greater than 1:2 in the
PT (381, 1086) (see above). Such a shift in stoichiometry
could be achieved by unveiling a cryptic HCO3⫺ cotransport
site (e.g., FIGURE 16, A–C versus E–G), or a cryptic H⫹ exchange site (e.g., FIGURE 16, A–C versus H–J). The mechanism(s) that control the apparent change in stoichiometry
from 1:2 to 1:3 and vice versa (reviewed in Ref. 349) are
unclear but have been suggested to involve a number of
factors, such as changes in [Ca2⫹]i (667), changes in the
phosphorylation state of the transporter (347), changes in
the direction of transport (750), the presence of an as-yetunidentified binding partner in PT epithelia (344), differences in cell type in which the transporter is being expressed
(346), and/or primary culture conditions in the case of
proximal tubules (666, 668).
III) Substrate specificity. When expressed heterologously,
NBCe1 does not require extracellular Cl⫺ to function (138,
339) and, as described above, is inhibited by stilbene disulfonates (140, 611, 853). The Km of the transporter for Na⫹
is ⬃20 –30 mM (634, 822, 853). Rat NBCe1 expressed in
Xenopus oocytes mediates a small amount of Li/HCO3
cotransport (estimated at ⬃3% of Na/HCO3 cotransport)
but does not mediate K/HCO3 cotransport (853). We estimate that human NBCe1, as expressed in oocytes, can support ⬃10% Li/HCO3 cotransport compared with Na/
HCO3 cotransport (Lee, Boron, and Parker, unpublished
data) but ⬃25% when expressed in a kidney cell line (39).
Similarly, NBCe1 assessed in basolateral membrane vesicles
from rabbit PT does not exhibit a strong Na⫹/Li⫹ selectivity
(897). It is unclear whether the poorer Na⫹/Li⫹ selectivity
in renal membranes versus oocyte membranes reflects differences in NBCe1 behavior, assay method, or contributions from other endogenous kidney transporters.
The Km of NBCe1 for HCO3⫺ is ⬃4 –10 mM (339, 543,
634). Preliminary studies indicate that at least rat NBCe1
expressed in Xenopus oocytes can also transport select anions other than HCO3⫺/CO32⫺, such as NO3– (852). Na⫹coupled, DIDS-sensitive HSO3⫺/SO32⫺ cotransport attributed to NBCe1 has been reported in rabbit PT vesicles (893)
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Na+
F
H+
MARK D. PARKER AND WALTER F. BORON
of NBCe1-B, -C, and -E in diverse cell types (FIGURE 17C).33
Translation of these variants begins at exon 2 (initiator
methionines are marked “M” in FIGURE 17C). The transcription of NBCe1-B from promoter P1 in mouse ameloblast-like LS8 cells is pH-dependent: transcript abundance
is increased in acid-incubated cells and decreased in alkaliincubated cells (706, 891). The human P1 region includes a
284 bp, “pH-responsive” sequence that ends 8 bp upstream
of the transcriptional start site (891). If this sequence is
placed upstream of a reporter gene that has a minimal promoter, the transcription of the reporter in LS8 cells is enhanced when the cells are maintained in media with an
acidic pH (pH 6.8 versus pH 7.4; Ref. 891). The action of
the “pH-responsive” enhancer requires DNA elements that
contain consensus binding sites for NF-␬B and p53 (891).
and in oocytes injected with rabbit kidney RNA (822), but
not in oocytes expressing human (339) or rabbit (Lee, Boron, and Parker, unpublished data) NBCe1, suggesting that
the observations from renal preparations could be complicated by the presence of other anion transporters, such as
Slc26a1 (FIGURE 1). Finally, according to one report,
NBCe1-A, at least at high extracellular pH, might mediate a
small degree of OH⫺ transport (39).
D) THE SLC4A4 GENE.
33
Only the ORFs, and not the 5= UTRs, have not been reported for
NBCe1-C, -D, and -E so the presence of exon 1 has not been
demonstrated in these transcripts. We cannot rule out the possibility that another promoter is present between exons 1 and 2.
The SLC4A4 gene has two distinct promoters (P1 and P2 in
FIGURE 17B, see Ref. 9). The first promoter, P1, is located
upstream of noncoding exon 1 and promotes transcription
A
Locus 4q21
50kb
DCK
B
SLC4A4
Gene structure
10 kb
P1
1
C
GC
2
P2
3
4
9
24
Transcript variation
P1
P2
M
4
NBCe1-A
NBCe1-B
5
6
7
23
24
*
25
26
24
*
25
26
24
25
*
25
26
24
*
25
26
M
1
2
3
4a
5
6
7
23
3
4a
5
6
7
23
5
6a
7
23
M
NBCe1-C
1
M
4
NBCe1-D
NBCe1-E
2
M
1
2
3
4a
5
6a
7
23
*
26
FIGURE 17. SLC4A4 gene structure and NBCe1 transcript variants. Scale diagrams showing the human
SLC4A4 gene locus together with the position of neighboring genes (A), the position of promoters (P1 and P2),
and the position of exons within SLC4A4 (B). Transcript variants are represented, not to scale, as numbered
boxes joined by a horizontal line (C). Each numbered box represents the inclusion of that exon in the mature
transcript. “//” denotes that all five transcripts include exons 7–23. Exons that include the initiator ATG codon
(“M”) and termination codon (“*”) are marked for each transcript. Sequences that are derived from part of a
larger exon sequence are labeled with an “a” (e.g., exon 4a is a subdivision of exon 4). Colored exons, or parts
of exons, correspond to the protein regions that each encodes, which are identically colored in FIGURE 18.
Uncolored exons, or parts of exons, denote untranslated 5= and 3= sequence. Exons that are connected with
a dashed line are predicted, but not demonstrated, to be included in the mRNA.
848
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The human NBCe1 gene maps to chromosomal locus 4q21 (6) and has at least 26 exons that
encompass ⬃390 kb of genomic DNA. As shown in FIGURE
17A, the upstream neighbor of SLC4A4 is DCK (deoxycytidine kinase) and the downstream neighbor of SLC4A4,
transcribed from the opposite DNA strand, is GC (groupspecific complement, vitamin D binding protein).
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
The second promoter, P2, is located upstream of exon 4
(FIGURE 17B) and promotes transcription of NBCe1-A, and
likely also NBCe1-D (FIGURE 17C). Translation of NBCe1A/D begins in exon 4 (initiator methionines are marked
“M” in FIGURE 17C). The P2 promoter is very active in
renal PT cells.
The 85-amino acid Nt appendage (FIGURE 18/blue module)
common to NBCe1-B, -C, and -E (encoded by exons 2 and
3 and beginning with the amino acid sequence “MEDE-”)
includes an autoinhibitory domain (AID) that inhibits
NBCe1 activity (634). The AID also includes binding determinants (IBD) for the NBCe1 activating protein IRBIT
(881).
E) STRUCTURAL FEATURES AND VARIANTS OF NBCe1.
B) Cassette I. In NBCe1-D/E, the excision of a 27 nt region,
homologous to cassette I of NBCn1, arises due to the use of
a cryptic splice site within exon 6 of the gene (see FIGURE
17C and Ref. 599). Omission of cassette I (FIGURE 18/purple module) is predicted to shorten the Nt loop region (see
FIGURE 15) by nine residues (loss of “RMFSNPDNG” in
mouse NBCe1). The effect of losing cassette I is unknown,
although cassette I does contain a consensus casein kinase II
phosphorylation site (599), indicating a regulatory role. Transcripts lacking cassette I appear to be widely distributed but
only account for a small fraction of the pool of total NBCe1
transcripts that had previously been identified as NBCe1-A/B
in any given organ (599). Omission of cassette I from NBCe1C-like transcripts has not been reported.
I) Sources of variation in coding sequence among NBCe1
variants. A) Alternative Nt appendages (“MSTE-” versus
“MEDE-”). The mechanisms that result in the production
of two alternative NBCe1 Nt appendages (FIGURE 18/blue
versus red modules) are shown in FIGURE 17C. The 41amino acid Nt appendage (FIGURE 18/red module) common
to NBCe1-A and NBCe1-D (encoded by exon 4 and beginning with the amino acid sequence “MSTE-” ) includes an
autostimulatory domain (ASD) that enhances NBCe1 activity (634).
C) Alternative Ct (“-HTSC” versus “-ETTL”). Alternative
splicing of exon 24 (the length of which is not a multiple
of 3 nt) in NBCe1 transcripts determines the reading
frame in which exon 25 is translated, impacting the remainder of the Ct sequence. In NBCe1-A/B/D/E transcripts, exon 24 (which encodes a 32-amino acid sequence) is spliced to exon 25 (which encodes a 14-amino
acid sequence, followed by a termination codon) produc-
TMD
Ct
ASD
NBCe1-A
AID
6–9
10–14
NBCe1-C
85
46
1,035
46
1,079
9
61
41
85
PDZ
85
NBCe1-E
1–5
41
NBCe1-B
NBCe1-D
C
as
se
tte
I
Nt
1,094
46
1,026
46
1,070
100 aa
FIGURE 18. NBCe1 protein variants. Scale diagram of protein variants that are encoded by the transcripts
represented in FIGURE 17C. Horizontal bars represent protein sequence laid out from Nt to Ct. Vertical bars
represent position of ␣-helical TMs. Protein cassettes are labeled with a number denoting their size in amino
acids and colored to denote their genetic origin as shown in FIGURE 17C. NBCe1-A and NBCe1-D include an
autostimulatory domain (ASD), whereas all other variants include an autoinhibitory domain (AID). NBCe1-C
terminates with a PDZ-domain binding sequence. A color-matched protein sequence alignment of the variants
is provided in Appendix V.
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The five distinct Slc4a4-encoded transcripts (FIGURE 17C) encode protein
products NBCe1-A through NBCe1-E (FIGURE 18). Variants
differ in the inclusion of one of two distinct Nt appendages,
the exclusion of a 9-amino acid cassette I within the Nt
domain, and the choice of one of two distinct Ct appendages. Below, we consider in detail the mechanisms that generate this diversity, the similarities and differences among
the variants and, anticipating the next section of this review,
briefly outline the distribution of each variant. The splicing
of NBCe1 along with that of other renal transporters has
been reviewed in Ref. 310.
MARK D. PARKER AND WALTER F. BORON
ing a 46-amino acid Ct appendage (FIGURE 18/green
module) that terminates with the sequence “-HTSC”.
The remainder of exon 25 and all of terminal-exon 26 of
NBCe1-A/B/D/E comprise the 3=-UTR (FIGURE 17C).
II) Cloned NBCe1 variants that are demonstrated or likely
to exhibit NCBT activity. A representation of the five variants NBCe1-A through NBCe1-E is shown in FIGURE 18,
and the composition of each is described below. Also listed
here are the major anatomical locations from which each
variant has been cloned as a full-length cDNA (the only
reliable demonstration of the presence of each in any preparation). Distribution of subsets of NBCe1 variants (such as
might be determined using an antibody that recognizes the
common Ct of NBCe1-A/B/D/E) are discussed separately in
section “Distribution of NBCe1” below. GenBank protein
accession numbers for the variants discussed in this section
are provided in Appendix IV.
A) NBCe1-A (NCBT activity demonstrated). This predominantly renal variant of NBCe1 (64, 138, 806) has a predicted nonglycosylated molecular mass of 116 kDa (190).
NBCe1-A includes 1) the 41-amino acid “MSTE-” Nt sequence that includes an ASD, 2) cassette I, and 3) the 46amino acid “-HTSC” Ct sequence. Due to the presence of
the ASD, NBCe1-A has a greater per-molecule activity than
either NBCe1-B or NBCe1-C (634). NBCe1-A has also been
cloned from testis, epididymis, and ovary (599).
B) NBCe1-B (NCBT activity demonstrated). This widely
expressed splice form of NBCe1 (6, 192) includes 1) the
85-amino acid “MEDE-” Nt sequence that contains an AID
and an IRBIT-binding sequence, 2) cassette I, and 3) the
46-amino acid “-HTSC” Ct sequence. Due to the presence
of the AID, NBCe1-B has a lower per-molecule activity than
NBCe1-A and a similar per-molecule activity to NBCe1-C
(634). Apart from the pancreas, where NBCe1-B transcripts
are most abundant, NBCe1-B has been cloned from the
850
C) NBCe1-C (NCBT activity demonstrated). This predominantly brain-expressed variant (79) includes 1) the 85amino acid “MEDE-” Nt sequence that constitutes an AID
and an IRBIT-binding sequence, 2) cassette I, and 3) the
61-amino acid “-ETTL” Ct sequence. NBCe1-C is uniquely
distinguished by the presence of the 61-amino acid Ct as it
is the sole variant that includes this Ct. NBCe1-C has also
been cloned from murine epididymis and testis (599) and
human heart. Due to the presence of the AID, NBCe1-C has
a lower per-molecule activity than NBCe1-A and a similar
per-molecule activity to NBCe1-B (634).
D) NBCe1-D (NCBT activity untested). NBCe1-D is identical
to NBCe1-A except for the absence of cassette I. Transcripts
lacking cassette I appear to be widely distributed but only
account for a small fraction of the pool of total NBCe1 transcripts that, until now, had been identified as NBCe1-A in any
given preparation (599). Full-length NBCe1-D cDNA has
been cloned from murine epididymis (599). We regard
NBCe1-D as likely to have NCBT activity because NBCn1-H,
which lacks the homologous cassette I, has NBCn1 activity.
E) NBCe1-E (NCBT activity untested). This variant is identical to NBCe1-B in its coding sequence except for the absence of cassette I (599). NBCe1-E transcripts only account
for a small fraction of the pool of total NBCe1 transcripts
that until now had been identified as NBCe1-B in any given
preparation (599). Full-length NBCe1-E cDNA has been
cloned from murine ovary, uterus, and epididymis (599).
We regard NBCe1-E as likely to have NCBT activity because NBCn1-H, which lacks the homologous cassette I,
has NBCn1 activity.
III) Predicted NBCe1 variants. NBCe1 variants that include
the 61-amino acid Ct of NBCe1-C and that also 1) include the
ASD of NBCe1-A or 2) lack cassette I have not been reported.
Possibly the splice machinery that excises exon 24 is absent
from the pool of cell types that promote NBCe1-A transcription. A lab-created, chimeric NBCe1 that includes the Nt of
NBCe1-A and the Ct of NBCe1-C is reported to exhibit an
activity that is slightly greater than NBCe1-A (634), consistent
with the mildly stimulatory effect of the NBCe1-C Ct in the
absence of an Nt AID (634).
IV) Other NBCe1 variants. We are not aware of any cloned
or predicted NBCe1 variants besides those mentioned
above.
F) DISTRIBUTION OF NBCe1.
The major organs most often associated with NBCe1 expression are the pancreas and kidney,
although NBCe1 is also abundant in many other organs.
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In NBCe1-C transcripts, exon 24 is omitted (FIGURE 17C).
Due to the resulting frame shift, exon 25 now encodes a
27-amino acid sequence and the terminal exon 26 encodes a
34-amino acid sequence followed by a termination codon
and the 3=-UTR. Thus it is that, in NBCe1-C, exons 25–26
encode a 61-amino acid Ct appendage (FIGURE 18/orange
module) that terminates with the sequence “-ETTL”. The
consequences of alternative Ct choice are unclear, but
“-ETTL” is a PDZ-binding domain interacting sequence
(79) and deletion of either the 46-amino acid or the 61amino acid Ct sequences results in reduced NBCe1 accumulation in the plasma membrane (276, 634). In the absence of
the Nt autoinhibitory domain, NBCe1-C has a greater activity than NBCe1-B, as if the 61-amino acid Ct appendage
is stimulatory, or the 46-amino acid appendage is inhibitory, in the absence of the Nt AID (634).
brain (79), cornea (922), heart (192), parotid salivary gland
(508, 710), ileum (64), and from diverse tissues within the
male and female reproductive tracts (599).
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
The distribution of NBCe134 in specific organ systems is
discussed below. The distribution of NBCe1 is summarized
and compared with that of other NCBTs in TABLE 5. In
instances where a detection method would not distinguish
between two variants, for example, use of an antibody
against the common Nt of NBCe1-B and NBCe1-C, we
refer to NBCe1-B/C. If it is unknown which variant is being
discussed, or in instance of organs in which there appears to
be no obvious bias in variant expression, we refer to NBCe1
without a variant designation.
34
Antibodies and most PCR probes used in these studies would not
be able to differentiate NBCe1-A from NBCe1-D, or NBCe1-B from
NBCe1-E, but NBCe1-D and NBCe1-E appear to only account for a
minor fraction of total NBCe1 product and are not considered here
in the discussion of reports of NBCe1-A and NBCe1-B.
35
NBCe1-A transcripts in mouse brain are nearly 50% as abundant as those encoding NBCe1-B/C (E. Roussa, personal communication).
As determined by in situ hybridization or immunohistochemistry, NBCe1-B and NBCe1-C are present throughout
the rat brain but exhibit particularly robust expression in
the dentate gyrus of the hippocampus, cerebellum, olfactory bulb, and piriform cortex (318, 624, 843), and in the
brain stem/diencephalon region (260, 1060). In general,
NBCe1-C transcripts appear to outnumber those encoding
NBCe1-B (624), although the ratio of NBCe1-A/B (likely
NBCe1-B) to NBCe1-C protein is greater in cerebellum
compared with other brain regions (261, 1060). In rats,
expression of NBCe1 transcripts (318) and protein (260) is
not detected in the brain until birth, whereupon NBCe1
levels increase gradually until an age of 4 wk (260).
36
The immunostaining of NBCe1-A protein throughout the mouse
brain must be interpreted with some caution: 1) the preimmune
serum from the rabbit used to generate the anti-NBCe1-A antibody
diffusely labeled tubules in the rat renal cortex (see Fig. 3C of Ref.
817); and 2) no preimmune controls are presented for the brain
sections (796).
Table 5. Sites of NCBT expression
Central nervous
system
Sensory organs
Peripheral nervous
system
Respiratory
system
Circulatory system
Musculoskeletal
system
Upper digestive
system
Lower digestive
system
NBCe1
NBCe2
NBCn1
NDCBE
NBCn2
Widespread, neurons
and astrocytes
Eye
Trigeminal ganglion
Blood-brain barrier
and elsewhere
Eye
Trigeminal ganglion
Widespread, neurons
Widespread, neurons
Widespread, mainly
neurons
Eye, ear
Nose and elsewhere
Lung
Trachea and lung
Trachea and lung
Vasculature
Heart
Heart
Osteoclasts, skeletal
muscle
Widespread
Skeletal muscle
Skeletal muscle
Cardiac myocytes and Heart
elsewhere
Skeletal muscle
Skeletal muscle
Widespread
Stomach
Widespread,
abundant in
pancreas
Widespread,
abundant in liver
Widespread
Widespread
Widespread
Spleen and
leukocytes
Thyroid
Kidney
Placenta and
testes
Spleen and
macrophages
Widespread
Spleen
Widespread
Kidney
Testes and elsewhere
Pituitary gland
Kidney
Testes
Lymphatic system
Endocrine system
Urinary system
Reproductive
system
Ear
Trigeminal ganglion
Thyroid and pancreas
Kidney
Widespread
Bladder and kidney
Widespread
Stomach
A more complete and detailed examination of each NCBT distribution is provided in text. A distribution of NCBT
expression based on the origins of corresponding expressed-sequence-tags is provided in Appendix VI.
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I) Central nervous system. A) General. The distribution of
NBCe1 variants in the central nervous system was recently
reviewed by Majumdar and Bevensee (623). Assessed by
PCR, NBCe1-B/C transcripts are abundant in rat brain,
whereas NBCe1-A is much less abundant (318). By immunoblot, NBCe1-C is abundant in rat brain versus kidney,
whereas NBCe1-A/B expression in brain is negligible compared with kidney (79). In mouse brain (796), NBCe1-A
can be identified by quantitative PCR,35 and the protein is
reported to be widespread by immunohistochemistry.36
However, in rat and human brain, northern blot and in
situ hybridization studies using NBCe1 variant-specific
probes indicate that expression of NBCe1-A transcripts
is insubstantial (6, 318, 624). Thus it would appear that
the predominant NBCe1 variants in mammalian brain
are NBCe1-B and NBCe1-C.
MARK D. PARKER AND WALTER F. BORON
B) Neurons versus glia. In primary cultures from cerebral
cortex, NBCe1-B protein is expressed mainly in astrocytes,
whereas NBCe1-C is expressed mostly in neurons (79).
However, the expression pattern appears to be just the opposite in situ (624), where immunohistochemistry and immuno-gold labeling reveals NBCe1-A/B (likely NBCe1-B)
inside neurons, and reveals NBCe1-C on the plasma membrane of astrocytes. A study on mouse or rat brain oligodendrocytes demonstrates NBCe1-A/B (likely NBCe1-B)
immunoreactivity in the dendrites of these cells (800).
D) Blood-brain barrier. NBCe1 protein has been detected in
basolateral membranes of choroid plexus epithelia (843).
Transcripts also are detected in the outer meningeal layer
(843).
II) Sensory Organs. A) Eye. NBCe1-B is often described as
the major NBCe1 variant expressed in the eye, although
most evidence relies on molecular tools that do not discriminate between NBCe1-B and NBCe1-C. To our knowledge,
an antibody specific for NBCe1-C has never been used to
examine the distribution of NBCe1 in the eye. In one study
on human corneal endothelium, a primer pair that should
amplify both NBCe1-B and NBCe1-C yielded one fulllength cDNA clone, which corresponds to full-length
NBCe1-B (922), but this does not exclude the presence of
NBCe1-C. In ciliary body, an antibody that recognizes both
NBCe1-A and NBCe1-B exhibits robust immunoreactivity
(1013), even though NBCe1-A is not abundantly expressed
in the eye (see below). Taken together, these data are consistent with the hypothesis that NBCe1-B is the dominant
NBCe1 variant in the eye.
NBCe1-B/C cDNA and protein are detected in a variety of
ocular tissues, namely the surface and wing cells, but not the
stroma, of the conjunctiva (94);37 the keratocytes of the
corneal stroma (94); the endothelial cells of the cornea (94,
248, 593, 922, 923, 989, 990), predominantly at the basolateral membrane (94, 248, 922, 923, 989; see cartoon in
FIGURE 19); the trabecular meshwork in the anterior chamber (989), responsible for draining aqueous humor; the pigmented epithelium of the ciliary body (94, 989) in the posterior chamber, specifically at the basolateral membrane of
the pigmented cells (94). Data conflict concerning the presence
of NBCe1 in the non-pigmented epithelia of the ciliary body
(94, 868, 989), which is responsible for secretion of the aque37
In a separate study, Turner and co-workers were unable to
demonstrate NBCe1-A/B immunoreactivity in the conjunctival epithelium of rats and pigs because their immunohistochemical studies
were hampered by “discernable nonspecific labeling” (985).
852
A small amount of NBCe1-A cDNA has been detected by
PCR in a human corneal endothelial cell line, but this is
swamped by a greater population of NBCe1-B/C cDNAs
(990). On the other hand, the cornea per se is reported to be
negative for NBCe1-A cDNA in cattle (923) and humans
(922), and corneal endothelium is negative for NBCe1-A
protein in rat (94). A report of NBCe1-A expression in rat
ciliary body was based on an antibody raised against an
epitope common to NBCe1-A and NBCe1-B (1013), and a
report of NBCe1-A expression in porcine nonpigmented ciliary epithelial cDNA depended on a primer pair that does not
distinguish among NBCe1 variants (868). Indeed, an antibody
study by Bok et al. (94) found only NBCe1-B, and not
NBCe1-A, expression in the ciliary body. An immunohistochemical study by the same workers did detect NBCe1-A in
the basal epithelium of rat conjunctiva (94). Only one study
reports appreciable NBCe1-A protein expression elsewhere in
the rat eye: using immunohistochemistry, Usui and co-workers found NBCe1-A protein together with NBCe1-B protein in
the ciliary body, lens, and cornea of the rat eye (990). However, the NBCe1-A immunoreactivity was diffuse, in contrast
to the clear membrane localization of NBCe1-B immunoreactivity in the study of Bok and co-workers.
Despite the relatively low abundance of NBCe1-A in the
eye, it is interesting to note that an individual with the
mutation Q29X, predicted to specifically eliminate
NBCe1-A (FIGURE 25 AND TABLE 6), has bilateral glaucoma
(412). Thus either NBCe1-A is expressed in tissues involved
in regulating anterior-chamber volume (e.g., nonpigmented
epithelium of the ciliary body, trabecular meshwork), presumably early in development, or the ocular phenotype is
secondary to the whole body acidosis caused by NBCe1-A
deficit in the kidney.
III) Peripheral nervous system. A) Trigeminal ganglion.
NBCe1-B/C, but not NBCe1-A, transcripts are detected by
reverse transcription polymerase chain reaction (RT-PCR)
in preparations of rat trigeminal ganglion neurons (408).
IV) Respiratory system. A) Nose. In human nasal mucosa,
NBCe1-A, but not NBCe1-B/C, transcripts are detected in
the epithelia and submuscosal gland cells of the inferior
turbinate mucosa, and in the superficial epithelia of nasal
polyps (558).
B) Lungs. NBCe1-B/C immunoreactivity is detected in
preparations of basolateral membrane proteins of the Calu
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C) Spinal cord. NBCe1 transcripts are detected in the developing rat spinal cord from embryonic day 19 (318), and NBCe1
protein has been noted in the spinal cord (both white and gray
matter; Ref. 843), according with a substantial presence of
NBCe1-B/C transcripts in spinal cord mRNA (6).
ous humor; the epithelium of the lens, in both apical and
basolateral membranes, and a human lens anterior epithelium
cell line (94, 875, 989); and the retina (30) including specifically the apical microvilli and end feet of Müller glial cells (94),
the apical membrane of retinal pigment epithelial cells (11,
94), and the choriocapillaris (1079).
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
Aqueous humor
Tight junction
Stroma
Na+
NBCe1
Na+
CFTR
2 HCO3–
Cl–
Na-K pump
3 Na+
2 K+
HCO3–
Stroma
NHE
H+
Na+
Lens
NKCC1
Corneal
endothelium
Na+
CA
K+
Aqueous Humor
2 Cl–
CO2
KCNQ1
H2O
K+
Corneal endothelium
FIGURE 19. Role of NBCe1 in the cornea. The corneal endothelium reabsorbs fluid from the collagen matrix
that constitutes the stroma, preventing corneal edema (see cartoon of eye). NBCe1-B in the basolateral
membrane supports transepithelial anion secretion. Note the similarities between this pathway and the
mechanism of NaCl secretion in dogfish salt glands (FIGURE 11).
cell line, which is derived from pulmonary airway submucosal-gland serous cells (515).
V) Circulatory system. A) Heart. Most northern blot analyses of human mRNAs and qPCR experiments indicate that
NBCe1 is expressed in the heart, although at a lower abundance than in kidney or pancreas (6, 31, 140, 192, 480,
684, 831). Full-length NBCe1-B has been cloned from human heart cDNA (192), and an antibody directed against
the third extracellular loop of NBCe1 immunoreacts with
protein in rat and human ventricular myocardial cells (481).
NBCe1-A/B
immunoreactivity,
likely
representing
NBCe1-B, is present in the left and right ventricles as well as
in the interventricular septum of rat heart (831). A preliminary immunocytochemical study of rat ventricular myocytes suggests that NBCe1 protein is located in the traverse
(T) tubules, in contrast to the predominantly surface-sarcolemmal distribution of NHE1 (311).
B) Capillaries. In the testes of rats, NBCe1 immunoreactivity is detected in capillary-lining endothelial cells (445).
VI) Musculoskeletal system. A) Skeletal muscle. NBCe1 immunoreactivity is detected in skeletal muscle homogenates
from humans and rats (518) and has been detected in soleus
and extensor digitorum longus (i.e., calf) muscles of rats (964).
Immunohistochemistry of rat muscle suggests that NBCe1 is
located in the sarcolemmal membrane and perhaps also, the
authors of the study suggest, in T tubules (518).
VII) Upper digestive system. A) Enamel organ. Ameloblasts
promote enamel deposition on developing teeth and
NBCe1-B, but not NBCe1-A, transcripts are detected in
preparations of microdissected ameloblasts from mice and
humans (538, 1099).38 NBCe1 transcripts are more abundant in mature than secretory ameloblasts (539, 540,
1099). Immunohistochemistry appears to demonstrate a
38
The report of human NBCe1-A, and not NBCe1-B, expression in
microdissected human enamel organ (1099) is probably incorrect.
The “human NBCe1-A” primer pair used in Ref. 1099 is actually
specific to NBCe1-B/C, whereas the “human NBCe1-B” primer pair
used in Ref. 1099 is actually specific for NBCe1-A.
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cAMP
HCO3–
Cornea
H+
++
MARK D. PARKER AND WALTER F. BORON
basolateral distribution of NBCe1-A/B protein in mouse
ameloblasts (538, 706), with an additional presence in the
adjoining stratum intermedium of the papillary cell layer
(538). However, high-resolution images presented in a
study of mouse dentition disclose NBCe1-A/B immunoreactivity only in the stratum intermedium, with no NBCe1
expression in the ameloblasts themselves (456), as depicted
in the cartoon in FIGURE 20.
strong basolateral NBCe1 immunoreactivity is present in
the parotid acini of humans (using an anti-NBCe1-B/C antibody; Ref. 710) and rats (anti-NBCe1; Ref. 818) as well as
in a rat parotid acinar cell line (anti-NBCe1-A/B; Ref. 740).
Taken together, these data suggest that NBCe1-B is the
major NBCe1 variant expressed in parotid acini. To our
knowledge, the presence of NBCe1-C in salivary glands has
not been examined.
Three factors could underlie the apparent discrepancy
among the above studies: 1) it is difficult to resolve the
ameloblast basolateral membrane from the membranes of
abutting papillary cells in the stratum intermedium, 2) the
studies were performed in different species, and 3) the studies employed different antibodies.
Apart from acinar cells, NBCe1 immunoreactivity is also evident in the basolateral membranes of striated and main duct
cells of rat parotid glands (818). In duct cells, NBCe1 would
act in parallel with NBCn1 (see cartoon in FIGURE 21B).
Interstitial
space
NBCe1-B
Na+
Na-K pump
2 HCO3–
2 K+
Papillary cell
3 Na+
Na+
HCO3–
NBCn1
H+
Gap junction
Tight junction
+ NHE
+
Basal
Lateral
Ameloblast
Na+
HCO3–
CO2
cAMP
CA
H+
H2O
Cl–
Cl–
Apical
Enamel surface compartment
HCO3– AE2
Slc26a4?
CFTR
Cl–
HCO3–
FIGURE 20. Role of NBCe1 in the enamel organ. Apatite formation in the enamel compartment generates
⫺
⫺
secreted from mature ameloblasts. NBCe1 mediates HCO3
influx in papillary
H⫹ that are neutralized by HCO3
⫺
⫺
⫺
cells, and the HCO3
is transferred to ameloblasts via connecting gap junctions. Transported HCO3
and HCO3
generated within ameloblasts by CA is secreting into the enamel compartment via lateral AE2 and perhaps
apical pendrin (Slc26a4). The presence of pendrin in the apical membrane of ameloblasts is controversial
(124, 125, 297). The extracellular face of AE2 is exposed to the enamel compartment by a rearrangement
of tight junctions (456).
854
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B) Salivary gland. In parotid salivary glands, the acinar cells
are a site of NBCe1 expression (see cartoon in FIGURE 21A).
Only NBCe1-B/C, and not NBCe1-A, is detected by PCR of
mouse and bovine parotid cDNA (490, 1065). NBCe1-B
was cloned from these cells in guinea pigs (508). Moreover,
Concerning the sublingual and submandibular glands,
NBCe1 transcripts are absent from cDNA prepared from
the sublingual salivary glands of mice (490), but are detected in the submandibular glands of guinea pigs (508).
Furthermore, two studies describe NBCe1 immunoreactivity in the basolateral membranes of rodent submandibular
gland duct cells (615, 818).
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
A
B
Lumen
Na+
H2O
Fluid secretion
TMEM16A
CFTR
Acinus
Cl–
Slc26a6
Slc26a3
Duct
–
HCO3
Cl–
H+
H2O
CAII
HCO3–
CO2
cAMP
Na+ 2 HCO3–
NHE1
Na+ 2 HCO3– Na+ HCO3–
Na+
++
Na+ K+ 2 Cl–
++
Cl–
H+
CO2
K+
HCO3–
AE2
NBCe1-B
H+
NKCC1
Salivary acinar cell
NHE1
NBCe1-B
NBCn1
Salivary duct cell
FIGURE 21. Role of NCBTs in exocrine glands. The inset in A displays a generic acinus (acinar epithelia,
blue) and duct (duct epithelia, yellow) for an exocrine gland such as the salivary gland or pancreas. NBCe1
activity regulates intracellular pH and could support transepithelial fluid and ion secretion by salivary gland
⫺
acinar cells (A). NBCe1 and NBCn1 support transepithelial HCO3
secretion by salivary gland duct cells (B),
⫺
-rich saliva. The presence of AE2 in the duct cells of salivary glands may
contributing to formation of a HCO3
be species specific [present in humans (998) but not in rats (818)]. Similarly, the presence of NKCC1 in
duct cells is not reported in all species (e.g., absent from mice in Ref. 279). The Na pump has been
omitted from both cell types for clarity. A similar mechanism for fluid secretion operates in pancreatic acini
and ducts.
C) Esophagus. NBCe1-A/B immunoreactivity is present in
the basolateral membranes of acinar and duct cells of
esophageal submucosal glands (3, 4). NBCe1 is also detected in enzyme-secreting serous cells, but here the polarity
of NBCe1 distribution is not evident (3, 4).
D) Stomach. NBCe1-B/C transcripts are present in stomach
preparations from rabbits (427), guinea pigs (508), and
humans (6). Northern blots and qPCR of rabbit gastric
mucosal cell preparations suggest that NBCe1 is more
abundant in mucous cells than chief or parietal cells (814).
NBCe1 transcripts are also present in a cell line derived
from rat gastric mucosa (369).
VIII) Lower digestive system. A) Intestines. At the level of
mRNA or cDNA, NBCe1-B (or NBCe1-B/C) is widely expressed in the lower digestive tract. Full-length NBCe1-B
has been cloned from rabbit duodenum (427). Intestinal
expression of NBCe1-B/C transcripts has also been demonstrated in 1) rabbit colonic mucosa, with lower levels of
expression in the ileum (427); 2) mouse duodenum (753)
and colon (1087) [in the mouse proximal colon, in situ
hybridization detects NBCe1 transcripts only in crypt epithelia (55)]; 3) rat small intestine and colon (318) [along the
rat distal colon, NBCe1 transcripts are more numerous in
the last quarter (furthest from the lymph node), than the
first quarter (closest to the lymph node; see Ref. 1059)];
4) guinea pig small intestine and proximal colon (508);
5) opossum ileum (64); and 6) human colon (6).
Aside from NBCe1-B/C, a small population of NBCe1-A
transcripts is detected in the ileum and colon of rabbits
(427) as well as in the duodenum (753) and colon (482) of
mice, but NBCe1-A is not present at appreciable levels in
the ileum of opossums (64). NBCe1-A is reportedly the
predominant form of NBCe1 expressed in the human cancer cell line HT29, although evidence for the assignment is
not provided (642).
At the level of protein, NBCe1 immunoreactivity is detected
in mouse duodenum in the basolateral membranes of enterocytes (see FIGURE 22 and Ref. 753). In the enterocytes of
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cAMP
Interstitium
Na+
H2O
Na+
Cl–
CAII
Na+
HCO3
H+
H2O
HCO3–
ENaC
–
MARK D. PARKER AND WALTER F. BORON
Lumen
Tight junction
Interstitium
Na+
Na+
NHE3
NBCe1-B
H+
2 HCO3–
cAMP
Na+
NBCn1
HCO3–
Cl–
CFTR
Na+
K+
Slc26a6
H+
Cl–
2
NKCC1
Cl–
HCO3–
Slc26a3
K+
Cl–
HCO3–
CA
Na+
cAMP
CO2
H2O
++
H+
Cl–
HCO3–
NHE
AE2
Duodenal villar enterocyte
FIGURE 22. Role of NCBTs in intestine. Shown is a duodenal villus enterocyte. The mechanism of transep⫺
ithelial fluid and HCO3
secretion is very similar to that shown in FIGURE 21 for salivary glands. The Na pump
is omitted for clarity.
rat proximal duodenum, NBCe1 immunoreactivity is strongest in villar enterocytes and decreased in abundance closer
to the crypts, such that NBCe1 immunoreactivity is not
detectable in goblet cells (430). A similar distribution is
detected in opossum ileum (64). NBCe1 immunoreactivity
is also evident in the enterocytes of the proximal jejunum
(villar and crypt enterocytes), ileum and proximal, but not
distal, colon (430). NBCe1 immunoreactivity has also been
detected in the basolateral membranes of brush cells from
rat cecum (696).
B) Liver. Northern blots indicate that the liver may be an
additional, albeit minor, site of NBCe1 expression (6, 806).
In rat bile duct, basolateral NBCe1 immunoreactivity is
detected in brush cells that are hypothesized to secrete
HCO3⫺ (695).
C) Pancreas. At the mRNA level, the pancreas is the single
most abundant site of expression of NBCe1, specifically
NBCe1-B (6). The role of NBCe1 in this organ is also reviewed in Reference 906. NBCe1-B has been cloned from
human pancreatic cDNA (6). Taken together, in situ hy-
856
bridization and immunohistochemical data demonstrate
that pancreatic NBCe1 is expressed in the acinar and duct
cells. NBCe1 is also expressed in insulin-secreting ␤ cells in
the islets.
In the acinar cells of mice, NBCe1-B transcripts have
been detected by in situ hybridization (6) and NBCe1-B
protein is located in the basolateral membrane of rat
acinar cells (817, 836, 962). However, no NBCe1-B immunoreactivity is detected in human pancreatic acini
(626, 836).
In the duct cells of the human pancreas, one study, using
antibodies to an Nt epitope or a Ct epitope that are common to all NBCe1 variants, demonstrated that NBCe1 colocalizes with Na-K pump (basolateral) but not with CFTR
(apical) (626). In other studies investigators variously report NBCe1-B immunoreactivity in the duct cells of both
human and rats as apical and/or basolateral, or as different
among duct types and even among cells in the same duct
(94, 817, 836, 962). In some cases this confusion may arise
from quality-control issues surrounding the use of antibod-
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HCO3–
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
ies raised to be specific to NBCe1-B.39 NBCe1-B is also
expressed in the pancreatic duct cell lines CAPAN-1 (883)
and mPEC1 (344), as well as in the cystic fibrotic pancreatic
duct cell line CFPAN-1 (883). Physiological data support an
exclusive presence of NCBT activity in the basolateral
membranes of duct cells (422, 883, 1096).
IX) Lymphatic and immune systems. As far as we are
aware, there are no reports of substantial NBCe1 expression in the lymphatic or immune systems. An NCBI-curated
database reports a small number of human NBCe1 ESTs
derived from bone marrow and spleen (Appendix VI).
However, NBCe1 transcripts are noted as undetectable by
northern blot of mouse spleen RNA (313).
XI) Urinary system. A) Kidney. The kidney is the major site
of expression for the NBCe1-A transcript (6). Renal
NBCe1-A transcripts have been cloned from many species
including humans (138) and rats (140, 806). NBCe1-A protein is expressed in the kidney cortex, specifically in the
basolateral membranes of the S1 (i.e., just distal to Bowman’s capsule) and early S2 PT segments in humans, rabbits, and rats (7, 273, 632, 829, 844, 1024, 1064), as depicted in the cartoon in FIGURE 23. The segmental distribution of NBCe1 along the nephron significantly overlaps
with that of protein 4.1B in the S1 and S2 tubules (958), and
with the Na/glucose cotransporter SGLT1 in the S2 tubule
(806).
A lesser amount of NBCe1 mRNA expression is detected in
the S3 proximal tubule segments of rabbits (7), as expected
from a tubule segment in which HCO3⫺ reabsorption is less
than for the S1 and S2 segments (7). Indeed, NBCe1 protein
is totally absent from the S3 segment of rats (632). Traces of
NBCe1 transcript expression have also been detected in the
renal medulla of rats (140) and in a mouse cell line from the
inner medullary collecting duct (35).
X) Endocrine system. A) Thyroid. NBCe1 transcripts are
detected in extracts prepared from human thyroid (309,
486).
B) Pancreas. NBCe1-B transcripts are not detected in pancreatic islet cells of mice (6). However, NBCe1-A and
NBCe1-B transcripts are detected in the pancreatic islet cells
of rats, although the immunoreactivity of anti-NBCe1-A
and -B/C antibodies are not robust at the level of western
blots (901). In immunohistochemical studies on rat using
those same antibodies, NBCe1-B immunoreactivity is detected in the ␤ cells that secrete insulin but not in the ␣ cells
that secrete glucagon (901). Moreover, NBCe1-A/B immunoreactivity is detected in insulin-positive cells of pancreatic
samples isolated from human cadavers (365). NBCe1-B immunoreactivity is also expressed in the insulin-secreting cell
Lumen
Some caution must be exercised when interpreting these studies, as the antibodies raised against epitopes in the common Nt of
NBCe1-B/C appear to be troublesome. For example, one NBCe1-B
antibody (796, 817) 1) exhibits more robust immunoreactivity with
renal protein extracts than with pancreatic protein extracts, opposite to the distribution of NBCe1-B transcripts; 2) immunoreacts
with a number of other proteins in pancreatic extracts, such that
full-length NBCe1-B is a minor target for this antibody in the pancreas; and 3) exhibits a staining pattern in renal sections that is not
different from the nonspecific staining produced using preimmune
serum from a rabbit used in the same study (See Fig. 3, C versus D,
in Ref. 817). Another NBCe1-B antibody, originally reported in Ref.
989, as expected, does not immunoreact with protein in human
kidney extracts but does immunoreact with a rat kidney protein
(273).
Interstitial
fluid
NHE1
NHE3
Na+
Na+
H+
++
cAMP
H+
cAMP
++
H+
H+
NBCe1-A
HCO3
HCO3–
H-pump
–
Na+
3 HCO3–
CAII
CAIV
CO2
+
39
Tight junction
H2O +
H2O
3 Na+
CO2
2 K+
AQP1
Na-K pump
Proximal tubule cell
FIGURE 23. Role of NBCe1 in the proximal tubule. H⫹ secreted by
proximal tubule (PT) epithelia into the PT lumen can either be titrated
by buffers such as phosphate or NH3, in which case they are excreted in the urine or, catalyzed by extracellular CA, they can be
⫺
. CO2 that enters PT epithelia from the lumen, and
titrated by HCO3
CO2 that is generated by PT metabolism, is hydrated by CAII into H⫹
⫺
⫺
and HCO3
. Reabsorption of HCO3
via NBCe1 drives H⫹ secretion
⫺
and supplies the blood with HCO3
, regulating whole body pH.
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Although, as we have just seen, NBCe1-B is undoubtedly
the major, pancreatic NBCe1 variant, it is perhaps not the
only one. A pool of NBCe1-A transcripts is detected by PCR
from pancreatic cDNA (135, 817, 836, 901) and antibodies
raised against an epitope in NBCe1-A immunoreact with a
diffuse population of protein in pancreatic duct (817, 836).
Considering their relative transcript levels (6), the functional significance of NBCe1-A is likely trivial compared
with that of NBCe1-B under basal conditions. To our
knowledge, the presence of NBCe1-C in pancreas has not
been examined.
line BRIN-BD11 (135). A diffuse staining of NBCe1-A is
detected in islet cells (901). In the exocrine pancreas,
NBCe1 is also present in acinar and duct cells.
MARK D. PARKER AND WALTER F. BORON
NBCe1-A is undoubtedly the major, but perhaps not the
only renally expressed NBCe1 variant. A small fraction of
NBCe1-B/C transcripts are detected by PCR from renal
cDNA (135, 318, 427, 817, 901), and antibodies raised
against an epitope in NBCe1-B/C immunoreact with a diffuse subapical population of protein in the rat PT (273,
817). Furthermore, on western blots, an NBCe1-C specific
antibody exhibits some immunoreactivity with a rat renal
protein extract (79). The expression level of these alternative variants is trivial compared with that of NBCe1-A under basal conditions, but their presence may be of importance during stressed conditions (117).
B) Male. NBCe1 expression is detected in testis (599), epididymis (445, 453, 599, 729), prostate (6, 684), sperm
(445), and vas deferens (152, 599). A combination of northern blotting and qPCR data indicate that the prostate is a
major site of NBCe1-B/C transcript expression in human
males (6, 684). Full-length NBCe1-B has been cloned from
human prostate cDNA (GenBank protein accession no.
AF053753). NBCe1-A/B immunoreactivity is present in
sperm extracts and in the basolateral membranes of apical
and principal cells of the epididymis (445).40 In the epididymis, NBCe1-A/B immunoreactivity is most pronounced in
the initial segments, growing progressively weaker towards
the cauda, a pattern matched by in situ hybridization results
using an anti-NBCe1 probe (445). In the testes of rats,
NBCe1 immunoreactivity is detected in smooth muscle cells
and in capillary-lining endothelial cells (445). The relative
abundance of the five NBCe1 variants throughout the
mouse reproductive tract is examined in Reference 599.
G) PHYSIOLOGICAL ROLES OF NBCe1. Its ability to transport
HCO3⫺ across membranes enables NBCe1 to play diverse
roles according to its location. In all of the cell types in
which NBCe1 is expressed, its action influences pH-sensitive processes within the cell and at the extracellular surface. In polarized epithelia, HCO3⫺ transport can also support HCO3⫺ secretion (i.e., away from the blood) or HCO3⫺
absorption (i.e., toward the blood). Here we first discuss
those processes that have general relevance to the function
of a number of systems, and then we discuss specialized
processes that are specific to certain organs and tissues.
I) General. A) Intracellular pHi regulation. NBCe1 presumably contributes to pHi regulation in every cell in which it is
expressed. However, the role played by NBCe1 would depend
critically on its stoichiometry. In cultured rat cerebellar (132)
40
A later review by the same group refers to epididymal NBCe1 as
NBCe1-A, although the immunohistochemistry by itself is not sufficient to support this specific assignment.
858
A word of caution is that one could easily be fooled by an
unanticipated combination of 1) an electroneutral acidbase transporter that requires Na⫹ and HCO3 (e.g., an
electroneutral NCBT, or a Na-H exchanger activated by a
CO2/HCO3⫺ receptor) and 2) a parallel though unlinked
electrogenic process (e.g., a pH-sensitive ion channel).
Thus, in reaching the conclusion that an electrogenic Na/
HCO3 cotransporter is responsible for a pHi change, it is
important that the investigator verify that the cells do indeed express the transporter, that the rate of pHi change
quantitatively matches some measure of electrogenic transport (e.g., a change in Vm but preferably a membrane current), and that the indexes of both transport and electrogenicity have the same ionic and pharmacological properties.
A case in point is a report that concluded that, in spinal cord
neurons of embryonic rats, an electrogenic NBC contributed to the observed, robust pHi recovery from an acid load
(118). The electrical link was a demonstration that an increase in [K⫹]o caused an abrupt pHi increase. However, in
salamander PTs, where such a depolarization-induced alkalinization (DIA) was first described, the DIA occurs in the
nominal absence of CO2/HCO3⫺ and, in fact, is mediated by
electroneutral Na/lactate cotransport across the apical
membrane, followed by H/lactate cotransport across the
basolateral membrane (884, 885). The spinal cord neuron
study did not include an analysis of the Na⫹ or HCO3⫺
dependence of the DIA, nor of its sensitivity to DIDS. Thus
one must exercise prudence in interpreting these data.
B) Possible role in cell migration. A localized regulatory volume increase (RVI) at the leading edge of lamellipodia in migrating cells is mainly mediated by NHE1 (910). However, on
the basis of a residual migratory capability of NHE-deficient
MDCK-F cells that is sensitive to the NCBT inhibitor S0859,
NBCe1 has been suggested to be capable of making a minor
contribution to migration (849). Because 1) S0859 has an untested specificity, 2) NBCe1 transcripts are scarce in these cells,
3) NBCe1 protein expression is undemonstrated in these cells,
and 4) other NCBTs aside from NBCe1 may be expressed in
these cells, the authors were not able to definitively link
NBCe1 activity with cell migration (849).
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XII) Reproductive system. A) Female. NBCe1 transcripts
are detected in mouse ovarian, uterine, and vaginal preparations (599, 1027).
and hippocampal (75) astrocytes, an electrogenic Na/HCO3
cotransporter enhances the pHi recovery from an acute intracellular acid load (i.e., the transporter functions as an acid
extruder, mediating the uptake of HCO3⫺ equivalents). Thus
this transporter, subsequently identified as NBCe1-B in cultured hippocampal astrocytes, must operate with a 1:2 stoichiometry. In renal PTs, NBCe1-A operates with an apparent
stoichiometry of 1:3 and thus mediates a net efflux of HCO3⫺
equivalents (i.e., it functions as an acid loader, mediating the
efflux of HCO3⫺ equivalents). In these cells, we would expect
that NBCe1-A would contribute to the pHi decrease following
an acute intracellular alkaline load, although, to our knowledge, this experiment has not been done.
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
Overexpression of CA IX on the extracellular surface promotes cell migration in MDCK cells (931). Furthermore,
NBCe1 and CA IX immunoreactivity colocalize in a hypoxic A549 lung-tumor cell line (254). Thus it has been
proposed that CA IX and NBCe1 form a “metabolon” in
which CA IX activity (CO2 ⫹ H2O ↔ HCO3⫺ ⫹ H⫹) is
promoted by the action of NBCe1 that removes HCO3⫺
from the cell surface (254, 931). The importance of CA IX
in tumor pH regulation is reviewed in Ref. 934.
NBCe1 in this regard could be minor compared with that of
NHE1.
A study of wound repair in monolayers of a rat gastric
epithelial cell line showed that the wound-healing process
(i.e., cell migration) could be inhibited by DIDS or by the
removal of Na⫹, Cl⫺, and/or HCO3⫺ (369). Although the
transport processes responsible for these phenomena remain unidentified, the authors detect both NBCe1 and AE2
transcripts in these cells (369).
In summary, although data are consistent with the appealing hypothesis that NBCe1 could support cell migration/
tumor metastasis, the data are not conclusive and the role of
A
Hippocampal neuron
41
Enhanced seizure resistance in a third, NBCe2-null, mouse
strain may be an indirect effect of altered CSF composition because
NBCe2 is not expressed in neurons.
Alkalinization
+
Ca2+ release
Neuron firing
+
Decreased [HCO3–]i
+
Cl–
NDCBE
2 HCO3– Na+
Acidification
Alkalinization
–
+
Ca2+
NBCn1 or
NBCn2
+
Na+
2 H+
Na+ 2 HCO3– HCO3– Na+
Depolarization
K+
PMCA
Increased [K+]o
+
NBCe1
Extracellular neuronal
microenvironment
3 Na+
NBCe1
+
+
Depolarization
K+
Decreased
Na+
NSS
+
[Na+]i
Decreased [Na+]i
–
2
2 HCO3
Alkalinization
B
Na-K pump
+
+
K+
Na+ NT–
ATP production
Glycolysis
Astrocyte
FIGURE 24. Role of NCBTs in excitable cells. Neuronal firing causes a depolarization induced alkalinization
(DIA), via NBCe1, that anticipates and counters the dampening of neuronal excitability by Ca2⫹ pump-mediated
H⫹ influx. The three electroneutral NCBTs also play critical roles in restoring neuronal pHi after a firing event
(A). K⫹ released by firing neurons is absorbed by astrocytes causing a DIA, via NBCe1, that stimulates glycolytic
ATP production (B), anticipating the increased energetic demand of the astrocyte for secondary active
neurotransmitter (NT) uptake via neurotransmitter/sodium symporters (NSS).
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II) Central nervous system. A) Enhancement of neuronal
excitability. Neuronal firing results in an intracellular acidification of neurons that tends to dampen neuronal excitability (reviewed in Refs. 76, 186, 187, and 898). The action of NCBTs in neurons, that as a population express at
least NBCe1, NBCn1, NBCn2, and NDCBE, re-alkalinizes
cells following firing events and thereby enhances the rate at
which excitability recovers (FIGURE 24A). Indeed, mice
lacking either of two other NCBTs, NDCBE and NBCn2,
exhibit signs of reduced neuronal excitability.41 In hippocampal neurons under high-[K⫹]o conditions (a mimic of
intense firing), the activity of NBCe1 is sufficiently strong
that NBCe1 (and other factors) produce a depolarization-
MARK D. PARKER AND WALTER F. BORON
induced alkalinization that overwhelms the natural tendency toward intracellular acidification (932).
III) Sensory organs. A) Transepithelial HCO3⫺ secretion
across corneal endothelium. Working with a 1:2 stoichiometry (543) and importing HCO3⫺ from the stroma (FIGURE
19) into the cell, NBCe1 in the corneal endothelium is in a
position to make a substantial contribution to the basolateral step of transepithelial HCO3⫺ secretion into the anterior
chamber (i.e., aqueous humor). It is thought that this transcellular HCO3⫺ movement drives fluid reabsorption from
the stroma into the anterior chamber, thereby maintaining
appropriate corneal hydration and transparency (579, 922,
923, 1035).42 The molecular mechanisms underlying this
process (shown in FIGURE 19) are reviewed in Reference 96.
Briefly, cytosolic HCO3⫺ accumulates either as HCO3⫺ enters
the cell directly across the basolateral membrane via
NBCe1-B, or as HCO3⫺ forms from cytosolic CO2 (catalyzed
by CA II) as Na-H exchangers extrude H⫹ across the basolateral membrane. Apical anion channels secrete HCO3⫺
into the anterior chamber. Thus the action of NBCe1-B
helps provide cytosolic HCO3⫺ for secretion and also regulates pHi. Consistent with a contribution to fluid secretion
by NBCe1, NBCe1 knockdown by siRNA reduces the
transepithelial HCO3⫺ flux in cultured bovine corneal endo42
Although one model proposed by Wiederholt et al. places an
electrogenic NCBT in the apical membrane, the authors state that
this is an assumption since their methods did not allow them to
determine the localization of NCBT in their cells (1035).
860
B) Potential to promote retinal attachment. In the retinal
pigment epithelium, NBCe1-B is present in the apical membrane (94). NBCe1-mediated uptake of HCO3⫺ across the
apical membrane would contribute to fluid absorption from
the subretinal space to blood, presumably minimizing subretinal edema, as has been proposed for an apical electrogenic NCBT activity in bullfrogs (FIGURE 13A). Subretinal
edema has not been described in Slc4a4-null mice nor in
patients with NBCe1-associated pRTA, although it is possible that the presence of edema is masked by other ocular
defects present in these individuals.
IV) Peripheral nervous system. A) Neuronal excitability. In
primary cultures of neurons from the rat trigeminal ganglion, application of anti-NBCe1-B/C siRNA results in a
⬃50% reduction of NBCe1-B/C protein abundance and,
following an NH4⫹ prepulse, causes a near total elimination
of HCO3⫺-dependent acid-extrusion in these cells (408).
Thus NBCe1-B/C is likely to be the major NCBT in these
cells. Furthermore, the frequency of action potential firing
in response to current-injection in these cells is reduced by
intracellular acidification and by DIDS treatment (408).
Taken together, these data indicate that NBCe1-B/C mediates an uptake of HCO3⫺ that counters the dampening effect
of intracellular acidification (408), FIGURE 24A pe and
thereby plays an important role in maintaining excitability
in these neurons.
V) Circulatory system. A) Myocardial contractility and
excitability. An NCBT-mediated increase in pH i enhances the contractility of myocardium in mammals
(160, 702, 972). Moreover, HCO3⫺-dependent alkalinization is enhanced by repeated depolarizations of cat
papillary muscle (147), consistent with the involvement
of electrogenic NBCs. The current carried by electrogenic
NCBT activity modulates the shape of myocyte action
potentials, shortening action-potential duration and contributing towards a hyperpolarized resting membrane
potential (14, 1006). Rat cardiac myocytes transfected
with an adenoviral vector designed to overexpress
NBCe1 are reported to exhibit an altered beat rate compared with nontransfected cells, although the direction of
the rate change is not reported, and overexpression of
NBCe1 transcripts or protein is not demonstrated (649).
The relative contributions of NBCe1 and NBCe2 to these
processes are unresolved. The influence of the action of
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B) Dampening of neuronal excitability by astrocytes. As
neurons fire action potentials, they release K⫹ into the extracellular microenvironment (FIGURE 24A). One effect of
the resulting elevated [K⫹]o would be to enhance neuronal
excitability. However, the action of the Na-K pump in astrocytes tends to remove this accumulated extracellular K⫹,
thereby dampening neuronal firing. The Na-K pump also
maintains a low astrocytic [Na⫹]i, thereby promoting Na⫹coupled neurotransmitter uptake. A second effect of the
elevated [K⫹]o is the stimulation of glycolysis in astrocytes
via a feed-forward mechanism that anticipates the energy
requirements of the astrocyte Na-K pump (83). The link
between elevated [K⫹]o and the stimulation of astrocyte
glycolysis appears to be NBCe1. Under conditions of intense neuronal activity, substantial K⫹ release would cause
an NBCe1-dependent DIA in astrocytes (825). The consequent pH-dependent increase in the activity of glycolytic
enzymes stimulates ATP production (FIGURE 24B). The importance of NBCe1 for this pathway is demonstrated by
1) the blockade of the pathway by S0859 and 2) the absence of
this pathway from astrocytes cultured from neonatal NBCe1null mice (825). As the action of NBCe1 produces the DIA in
astrocytes, the concomitant decrease in extracellular pH
would dampen neuronal excitability and decrease the ability
of neuronal NCBTs to counter intracellular acidification,
thereby preventing excessive firing (as in FIGURE 24A).
thelium (579). In one study, a partial in-vivo knockdown
(i.e., 25%) of NBCe1 by shRNA in rabbit eyes was not
sufficient to produce the expected corneal thickening without the additional pharmaceutical inhibition of carbonic
anhydrases (593). Given the mild knockdown, perhaps this
result is not surprising. However, even in NBCe1-null mice,
the effect of NBCe1 deficiency on HCO3⫺ secretion by colonic mucosa is detectable only following CA inhibition,
even under secretagogue stimulated conditions.
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
NBCe1 and other acid-base transporters on cardiac myocyte function is reviewed in Reference 996.
VI) Musculoskeletal system. A) Myocyte contractility. By
contributing towards pHi regulation in myocytes, NBCe1
likely contributes towards maintenance of contractility and
excitability, as it does in cardiac myocytes (see above).
NBCe1 dysfunction is associated with enamel defects. Two
alternative models have been proposed to explain how
NBCe1 contributes to enamel formation. Both models posit
that NBCe1 supports AE2-mediated HCO3⫺ secretion into
the enamel surface compartment to buffer the H⫹ formed
by apatite formation (706). However, the models differ in
the location of NBCe1 and AE2, as well as in the consideration of how the enamel organ cells interact.
In the first model (not shown), ameloblasts express NBCe1
in their basolateral membrane and AE2 (unusually for an
Slc4) in their apical membrane. The concerted action of
NBCe1-mediated HCO3⫺ influx and AE2-mediated HCO3⫺
efflux are proposed to form a pathway that secretes HCO3⫺
into the enamel-surface compartment (706).
The second model (shown in FIGURE 20) is based on an
alternative distribution of NBCe1 and AE2, and considers
that the papillary cells and ameloblasts form a syncytium
(456). In this model, NBCe1 is present in the membranes of
papillary cells that abut the basal surface of ameloblasts,
and AE2 is located in the basolateral membranes of ameloblasts (126, 456, 617). During the morphological switch of
ruffle-ended ameloblasts to smooth-ended ameloblasts (associated with neutralization of the enamel-surface compartment acidity) a rearrangement of tight junctions exposes the
lateral, but not the basal, surface of these cells to the enamelsurface compartment. Thus the action of papillary cell
NBCe1, translated via gap junctions to the cytoplasm of the
ameloblasts, is still in a position to support ameloblast HCO3⫺
secretion into the enamel fluid via AE2 (456). In an update to
the second model, pendrin (Slc26a4) has been immunolocalized to the apical membranes of ameloblasts, providing an
apical exit route for HCO3⫺ (125). However, unlike mice with
AE2 or NBCe1 dysfunction, mice with a pendrin deficiency do
not exhibit obvious defects in enamel deposition (125).
HCO3⫺
secretion in the parotid
B) Transepithelial fluid and
salivary gland. Acinar cells in the parotid glands secrete an
In the duct cells, where basolateral NBCn1 is abundant
ductal NBCe1 could contribute to the support of transepithelial secretion of HCO3⫺, via a mechanism
similar to that described for NBCe1 in corneal endothelia
(FIGURE 19). The role of basolateral NCBT activity in
HCO3⫺ and fluid secretion in salivary glands is reviewed in
Reference 555.
(FIGURE 21B),
C) Protection of gastric mucosa from acid attack. The presence of NBCe1 transcripts in mammalian stomach preparations suggests that NBCe1 could, as has been proposed
for an electrogenic NCBT in amphibian gastric mucosa (FIG⫺
URE 13B), support HCO3 secretion into the mucus layer
that covers the stomach lining, thereby protecting gastric
epithelia from acid attack.
VIII) Lower digestive system. A) Transepithelial HCO3⫺
secretion across pancreatic duct cells. Working with a 1:2
stoichiometry and importing HCO3⫺ into a cell, NBCe1 in
pancreatic duct cells can make a substantial contribution to
the basolateral step of transepithelial HCO3⫺ secretion, and
thus fluid secretion (422, 883, 1096). The contribution of
NBCe1 towards the formation of pancreatic juice by acinar
and duct cells is likely identical to that shown in FIGURE 21
for salivary glands. Cytosolic HCO3⫺ enters the cell either
through NBCe1-B or is generated de novo by CA II action
upon CO2. Apical Slc2643 proteins secrete HCO3⫺ into the
duct lumen in exchange for Cl⫺. When fully stimulated by
secretagogues (e.g., secretin), the luminal fluid in humans
can be near-isotonic NaHCO3. The alkaline duct fluid
keeps the pancreatic digestive enzymes in an inactive state
and flushes them from the ducts, both of which protect from
pancreatitis. In addition, pancreatic juice neutralizes acidic
gastric chyme. NBCe1 has also been suggested to contribute
towards the endocrine function of the pancreatic islets. The
role of basolateral NCBT activity in ductal fluid and HCO3⫺
secretion is reviewed in Ref. 555.
B) Transepithelial HCO3⫺ secretion across intestinal enterocytes. HCO3⫺ secretion across duodenal enterocytes plays a
major role in protecting mucosa from acid attack (17). Con43
Anion secretion across the apical membrane of pancreatic duct
epithelia is likely mediated by the concerted actions of CFTR, Ca2⫹activated chloride channels, and Slc26a6 (aka PAT1) with Slc26a3
(aka DRA1) playing a supporting role (333, 421, 628, 909, 1028).
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VII) Upper digestive system. A) Role in enamel deposition.
The role of enamel organ epithelia in the formation of
enamel in the enamel-surface compartment is still largely
unknown. The apical membranes of ameloblasts face the
enamel-surface compartment and, when mature, form alternating zones of ruffle-ended cells (facing enamel fluid
that is acidic) and smooth-ended cells (facing enamel fluid
that has a neutral pH).
isotonic fluid. The composition of the fluid is modified by
duct cells that, among other functions, secrete proteins and
HCO3⫺ to produce the HCO3⫺-rich saliva that acts to optimize
amylase activity and to buffer gastric juices. Working with a
1:2 stoichiometry and importing HCO3⫺ into a cell, basolateral
NBCe1 in parotid acinar cells is in a position to regulate pHi.
Moreover, in concert with AE2, which would recycle HCO3⫺
back into the interstitium, the NBCe1 could make a contribution to the basolateral step of transepithelial NaCl and fluid
secretion (Ref. 710, as shown in FIGURE 21A).
MARK D. PARKER AND WALTER F. BORON
C) Potential role in drug resistance of colon cancer cells.
siRNA suppression of NBCe1 expression in a human colon
carcinoma cell line increases sensitivity of the cells to the
anti-cancer agent methotrexate (642), a phenomenon that
the authors of the study hypothesize to be due to pH dependence of methotrexate uptake transporters.
D) Potential role in transepithelial HCO3⫺ secretion across
cholangiocytes in the liver. In cholangiocytes, immunolocalization studies seem to indicate that AE2 has an unusual apical
disposition (46, 631, 855, 902, 971). Moreover, it has been
proposed that this apical AE2 mediates HCO3⫺ secretion into
the bile duct lumen (46, 631, 902), thereby protecting the liver
from bile acid attack (390). In the cholangiocytes of mice that
are unable to express the a and b variants of AE2, NBCe1
transcript and protein abundance are increased compared
with control cells from wild-type mice, as is an electrogenic
NBC activity (987). As cholangiocytes of AE2a,b-null mice are
able to compensate for their HCO3⫺ secretion deficit via a
Na⫹-dependent mechanism, it is suggested that NBCe1, again
targeted to the apical rather than the basolateral membrane,
might be able to compensate for a HCO3⫺ secretion defect by
operating with a 1:3 stoichiometry (987).44 Neither the apical
presence of NBCe1 protein in mouse cholangiocytes nor the
stoichiometry of the transport process in these cells has yet
been demonstrated, although NBCe2 immunoreactivity has
been detected in the apical membrane of rat cholangiocytes (8).
44
Although rat cholangiocytes express NBCe2 in their apical membranes (8), mouse cholangiocytes are reported to lack NBCe2
(987).
862
IX) Lymphatic and immune systems. The lymphatic and
immune systems are not major sites of NBCe1 expression.
We are unaware of any reports that assign a physiological
role to NBCe1 in these systems.
X) Endocrine system. A) Possible role in HCO3⫺ exit from
pancreatic islet cells. Insulin-producing cells generate a substantial amount of CO2 that is linked to the production of
insulin. One group suggests that the CO2 generated from
nutrient insulin secretagogues (e.g., glucose) first is converted to HCO3⫺ for exit across the plasma membrane
(863). NBCe1 is expressed in both pancreatic islet cells and
a related tumor cell line (135, 901). Although both
NBCe1-A and NBCe1-B are present in islets, NBCe1-B predominates in the insulin-producing ␤ cells. Treatment with
tenidap (an inhibitor of NBCe1) reduces glucose metabolism, reduces glucose-stimulated insulin secretion, and also
lowers pHi. The last observation is consistent with the hypothesis that NBCe1-B normally functions as an acid extruder (i.e., mediates HCO3⫺ uptake) in these cells. However, tenidap also increased 22Na uptake and hyperpolarized the cells, which would be consistent with the opposite
hypothesis: that NBCe1-B normally operates as an acid
loader (i.e., mediating HCO3⫺ efflux). It seems clear that
NBCe1 is important for maintaining insulin secretion from
pancreatic tissue, by promoting fluid secretion. However,
the tenidap (which was developed by Pfizer as a nonsteroidal anti-inflammatory drug) probably has complex actions
in these cells, witness the effects on 22Na fluxes and Vm. In
any case, we would not expect the CO2 generated from the
metabolism of nutrient secretagogues to exit the cell via
NBCe1, which is presumably mediating the net uptake of
HCO3⫺. Even in the renal PT, which generates large
amounts of CO2 and in which NBCe1-A mediates HCO3⫺
efflux, the most straightforward mechanism for the disposal
of metabolically generated CO2 is the same as for other cells
in the body: CO2 in the steady state moves passively across
the cell membrane, perhaps via gas channels, and diffuses
into systemic capillaries for disposal in the exhaled air. For
a discussion of the contribution of NBCe1 to the digestive
role of the pancreas, see above.
XI) Urinary system. A) HCO3⫺ reabsorption across proximal
tubule epithelia. As illustrated in FIGURE 23, NBCe1-A
plays a central role in the transepithelial secretion of H⫹
by the renal PT. H⫹ extruded across the apical membrane
has three fates, titrating: 1) HCO3⫺ (filtered from blood in
the glomerulus) to CO2 ⫹ H2O, 2) NH3 to NH4⫹, and
3) HPO42– (and weak bases other than HCO3⫺ and NH3) to
H2PO4– (and the conjugate weak acids of the other weak
bases), the so-called titratable acidity. In the case of HCO3⫺
reabsorption, the newly formed CO2 and H2O enter the PT
and form HCO3⫺. In the case of NH4⫹ excretion and formation of titratable acidity, the intracellular HCO3⫺ forms
from CO2 that originates either from PT oxidative metabolism or from the blood. The common denominator is that
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sistent with a role for NBCe1-B in HCO3⫺ secretion
throughout the gut (via a mechanism such as that shown for
a duodenal villar enterocyte in FIGURE 22), the secretagogues carbachol and forskolin increase the basolateral
abundance of NBCe1 protein in rat proximal jejunum enterocytes (430) and in murine colonic crypts (1087). Furthermore, both DIDS and siRNA knockdown of NBCe1
inhibit parathyroid-hormone–stimulated short-circuit currents, a measure of transepithelial anion secretion, across
monolayers of a human intestinal epithelial-like cell line
(172). On the other hand, compared with tissues from wildtype mice, proximal colons from NBCe1-null mice exhibit a
reduced cAMP-stimulated HCO3⫺ secretion only under conditions in which blockade of CA II severely curtails the
generation of intracellular HCO3⫺ from CO2 (313). At face
value, these data are inconsistent with the idea that NBCe1
plays a major role in HCO3⫺ secretion under physiological
conditions. On the other hand, it is possible that the
NBCe1-null mice may have upregulated the CA-dependent
pathway by enhancing basolateral H⫹ extrusion via NHEs
or NBCn1. At least in the duodena of mice, NBCn1 makes
a more substantial contribution to HCO3⫺ secretion than
NBCe1.
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
NBCe1-A exports the HCO3⫺ across the basolateral membrane.
The role of an electrogenic NCBT as the basolateral step in
the pathway that reabsorbs HCO3⫺ from the PT lumen was
first demonstrated in salamanders in 1983 (103). Demonstration of the equivalent activity in mammals, namely, rabbits and rats, followed in an array of papers published between 1985 and 1987 (28, 80, 81, 331, 514, 832, 896,
1085). The subsequent cloning and characterization (809)
as well as the immunolocalization of NBCe1 to the basolateral membranes of PT epithelia (632, 844) demonstrated
that NBCe1 is indeed the transporter responsible for this
activity.
45
Because NBCe1 knockout mice have not survived to a breeding
age (313), it is not known whether NBCe1 is necessary for fertility.
B) Transepithelial HCO3⫺ secretion across uterine epithelia.
Basolateral NBCe1 is in a position to support HCO3⫺ secretion across endometrium epithelia (1027) that secrete a
HCO3⫺-rich uterine fluid, which is important for sperm capacitation and egg fertilization (e.g., see Ref. 554).
H) CAUSES OF NBCe1 UPREGULATION.
In this section we consider
disturbances that result in upregulation of NBCe1 at the
level of transcript abundance, protein abundance, translocation to the plasma membrane, or transporter activity.
Note that an increase in any one of these factors need not
necessarily correlate with an increase in the others.
The plasma-membrane abundance, as well as per-molecule
activity, of NBCe1-B/C can be increased by activation of the
soluble binding partner IRBIT. However, the physiological
cues that activate IRBIT have not been described.
In the following discussion, we have omitted cellular studies
that report only indirect evidence of NBCe1 upregulation
(e.g., upregulation of HCO3⫺ reabsorption) because such
observations might at least in part be explained by effects
on other proteins. We have arranged the reports in the order
of the organ in which each observation was made and then
in order of disturbances that are shown to increase NBCe1
transcript abundance, increase NBCe1 protein abundance,
increase NBCe1 abundance in the plasma membrane, and
stimulate NBCe1 activity.
I) Central nervous system. A) Increased transcript abundance following cerebral arterial occlusion. In rats subjected to permanent cerebral-artery occlusion, the abundance of NBCe1 protein in the ischemic penumbra is
more than twice as great as in sham-operated controls
(458). It is reasonable to suggest that this upregulation of
NBCe1 leads to an increase in [Na⫹]i that could contribute to edema as well as other secondary brain injuries.
Thus NBCe1 inhibitors have the potential to limit such
ischemic damage (458).
B) Increased protein abundance following seizure induction. Seizure-sensitive and seizure-resistant gerbils exhibit similar expression patterns for NHE1 and NBCe1
immunoreactivity. However, 30 min and 180 min after
the induction of seizures in the SS gerbils, the expression
of both proteins increased markedly in the hippocampal
CA1–3 regions and granule layer of the dentate gyrus
(467). Also, NBCe1 protein levels are elevated in the
hippocampi of gerbils 4 h after administration of the
GABAB receptor agonist baclofen but not after administration of the GABAA receptor agonist muscimol (466).
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XII) Reproductive system. A) Possible role in HCO3⫺ reabsorption and/or secretion in the epididymis. The lumen of
the epididymis is a site of Na⫹ reabsorption and H⫹ secretion (572), with low luminal pH being a requirement for
storage of viable sperm. NBCe1-B is present in the cells of
the epididymis, and cultured epididymal cells exhibit a
DIDS-sensitive, Na⫹- and HCO3⫺-dependent pHi recovery
from an acid load, leading several groups to suggest that
NBCe1 might be involved in H⫹ secretion/HCO3⫺ reabsorption by these cells (164, 445, 729, 1119). Mice deficient in
the estrogen receptor ESR␣ (or ESR1) are defective in their
ability to acidify the epididymal lumen. In the initial segment of the epididymis, these mice exhibit a ⬃50% reduction in protein abundance of apical NHE3 and CA XIV, as
well as basolateral NBCe1 (453). At present there is no
direct evidence that NBCe1 plays a substantial role in epididymal HCO3⫺ reabsorption in these cells. Relevant issues
include: 1) the direction of NBCe1-mediated transport can
vary in a tissue-specific manner (346) and physiological
data to support an outwardly directed basolateral NCBT
activity in these cells is presently lacking. 2) In order for a
basolateral NBCe1-B to contribute to HCO3⫺ reabsorption,
it would presumably have to operate with a 1:3 stoichiometry, rather than the 1:2 stoichiometry that it has in pancreatic ducts (344). On the other hand, one report suggests that
the stoichiometry of NBCe1-B might depend on the celltype in which it is expressed (346). 3) NBCe1 immunoreactivity in the epididymis is not restricted to the acid-secreting
narrow or clear cells (729). 4) AE2, a related acid-loading
transporter, is also expressed in the basolateral membranes
of epididymal epithelia (446). Mice that are unable to express the a, b1, and b2 variants of AE2 are infertile (638).
Thus NBCe1-B is unable to compensate sufficiently in these
knockouts.45 On the other hand, luminal H⫹ secretion from
epididymal epithelia is stilbene-sensitive and independent
of Cl⫺ (120, 164, 1119). We conclude that the physiological role of NBCe1-B in epididymal H⫹ secretion/HCO3⫺ reabsorption remains open. One possibility is that NBCe1-B plays
a role in pHi regulation in epididymal epithelia. Another
possibility is that NBCe1-B supports regulated HCO3⫺ secretion in epididymal epithelia (152, 164), which could activate sperm mobility prior to ejaculation (697, 937).
MARK D. PARKER AND WALTER F. BORON
II) Circulatory system. A) Increased transcript abundance and activity in heart following abdominal aortal
constriction. NBCe1 (and NBCn1) transcript abundance
increases in a rat model of ventricular hypertrophy
(1071), generated by constriction of the abdominal
aorta, and is accompanied by an increase in HCO3⫺-dependent acid extrusion in myocytes isolated from the
hypertrophic ventricles. The authors suggest that NBCe1
contributes to an increased Na⫹ load in hypertrophic
myocytes, promoting arrhythmia and reperfusion injury
via activation of the Na-Ca exchanger (1071). The action
of NBCe1 also could contribute towards the severity of
the hypertrophy in myocytes, as described for NHE1
(676, 1061). Indeed, overexpression of cardiac NBCe1
may exacerbate reperfusion injury by contributing to
ischemic [Na⫹]i overload (956).
B) Increased transcript and protein abundance in heart following terminal heart failure. Cardiac NBCe1 transcript
and protein levels are both elevated in preparations from
individuals that suffered terminal heart failure, although
whether this is a cause or consequence of heart failure has
yet to be established (481).46
C) Increased protein abundance in heart by chronic hypercapnia. In neonatal, but not adult mice, chronic (2 wk)
exposure to 12% CO2 causes NBCe1 protein abundance to
increase by ⬃40% in heart, reflecting a general pattern of
46
The authors of another study report a significant difference in
NBCe1 mRNA expression levels between normal and failing hearts
based on microarray data, but do not report the direction of the
change (649). Transfecting cultured rat cardiomyocytes with an
adenoviral vector that is designed to overexpress NBCe1 “modified
the beating rate” and “lowered the viability,” although no confirmation of NBCe1 overexpression or primary data were provided (649).
Mice genetically modified with an NBCe1 transgene (to mimic
NBCe1 overexpression) exhibited no cardiac detectable or bloodpressure phenotype, although neither the identity of the splice variant nor confirmation of NBCe1 overexpression was provided (649).
864
increased abundance of acid extruders (e.g., NBCn1 and
NHE1), which may help to counter the acidifying effects of
hypercapnia (463). Also in the kidney, hypercapnia increases NBCe1 protein abundance.
D) Potential stimulation of NBCe1 in response to ethanolinduced acidosis. The application of 30 –1,000 mM ethanol
to human atrial cardiac myocytes causes a graded fall in pHi
and a modest stimulation of an unidentified HCO3⫺-dependent acid extruder (979), likely NBCe1. Note that even 30
mM ethanol is about twice the legal limit for alcohol intoxication in many jurisdictions. Moreover, the study did not
take into consideration either the osmolality or reflection
coefficient of ethanol.
E) Increased activity in cardiac myocytes in response to
acidosis and/or angiotensin II. In cardiac myocytes
(1072), acute intracellular acidosis stimulates an unidentified electrogenic NCBT, likely NBCe1, that contributes
to pHi recovery. In infarcted rat hearts, acidosis increases
the abundance of NBCe1 transcripts and protein in the
left ventricular free wall via a pathway that involves a
local renin-angiotensin system, including angiotensin
converting enzyme, angiotensin II (ANG II), and stimulation of AT2 receptors (831). Stimulation of NCBT activity by 10⫺7 M ANG II via an AT2-dependent pathway
has also been demonstrated in neonatal rat cardiac myocytes in which the stimulation can be mimicked by application of arachadonic acid (503).
Other studies report that the stimulatory effect of 10⫺7 M
ANG II upon NCBT activity in rat (313)and cat cardiac
myocytes is mediated by the AT1 receptor (58, 224), similar
to the stimulation of NBCe1 functional expression in the
proximal tubule. However, in cat cardiac myocytes, the
phenomenon stimulated by ANG II is reported to represent
stimulation of NBCn1 and inhibition of NBCe1.
We note in summary that the study of Sandmann and coworkers (831), in which ANG II stimulates NBCe1, is corroborated by molecular evidence of the increased NBCe1
abundance. On the other hand, the presence of NBCn1 in
cardiac myocytes is not well demonstrated. Without invoking species differences, these studies are not readily reconciled.
III) Musculoskeletal system. A) Increased protein abundance in skeletal muscle following training. NBCe1 protein
abundance is doubled in the soleus muscle (a predominantly
oxidative organ), but not the extensor digitorum longus
muscle (a predominantly glycolytic organ), of rats after 5
wk of interval training on a treadmill (964).
B) Increased protein abundance in skeletal muscle at high
altitude. Human subjects that live at high altitude, or those
who normally live at low altitude but move to high altitude
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An elevation of NBCe1 protein abundance also occurs in
gerbils treated with the GABA degradation inhibitor vigabatrin (466). Two critical issues not addressed in the
aforementioned studies are the identity of the upregulated NBCe1 splice variant (i.e., NBCe1-B versus -C) and
the identity of the cells in which it was upregulated. For
example, in rat hippocampus, NBCe1-C is abundant in
the astrocytes that surround the neuronal cell bodies in
the pyramidal cell layer (624). If the seizure activity leads
to an increase in NHE1 and NBCe1-C activity in astrocytes, that would lower extracellular pH and reduce neuronal excitability (reviewed in Refs. 186, 187, and 898).
However, if the seizure activity leads to an increase in
NHE1 and NBCe1-B in neurons, that would tend to increase neuronal excitability, which would be a maladaptive consequence of an attempt to protect neurons from
acidosis.
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
for 8 wk, have double the abundance of NBCe147 protein in
their skeletal muscle compared with individuals who live at
sea level (457). The authors of the study suggest that this
upregulation of NBCe1 as an acid extruder may reflect a
mechanism that compensates for lower-than-normal arterial HCO3⫺ (at rest), itself a compensation for the respiratory alkalosis produced by hyperventilation, measured in
these individuals at high altitudes (457). If it turns out that
CO32⫺ is the substrate of NBCe1 (p. 56), then a critical
question is whether the combination of the uncompensated
alkalosis (i.e., high pHo) and compensatory low [HCO3⫺]o
results in a depressed [CO32⫺]o.
B) Increased transcript abundance during dark periods. Investigators studying the circadian rhythm of transcript
abundance in mouse molars determined that NBCe1 transcript abundance is nearly doubled during dark periods
compared with light periods (537). The authors suggest that
this observation correlates with periods of enhanced enamel
deposition by ameloblasts (537).
V) Lower digestive system. A) Increased protein abundance
in the plasma membrane of colonic mucosa by secretagogues. The application of forskolin, which raises [cAMP]i,
stimulates colonic HCO3⫺ secretion in vivo, without increasing NBCe1 transcript abundance (55), by enhancing
the accumulation of NBCe1-B protein in the plasma membrane (1087). In contrast, forskolin inhibits heterologously
expressed NBCe1-B activity in a renal cell line (54).
VI) Urinary system. A) Increased transcript abundance by
chronic dexamethasone treatment. Glucocorticoid excess
causes a metabolic alkalosis associated with enhanced renal
47
In this report, the authors state that the antibody used in this
study (no. 3212; Chemicon) does not discriminate between NBCe1,
NBCe2, or NBCn1. The antibody was raised against an 54-amino
acid epitope in the soluble Nt domain of rat NBCe1 and appears, as
evidenced by lack of immunoreactivity with the mTAL in rat kidney
section (844), to be at least unreactive towards NBCn1. A later
paper by the same group demonstrated that this antibody and an
NBCe2-specific antibody recognize proteins of different molecular
weights in rat muscle preparations, indicating that this antibody is
likely to be specific for NBCe1.
B) Increased transcript and protein abundance following
birth. In mice, NBCe1 transcript and protein abundance
increases in the PT following birth (in this study, day 3 to
day 18), coordinated with the upregulation of other renal
ion transporters, such as NHE3, and concomitant with a
drop in urinary pH over the same time period (97).
C) Increased transcript and protein abundance following
renal transplant rejection. NBCe1 transcripts and protein
levels are increased in PTs of transplanted rat kidneys following acute rejection (999). The significance of these findings is presently unclear.
D) Increased transcript abundance in Aadc-null mice. Mice
with a PT-specific deletion of aromatic amino acid decarboxylase (AADC) exhibit elevated NBCe1 mRNA abundance (1094). Because AADC catalyzes the final step in
dopamine synthesis, and because dopamine reduces Na/
HCO3 cotransport activity, these observations suggest that
it is intrarenal dopamine-signaling pathways that normally
limit NBCe1 abundance.
E) Increased transcript abundance and stimulation of activity in K⫹-deprived rats. Increased NBCe1 transcript abundance and elevated NCBT activity has been described in the
PT and medullary thick ascending limb (mTAL) of K⫹deprived (KD) rats. Upregulation of HCO3⫺ reabsorption by
these tubule segments has been implicated in the pathogenesis of whole-body alkalosis in KD animals (38, 894). For
the mTAL, this hypothesis requires that the NBCe1, the
splice variant of which is unknown, be basolateral and operate in the HCO3⫺-outward direction.
The medulla is not usually associated with NBCe1 expression (38); mTAL epithelia normally only express the relatively DIDS-insensitive NBCn1. In the renal medulla of KD
rats, NBCe1 transcript abundance is increased approximately fourfold, and an unusual DIDS-sensitive NCBT activity is detected in mTAL epithelia (38). A whole-kidney
intracellular acidosis, measured by 31P-NMR, has also been
noted in KD rats (10). To the extent that this “renal” pHi
decrease reflects a fall in the pHi of PT cells, it would be
consistent with an increase in basolateral NBCe1-A activity, which would in turn tend to alkalinize the blood.
F) Increased protein abundance by chronic norepinephrine
treatment. Consistent with a potentially causative role in
norepinephrine-promoted Na⫹ retention, NBCe1 protein
abundance is doubled in the renal cortex of norepinephrineinfused rats, as is the abundance of two other Na⫹ trans-
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IV) Upper digestive system. A) Increased transcript abundance in ameloblast-like cells maintained at acidic pH. LS8
cells are derived from the enamel organ of embryonic mice.
In LS8 cells maintained for 24 h in acidic medium, NBCe1
transcripts are more abundant than in LS8 cells maintained
in an alkaline medium (706, 891). This phenomenon is
controlled by a pH-responsive enhancer region in the
NBCe1-B/C promoter. If the increased abundance of
NBCe1-B transcripts in LS8 cells translates to an increase in
NBCe1 acid-extruding activity (see FIGURE 20), enamelorgan cells exposed to acid should be adapted to 1) defend
pHi from acidosis and 2) secrete HCO3⫺ into acidic enamel
fluid.
HCO3⫺ reabsorption. Consistent with this phenomenon, a
4-day period of dexamethasone treatment results in a doubling of NBCe1 transcript abundance in the renal cortex of
rats (21).
MARK D. PARKER AND WALTER F. BORON
porters, namely, the Na/K/Cl cotransporter 2 (NKCC2) and
NHE3 (899).
G) Increased protein abundance in hypovolemic rats. The observed increase in NBCe1 and NHE3 protein abundance in the
PT epithelia of hypovolemic (i.e., volume depleted) rats might
contribute to the increased urine acidity in these animals (620).
These changes would promote fluid reabsorption and thus be
a reasonable adaptation to hypovolemia.
According to the first report, NBCe1 protein abundance is
doubled in the renal cortex of SHR compared with control
rats (900). With the assumption that this change corresponds to an increase in functional NBCe1 activity, the
result would be increased Na⫹ reabsorption, which would
be expected to contribute toward a hypertensive phenotype.
According to the second report, a study of immortalized PT
epithelia from SHRs, NBCe1 transcript abundance and
NCBT activity are reduced (731).48 These results may seem
counterintuitive because downregulation of NBCe1 would
tend to reduce, not increase, Na⫹ reabsorption. However,
reduction of NBCe1 in these immortalized cells may reflect
an adaptation to hypertension that occurred in the donor
SHR rat.
Possible explanations for the apparent discrepancy between
the two studies include 1) differences between rat tissue and
immortalized cell lines, 2) variability in the genetic basis of
hypertension (e.g., in one case NBCe1 contributes to hypertension whereas in the other it opposes it) between
populations of SHR rats (672), and 3) an increase in
NBCe1 protein abundance may not result in an increase
in NCBT activity.
I) Increased protein abundance in the kidney during chronic
hypercapnia. In adult rats, chronic (10 day) exposure to 8%
CO2, 13% O2 (i.e., hypoxic hypercapnia) results in a near
48
Interestingly, the characteristics the NCBT activity in immortalized SHR cells differ from the NCBT activity in control cells in two
ways (731). In SHR cells, NCBT activity is 1) more sensitive to
stimulation by acidosis and 2) poorly DIDS-sensitive (50% blockade
by 1 mM DIDS). These features are reminiscent of NBCn1 which is
strongly upregulated by acidosis and poorly sensitive to DIDS, leaving
open the possibility that NBCn1 is expressed in these immortalized
SHR cells.
866
J) Increased plasma membrane abundance in the kidney in
response to ANG II. In the proximal tubule, low doses of
ANG II stimulate reabsorption of HCO3⫺ (JHCO3) and Na⫹
(e.g., see Refs. 367, 394, and 1106). A 15-min application
of 10⫺10 M ANG II to a polarized monolayer of immortalized renal epithelial cells from opossums (OK cells) increases the basolateral plasma membrane abundance of
NBCe1 (802). The enhancement of NBCe1 functional expression by ANG II in these cells is blocked by antagonists
of the AT1 receptor, blockers of Src family tyrosine kinases,
and blockers of the mitogen-associated protein kinase
(MAPK) signaling pathway (802).49 Furthermore, the ANG
II–induced increase in JHCO3 is absent in AT1A receptor-null
mice (394, 1100). As shown in studies of perfused tubules,
and as modeled in Xenopus oocytes, the effects of ANG II
are biphasic; low concentrations (10⫺10 and 10⫺11 M) of
ANG II are stimulatory to NBCe1 functional expression,
whereas higher concentrations are inhibitory (367, 394,
738, 739).
In isolated perfused rabbit proximal tubules, acute isolated
increases in basolateral [CO2] or isolated decreases in basolateral [HCO3⫺] cause an increase in JHCO3 (1108). This
response requires the secretion of local ANG II into the
tubule lumen (1104) and is blocked by inhibition or knockout of luminal AT1 receptors (1107).
Note that agonism of the M1 muscarinic receptor, another
G protein-coupled receptor, is associated with an increase
in Na⫹ and HCO3⫺ reabsorption by the PT (823). The action of the non-receptor tyrosine kinase Pyk2 appears to be
a common factor in the costimulation of NBCe1 and NHE3
activity by GPCR agonists and by acidosis (277, 582).
ANG II also enhances functional expression of NBCe1 in
the heart via an AT2-dependent pathway that appears to
share commonality with the pathway that upregulates
NBCe1 in response to acidosis (see p. 866).
49
Src and MAPK phosphorylation are also implicated in a PPAR␥associated pathway that stimulates NBCe1 functional expression in
response to thiazolidinedione treatment.
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H) Increased protein abundance in some spontaneously hypertensive rats. Dopamine normally reduces Na/HCO3
cotransport activity in the proximal tubules of rabbits and
rats (524). In a particular strain of spontaneously hypertensive rats (SHRs), an undetermined defect in the DA1 dopamine receptor leaves presumed NBCe1 activity unresponsive to downregulation by dopamine (524), thereby limiting
the ability of the PT to reduce Na⫹ reabsorption (405). Two
other reports that directly address perturbation of NBCe1
in SHRs appear to differ in their findings.
doubling of NBCe1 protein abundance in the PT (226).
This observation is consistent with the findings of an earlier
study of cultured rat proximal tubules cells in which stimulation of Na/HCO3 cotransport activity by respiratory acidosis was prevented by treatment with inhibitors of protein
synthesis (824). In neonatal, but not adult mice, chronic (2
wk) exposure to 12% CO2 causes NBCe1 protein abundance to increase by ⬃20% in kidney (463). These responses reflect a general pattern of increased abundance of
acid extruders (e.g., NBCn1 and NHE1), which may help to
counter the acidifying effects of hypercapnia (463). Hypercapnia also increases NBCe1 protein abundance in the heart
(p. 877).
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
K) Stimulation of activity in response to acidosis. In the
renal cortex of rats, NBCe1-A transcript (140) and protein
(36, 484, 530) levels are unperturbed by NH4Cl-induced
acidosis. However, consistent with a model in which existing NBCe1 protein is activated, NBCe1 activity is increased
in acidotic rats (757) and rabbits (15), isolated basolateral
membrane vesicles prepared from suspensions of rabbit PTs
subjected to metabolic acidosis (895), and immortalized rat
PT epithelia treated with NH4Cl (731).
Transcript abundance of an unidentified NBCe1 variant in
an inner medullary collecting duct (IMCD) cell line is decreased by acid stress (1029); however, the IMCD is not a
site of substantial NBCe1 expression. Acidosis also stimulates NBCe1 activity in the heart.
XIII) Stimulation of functional expression in PT by thiazolidinediones. Drugs such as pioglitazone (PGZ) and rosiglitazone (RGZ), agonists of peroxisome proliferator-activated receptor gamma (PPAR␥), are used to increase insulin
sensitivity in patients with type II diabetes. Thiazolidinedione (TZD) use is associated with an expansion of plasma
volume that is hypothesized to be due to increased renal
solute reabsorption (272). Endo and co-workers (272) report that PGZ and RGZ stimulate basolateral HCO3⫺ transport in rabbit PTs via a PPAR␥-dependent pathway that
also increases the phosphorylation of Src family kinases and
MAPK. Although the mechanism of increased basolateral
HCO3⫺ transport in response to TZD treatment is untested
in this instance, other studies link the activation of Src and
MAPK pathways with an increase in the plasma membrane
abundance of NBCe1 in the PT (see p. 868).
I) CAUSES OF NBCe1 DOWNREGULATION. In this section we consider disturbances that result in downregulation of NBCe1
either at the level of transcript abundance, protein abun-
50
Conversely, NaCl feeding, a maneuver that would tend to lower
the glomerular filtration rate, results in a reduction of NBCe1 protein
abundance.
I) Central nervous system. A) Reduced protein abundance
in brain in response to intermittent hypoxia. Chronic intermittent hypoxia (CIH), a model for sleep apnea, appeared
to decrease NBCe1 protein abundance in the cerebellum as
assessed with an antibody that should not discriminate
among NBCe1 variants (261). On the other hand, antibodies specific for NBCe1-A/B and NBCe1-C did not reveal
statistically significant changes in the cerebellum.
II) Circulatory system. A) Potentially reduced activity in
cardiac myocytes in response to angiotensin II. Although
studies from two groups of investigators are consistent with
upregulation of NBCe1 in rat cardiac myocytes by ANG II
(via an AT2-dependent pathway), studies by a third group
are consistent with downregulation of an NBCe1-like activity by the same concentration of ANG II in cat cardiac
myocytes (via an AT1-dependent pathway). These observations are not readily reconciled, but could be explained by
species differences.
III) Upper digestive system. A) Reduced transcript abundance in ameloblast-like cells maintained at alkaline pH.
NBCe1 transcripts are less abundant in LS8 cells (derived
from the enamel organ of embryonic mice) that are maintained for 24 h in alkaline medium (891). This adaptation is
the counterpart to the upregulation of NBCe1 in response
to decreased extracellular pH, which is proposed to support
ameloblast function.
IV) Lower digestive system. A) Downregulation in jejunum
by chronic gamma-irradiation. Diarrhea such as that associated with radiation exposure follows a decrease in anion
(and thus fluid) absorption and/or an increase in anion/fluid
secretion. In the case of secretagogue-induced diarrhea, anion secretions are rich in Cl⫺ and HCO3⫺, supported by
upregulation in the gut of NKCC1 (211) and NBCe1. However, in gamma-irradiated mice, increased anion secretion is
effected with a seemingly counterintuitive reduction in
HCO3⫺ secretion (1093). In the jejuna of these mice,
NBCe1-A/B immunoreactivity is lost, reducing support for
HCO3⫺ secretion from jejunal enterocytes (1093). An accompanying increase in NKCC1 immunoreactivity in the
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L) Stimulation of activity in PT by chronic hyperfiltration.
In rats, following removal of a kidney, the remaining kidney
experiences a ⬃50% increase in glomerular filtration rate.
In addition, the PTs from the remnant kidney adapt (2 wk
after surgery) by doubling the functional expression of both
apical Na-H exchange and basolateral Na/HCO3 cotransport (758).50 This adaptation does not appear to be an
acute effect of acidosis inasmuch as blood pH was not different from that of control rats (758). Whether this upregulation of NBCe1 activity is accompanied by an increase in
the abundance of NBCe1 transcripts and/or protein is not
documented in this study. However, in rats in which one
ureter is partially obstructed within 48 h of birth, NBCe1
protein abundance is doubled after 7 wk in both the obstructed kidney and unobstructed kidney (1025).
dance, translocation to the plasma membrane, or transporter activity. Note that a decrease in any one of these
factors need not necessarily correlate with a decrease in the
others. We have omitted cellular studies that report only
indirect evidence of NBCe1 downregulation (e.g., inhibition of HCO3⫺ reabsorption) because such observations
might at least in part be explained by effects on other proteins. We have arranged the reports in the order of the organ
in which each observation was made and then in order of
disturbances that are shown to reduce NBCe1 transcript
abundance, reduce NBCe1 protein abundance, reduce
translocation to the plasma membrane, and reduce NBCe1
activity.
MARK D. PARKER AND WALTER F. BORON
jejuna of irradiated mice suggests that increased Cl⫺ uptake
across the basolateral membrane may compensate for decreased anion secretion support from NBCe1 insufficiency,
producing Cl⫺–rich secretions (1093).
C) Downregulation by microRNAs in colon carcinoma
cells. In the human colon carcinoma cell line HT29 (642),
the microRNA miR-224 is under-represented. The 3=-UTR
of NBCe1 transcripts is a potential target of miR-224 (642).
Consistent with this hypothesis, NBCe1 transcript abundance are unusually abundant in HT29 cells.
V) Endocrine system. A) Decreased transcript abundance in
thyroid cancer. Two studies report a greater than threefold
reduction in NBCe1 transcript abundance in papillary thyroid carcinoma compared with normal thyroid tissue isolated from the same patients (309, 486). With the assumption that this downregulation correlates with a decrease in
functional expression, and that in these cancer cells NBCe1
operates in the HCO3⫺-inward direction, these changes,
which would tend to lower pHi, might be predicted to harm
the cancer cell rather than encourage tumor proliferation
(see below for a discussion of the reported downregulation
of NBCn1 in breast cancer).
VI) Urinary system. A) Decreased transcript and protein
abundance and reduction of activity in kidneys of Na⫹loaded and alkalotic animals. In the PT epithelia of rats
whose drinking water is spiked with NaHCO3 or NaCl
(thereby producing a Na⫹ load that could make the animal
hypervolemic), NBCe1 transcript (140) and protein (36,
620) abundance is reduced. These adaptations would tend
to reduce Na⫹ reabsorption and thus tend to oppose the
development of hypertension. The adaptations would also
tend to reduce HCO3⫺ reabsorption, which in the case of
NaCl feeding might render the animals less able to respond
to an acute acid load. In the case of NaHCO3 feeding, the
adaption would oppose whole body alkalosis (37). In rabbits, Cl⫺-depletion alkalosis (CDA), which presumably re-
868
B) Decreased transcript abundance and reduction of activity in PT of some spontaneously hypertensive rats. As discussed and contrasted above, although some spontaneously
hypertensive rats exhibit increased NBCe1 protein abundance, authors of a separate study report reduced NBCe1
transcript abundance and reduced NBCe1 activity in another population of SHRs. Inasmuch as reduced NBCe1
activity would tend to counter hypertension by reducing
Na⫹ reabsorption, these reductions are more likely to be a
consequence than a cause of hypertension in these animals.
C) Reduced protein abundance in response to hypoxia. An
immunohistochemical study of mouse kidney slices appears
to show, although the authors of that study do not specifically comment on this phenomenon, a reduced abundance
of NBCe1 protein in the basolateral membranes of PT epithelia in slices that are briefly (10 s to 2 min) exposed to
hypoxia prior to cryofixation (829).
D) Decreased protein abundance in kidney following ureteral obstruction. Following a 24-h bilateral ureteral occlusion in rats, NBCe1 and NHE3 protein abundance is substantially decreased in the PTs (1024). This downregulation
may partly explain the phenomenon of obstruction-induced
renal tubular acidosis. In rats in which only one ureter is
partially obstructed within 48 h of birth, NBCe1 protein
abundance is doubled after 7 wk in both the obstructed
kidney and unobstructed kidney (1025). On the other hand,
after 14 wk, the obstructed kidney exhibits a ⬃40% decrease in NBCe1 protein abundance, whereas the abundance of NBCe1 in the unobstructed kidney appears to be
close to normal (1025).
E) Decreased protein abundance in kidney during gentamycin-induced nephropathy. Treatment of bacterial infections
with gentamycin can cause PT dysfunction in humans (reviewed in Ref. 308). In rats treated with gentamycin for 7
days, renal NBCe1 protein abundance is decreased by half
(57). The abundance of other renal Na⫹ transporters,
namely, NHE3 and the Na-K pump, and the water/carbon
dioxide channel AQP1 are also reduced under these conditions (57).
J) CONSEQUENCES OF NBCe1 DYSFUNCTION. In this section we
consider the pathologies that are associated with genetic
ablation of, genetic alterations in, and dysfunction of
NBCe1 in the brain, eye, heart, enamel organ, lower diges-
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B) Suggested reduction of activity in pancreas by elevated
glucose levels. In isolated, perfused pancreatic ducts, high
levels of luminal glucose lead to an accumulation of Na⫹
and membrane depolarization, via the action of the apical
Na/glucose cotransporter SGLT1, in duct cells (307). The
authors of the study suggest that elevated [Na⫹]i inhibits
NBCe1-mediated influx of Na⫹ and HCO3⫺ at the basolateral membrane, thereby contributing to the decreased pancreatic HCO3⫺ secretion associated with diabetes. On the
other hand, the authors suggest that the depolarization, via
an inhibitory effect upon CFTR, could be a more important
factor in reducing HCO3⫺ secretion. Depolarization would
be expected to enhance NBCe1 activity. This model requires that elevated serum [glucose] results in an elevated
luminal [glucose] via an undetermined transcellular glucose
transport pathway.
sults in volume contraction, causes a fall of NBCe1 activity
in basolateral renal cortical membrane preparations (15).
On the other hand, in rats, a similar stress has no effect on
NBCe1 transcript levels in the PT (140). If these two observations can be taken together, they are consistent with the
hypotheses that 1) the regulation of NBCe1 activity by alkalosis is purely posttranslational or 2) the response to
CDA is species specific.
⫺
TRANSPORTERS
Na⫹-COUPLED HCO3
tive system, and kidney. The primary syndrome associated
with NBCe1 dysfunction is proximal renal tubular acidosis.
These data are mostly obtained from clinical studies of individuals with defects in the SLC4A4 gene, genetic linkage
studies, and studies of genetically altered mice.
I) Central nervous system. A) Mental retardation, migraine,
and epilepsy. Many individuals with defects in the SLC4A4
gene present with mental retardation (see TABLE 6). The presence of this trait in individuals with the Q29X mutation,
which is predicted to abrogate renal (i.e., NBCe1-A) but not
neuronal or glial NBCe1 expression (i.e., NBCe1-B and -C),
suggests that mental retardation can be secondary to whole
body acidosis that is the signature of pRTA.
In a separate study, genetic-linkage analysis mapped a familial temporal lobe epilepsy (including “déjà vu” auras) to
a chromosomal locus that includes SLC4A4, although sequencing of SLC4A4 exons in two affected individuals did
not reveal any genetic abnormalities (374). However, the
possibility of a linkage remains open as the promoter regions of SLC4A4 were not included in this analysis and it is
not disclosed whether the exons of all NBCe1 variants were
sequenced.
II) Sensory organs. A) Glaucoma, band keratopathy, cataracts, and corneal edema. Many individuals presenting with
pRTA have ocular defects ranging from glaucoma and band
keratopathy to total blindness (TABLE 6). The widespread
expression of NBCe1 throughout the eye as well as the
absence of certain ocular phenotypes in some individuals
with pRTA indicate that ocular phenotypes need not necessarily be secondary to whole body acidosis.
In these patients, it is not clear what causes the high-tension
glaucoma, a buildup of aqueous humor that causes an increase in intraocular pressure. The two major forms of glaucoma in the general population are due to decreased disposal of the aqueous humor, ultimately via the trabecular
meshwork in the anterior chamber. As noted earlier,
NBCe1 has been detected in the trabecular meshwork
(989), but the role of NBCe1 in this tissue is untested. Glaucoma is not a feature associated with pRTA in individuals
with either the T485S or G486R mutations (TABLE 6).
The pathogenesis of cataracts in some individuals with
SLC4A4 defects could be caused by defective Na⫹ transport
or pHi regulation in lens epithelial cells. Perturbations of
either process are associated with increased lens opacity
Band keratopathy, corneal opacity caused by the deposition
of Ca2⫹ salt can be secondary to renal failure (briefly reviewed in Ref. 149). In one case, a 12-yr-old girl with a
defect only in (renal) NBCe1-A variant did not have band
keratopathy (412), consistent with variable penetrance or
with the hypothesis that it is specifically a defect in
NBCe1-B and -C in the corneal endothelium that causes the
band keratopathy. If HCO3⫺ secretion across the corneal
endothelium and into the aqueous humor were compromised, a localized elevation of [HCO3⫺] in the subepithelial
region of the corneal stroma might be expected to enhance
the deposition of Ca2⫹ salts (928, 989). Band keratopathy is
not a feature associated with pRTA in individuals with the
Q29X or R881C mutations (TABLE 6).
The eyes of NBCe1-null mice appear normal. These mice
die at 4 wk (313) before any ocular defects are evident.
However, in a line of transgenic mice that express the
mouse ortholog of the human NBCe1/W516X pRTA mutant (FIGURE 25 and TABLE 6), corneal opacity and edema
are evident when these mice are kept alive beyond week 7 by
administration of HCO3⫺ (602).
III) Peripheral nervous system. We are unaware of any
reports of peripheral nervous system dysfunction associated
with NBCe1 mutations.
IV) Circulatory system. A) Possible contribution to ischemic and reperfusion injury in the heart. As discussed earlier, humans with cerebral artery occlusion, abdominal
aorta constriction, or heart failure all appear to have elevated NBCe1 abundance. It is undemonstrated whether
ischemic and reperfusion injuries are a cause or consequence of NBCe1 upregulation, but it is possible that elevated NBCe1 levels could increase the risk factor for ischemic injury and heart failure. Indeed, Giffard and co-workers (318) observed that overexpressing NBCe1-B in the 3T3
fibroblast cell line renders these cells susceptible to acid
injury in the presence of HCO3⫺, perhaps via Na⫹-loading
and thence Ca2⫹-loading via a Na-Ca exchanger (318).
Many studies have suggested that NBCe1 inhibitors could
be cardioprotective (225, 481, 840).
V) Musculoskeletal system. A) Growth retardation. Individuals with defects in NBCe1 typically exhibit below average height and weight (see TABLE 6), and mouse models of
NBCe1-associated pRTA exhibit bone dysplasia (313, 602)
and reduced muscle mass (602). NBCe1 is not known to be
directly involved in bone remodeling, and it is possible that
these signs are secondary to the whole body acidosis that
accompanies pRTA.
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Migraine is a symptom associated with many different
NBCe1 mutations that cause pRTA (930), although the
precise molecular mechanism underlying this pathology is
unknown. At least in one case of two sisters, it appears that
a 65-bp deletion affecting the Ct of all NBCe1 variants is
also associated with epilepsy (72, 930).
(65, 240, 732). Cataracts are not a feature associated with
pRTA in the individual with the Q29X mutation that is
specific to NBCe1-A (TABLE 6).
MARK D. PARKER AND WALTER F. BORON
Table 6. SLC4A4 mutations in individuals with proximal renal tubular acidosis
Label in
Figure 25
Trivial Name and
Original Report
Predicted Protein
Producta
DNA
b
Likely Molecular Basis for
pRTA
Q29X (412)
p.Gln29X
c.85C⬎T
Protein not translated
(929)
2
R298S (411)
p.Arg298Ser
c.894A⬎C
3
S427L (253)
p.Ser427Leu
c.1280C⬎T
Partial mistargeting to
apical membrane with
some cytosolic
retention (929)d
combined with a
approximately 25%
reduction in permolecule function
(166).
Partial mistargeting to
apical membrane
(577) combined with
a likely reduction in
per-molecule function
(253, 577).
4
T485S (393)
p.Thr485Ser
c.1453A⬎T
5
G486R (929)
p.Gly486Arg
c.1456G⬎A
6
R510H (411, 879)
p.Arg510His
c.1529G⬎A
7
W516X (602)
p.Trp516X
Not reportedg
Complete loss of protein
(602)
8
L522P (241)
p.Leu522Pro
c.1565T⬎C
Intracellular retention of
mutant (241, 929,
930)
Mutant traffics normally
thus loss of function is
likely explained by
impaired per-molecule
activity (393, 576,
929, 930).
Mutant traffics
normally;e thus loss of
function is likely
explained by impaired
per-molecule activity
(929).
Intracellular retention of
mutant (577, 930).
Additional
per-molecule activity
defects not reported.f
Mental retardation,
growth retardation,
glaucoma. No evidence
of cataracts or band
keratopathy.
Mental retardation,
growth retardation,
glaucoma, cataracts,
and band keratopathy.
Elevated serum
amylase. Calcification of
basal ganglia (414).
Growth retardation,
glaucoma, and
cataracts. Poor
dentition. Normal
intelligence. No specific
mention of band
keratopathy, but
corneal clouding was
evident. Also some
evidence of respiratory
acidosis (i.e., elevated
Pco2).
Growth retardation,
cataracts, and band
keratopathy. No specific
mention of mental
retardation or
glaucoma.
Growth retardation,
cataracts, and band
keratopathy. Normal
intelligence and no
glaucoma.
Growth retardation,
glaucoma, cataracts,
and band keratopathy.
Delayed neurological
and motor
development. No
mention of mental
retardation. Migraines
(930).
Growth retardation,
glaucoma, cataracts,
and band keratopathy.
Calcification of basal
ganglia. No mention of
mental retardation.
Motor and mental
retardation, growth
retardation, glaucoma,
cataracts, and band
keratopathy. Dental
abnormalities.
Migraines.
Continued
870
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1
Pathological Features From
Original Report
(Other Than pRTA)c
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
Table 6.—Continued
Label in
Figure 25
Trivial Name and
Original Report
Predicted Protein
Producta
DNA
Likely Molecular Basis for
pRTA
b
p.Asn721ThrfsX30i
c.2162delA
Complete loss of protein
(416)
10
A799V (393)
p.Ala799Val
c.2396C⬎T
11
R881C (393)
p.Arg881Cys
c.2641C⬎T
Intracellular
accumulation of
mutant, reduced
per-molecule function,
⫺
and a HCO3
independent
conductance (721).
Intracellular retention of
protein (576, 930,
977, 1113). Protein
has close to normal
per-molecule activity
(977).
12
⌬65bp (413, 930)
p.Ser982AsnfsX4k
c.2944_2967 ⫹
42dell
Intracellular retention of
mutant (930), but
mutant protein
functions normally
when expressed in
oocytes (930).
Growth retardation,
cataracts, band
keratopathy. Dental
abnormalities.
Calcification of basal
ganglia. Normal
intelligence. No
glaucoma, but elevated
ocular pressure.
Elevated serum lipase
and amylase.j
Motor and mental
retardation, growth
retardation, low weight,
glaucoma, cataracts,
band keratopathy, and
calcification of basal
ganglia (231).
Growth retardation,
glaucoma, cataracts.
No specific mention of
mental retardation or
band keratopathy.
Elevated serum
amylase. Migraines
(930).
Glaucoma, cataracts, and
band keratopathy.
Migraines. Normal
intelligence and stature.
Epilepsy. Nausea.
A database of human SLC4A4 mutations is curated at the Leiden Open Variation Database (https://
grenada.lumc.nl/LOVD2/shared1/home.php?select_db⫽SLC4A4). The position of the mutated residues
within NBCe1 is depicted in Figure 25 and shown on sequence alignments in Appendix I. aBased on
NP_003750. bBased on NM_003759.3 (“A” of initiating ATG codon is counted as nucleotide 1). cFeatures not
described in the original report are provided together with a reference to the paper in which the feature was
described. dReports conflict as to whether the equivalent mutation in NBCe1-B (p.Arg324Ser) causes the
protein to be mistargeted. The mutant is reported to accumulate normally in the plasma membrane of a
human-bladder-endothelium cell line (836) and a rat glioma cell line (930) but to be retained in the cytosol of
a canine-kidney-epithelium cell line (577). Interestingly, an artifical mutant—R298C— created in a version of
NBCe1-A that lacks the five endogenous, cytoplasmic cysteine residues traffics efficiently to the plasma
membrane in a human-kidney-epithelium cell line (1113). eIn a rat glioma cell line, the equivalent mutant of
NBCe1-B (p.G530R) appears to have an increased intracellular presence compared with the wild-type transporter (930). fOne study finds that the equivalent mutation in NBCe1-B (p.Arg554His) exhibits a loss of function
but not reduced accumulation of transport protein in the plasma membrane of human endothelial cell line
(836). gLikely c.1547G⬎A or c.1548G⬎A. hThis designation counts the first base of the 5=-UTR as nucleotide
1. iFirst affected amino acid is Asn721, which changed to Thr. Thr becomes residue #1 of the frame shifted
reading frame (fs) that has a termination codon at position #30. The unique 29-amino acid appendage is
predicted to have the sequence TEVGSFHRLEKTPGGCALLLLSRLCWSLY. jThe authors mention only elevated
lipase in their clinical description of the patient, but refer also to elevated amylase in the discussion of their
findings. kThe unique 3-amino acid appendage is NKF (930). l2944 –2967 of the exon 23 are missing plus 42
of the following intron.
B) Hypokalemic paralysis. An individual with the A799V mutation in NBCe1 exhibited hypokalemic paraplegia (231). It is
notable that the mutant NBCe1, when expressed in Xenopus
oocytes exhibits a HCO3⫺-independent ion leak (721). Such a
leak could in principle contribute towards the observed paralysis, as has been suggested in the case of unusual ion leaks
though Na⫹ and Ca2⫹ channels (293, 1047). Under hypokalemic conditions, the conductance of inward-rectifier K⫹
channels in muscle cells is reduced, destabilizing their membrane potential. In this situation, small pathological currents
can make a disproportionately large contribution to resting
Vm, and the prolonged depolarization of the cells that results,
inactivates voltage-gated Na⫹ channels (820).
VI) Upper digestive system. A) Compromised enamel deposition. The teeth of humans with a mutant SLC4A4 gene
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nt2311h (416)
9
Pathological Features From
Original Report
(Other Than pRTA)c
MARK D. PARKER AND WALTER F. BORON
NBCe1-A
EL3
9
6
7
8
1
2
4/5
3
4
Lumen
5
6
7
8
9 10
10
11
E 13
14
3
Cytosol
11
2
Ct
Nt
H2N
12
1
FIGURE 25. Location of disease-associated mutations in NBCe1-A. Representation of human NBCe1-A
topology from FIGURE 2A. Numbered circles show the positions of SLC4A4 mutations (numbers 1–12 match
the numbered mutant descriptions in TABLE 6) that cause proximal renal tubular acidosis. Nonsense
mutations that result in premature translational termination are colored red, and missense mutations are
colored green.
(TABLE 6),
and of mice with a disrupted Slc4a4 gene (313,
538), exhibit signs of defective enamel deposition. NBCe1 is
expressed in the enamel organ (456, 538, 706), and CO32⫺ is
an important constituent of enamel (reviewed in Ref. 564).
However, the precise role of NBCe1 in the process of
enamel remodeling (see FIGURE 20) has yet to be elucidated.
It is possible that the defect is not secondary to whole body
acidosis, inasmuch as unusual dentition was not noted in
the description of a 12-yr-old girl who was predicted to lack
only the renal variant of NBCe1 and unaffected ameloblastic NBCe1 (412). Furthermore, the defect is unlikely to be
secondary to a reduced salivary pH because, in mice, the
defect is noted in preerupted teeth (538). The likely importance of NBCe1 for correct enamel deposition was recently
reviewed by Urzúa et al. (988).
VII) Lower digestive system. A) Intestinal obstruction. Although individuals with SLC4A4 defects are not reported to
have intestinal problems, genetic linkage analysis has indicated that NBCe1 could contribute to the severity of ileal
obstruction in newborns with cystic fibrosis (257). Indeed,
NBCe1-null mice have small ceca and those that survive
beyond 20 days have impacted terminal ilea, ceca, and colons (313), a phenomena also reported in the W516X
mouse model of pRTA (602). However, the defective net
ion secretion that is observed in isolated colons from
NBCe1-null mice could be explained by increased fluid absorption due to dysregulation of other ion transporters such
as ENaC and NKCC, rather than by decreased fluid secretion due to a lack of NBCe1 per se (313).
B) Possible signs of pancreatitis. The abundance of NBCe1-B
in the pancreas suggests that this protein plays a major role
872
in HCO3⫺, and consequently fluid, secretion by this organ.
Dysfunction of NBCe1-B, like dysfunction of CFTR (e.g.,
Ref. 845), might therefore result in pancreatitis. Although
some individuals with NBCe1-associated pRTA exhibit
molecular signs of pancreas dysfunction, such as elevated
serum amylase and lipase (TABLE 6), clinical signs of pancreatitis have not been reported. NBCe1-null mice have a
normal pancreatic histopathology, although, as the authors
of that study note, these young mice51 may have been examined prior to development of pancreatic pathology. In
addition, the mouse pancreas is known to be a poor model
for human pancreatic insufficiency (313). Taken together,
these observations suggest that development of pancreatitis
due to NBCe1 dysfunction may be age-dependent, or prevented by an as-yet-unidentified mechanism. For example,
NBCn1 could compensate for NBCe1 loss in the basolateral
membranes of exocrine duct cells (FIGURE 21).
C) Potential contribution to diarrhea. In the colon, NBCe1
action could support HCO3⫺ secretion and thereby promote
Cl⫺ absorption via the apical Cl-HCO3 exchanger Slc26a3
(DRA, see Ref. 388) and the basolateral chloride channel
ClC-2 (see Ref. 159). If this hypothesis is correct, then
NBCe1 dysfunction is expected to reduce fluid absorption
and promote diarrhea. However, the colons of NBCe1-null
mice do not exhibit fluid absorption defects perhaps due to
the documented dysregulation of other ion transporters in
the colons of these mice (313).
51
NBCe1 null-mice rarely survive beyond weaning age (313).
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HOOC
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
VIII) Lymphatic and immune systems. Mice with a disrupted Slc4a4 gene exhibit severe splenomegaly with an
increased count of nucleated red blood cells (313, 602).
However, the spleen is not a major site of NBCe1 expression, and both phenomena are considered to be secondary
to whole body acidosis (313, 602).
B) Hypertension. Two independent genome-wide association studies reported in Reference 1077 link the SLC4A4
gene locus with hypertension in individuals from China.
However, the precise location of hypertension-associated
markers within SLC4A4 is not stated.
Mice that lack aromatic amino acid decarboxylase (AADC) in
their PTs develop salt-sensitive hypertension. The kidneys of
these mice exhibit elevated mRNA abundance of a number of
transporters involved in Na⫹ reabsorption, including NBCe1
mRNA (1094). However, because of the complex effects of
AADC disruption, the specific contribution of NBCe1 to hypertension in these mice is difficult to assess. It is noteworthy
that one strain of spontaneously hypertensive rat exhibits elevated renal NBCe1 abundance (900).
X) Reproductive system. A) Expected, but not demonstrated, reduction in fertility. Based on the putative role of
NBCe1 in the reproductive tracts of males (i.e., maintenance and activation of sperm) and females (i.e., enhancing
sperm fertilizing capacity), it is possible that NBCe1 defects
could be associated with a loss of fertility. However, this
remains unverified as Slc4a4-null mice do not survive to
2. NBCe2 (Slc4a5)
A) SUMMARY. The electrogenic Na/HCO3 cotransporter
NBCe2 (encoded by the Slc4a5 gene) is present in many
organ systems throughout the body but is notably abundant
in the choroid plexus, where NBCe2 contributes towards
HCO3⫺/fluid (i.e., CSF) secretion into the brain ventricles,
and the liver, where robust expression of NBCe2 likely
maintains hepatocyte pHi.
Consistent with its proposed role in the choroid plexus, one
strain of Slc4a5-null mouse exhibits a CSF secretion defect
that likely contributes to the reduced neuronal excitability
observed in these animals. In humans, multiple studies report genetic linkage between the SLC4A5 locus and blood
pressure traits. Although the physiopathology underlying
this linkage is presently unclear, Slc4a5-null mice exhibit
elevated blood pressure. Little is known about the regulation of the Slc4a5 gene or products and is the only one of the
five NCBTs that has not been demonstrated to be stimulated by the cytosolic protein IRBIT. NBCe2 has only one
variant, NBCe2-c, that is known to be functional.
B) NOMENCLATURE OF Slc4a5 PRODUCTS. The Slc4a5 gene product was initially called NBC4 (767), being the fourth member of the gene family to be identified at the molecular level.
The gene product has since been renamed NBCe2 (1009) to
reflect its characterization as the second electrogenic member of the family (835, 1009).
Six variant products have been reported (NBC4a-f), but
only two (NBC4a and NBC4c) seem likely to produce a
functional transporter. We provisionally refer to these as
NBCe2-a and NBCe2-c (note the lowercase “a” and “c”).
C) MOLECULAR ACTION OF NBCe2. Overexpressed in HEK-293T
or mPCT renal cells (835) and Xenopus oocytes (1009),
NBCe2-c mediates a reversible, DIDS-sensitive, and Na⫹dependent HCO3⫺ transport that is accompanied by a Na⫹and HCO3⫺-dependent conductance. Thus NBCe2 is an
electrogenic Na/HCO3 cotransporter (FIGURE 16). Furthermore, in oocytes, NBCe2-mediated HCO3⫺ efflux does not
require extracellular Cl⫺ and thus NBCe2 is not a Na⫹driven Cl-HCO3 exchanger (1009). In mPCT cells (a mouse
PT cell line) that are overexpressing NBCe2 (835), and in
mouse choroid plexus epithelia (645), NBCe2 operates with
a Na⫹:HCO3⫺ stoichiometry of 1:3. However, when heterologously expressed in oocytes (1009) or HEK-293 cells
(869), NBCe2 operates with a 1:2 stoichiometry. This celldependent stoichiometry is also characteristic of NBCe1. As
well as being blocked by DIDS, NBCe2 is inhibited by the
NBCe1 blocker tenidap, as demonstrated for NBCe2 expressed in HEK-293 cells (869).
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IX) Urinary system. A) Proximal renal tubular acidosis.
Twelve homozygous mutations have been described in the
SLC4A4 gene in individuals with proximal renal tubular
acidosis (pRTA; see TABLE 6 AND FIGURE 25). As expected,
considering the role of NBCe1 in the kidney, pRTA is characterized by an impaired ability of the PT epithelium to
reabsorb HCO3⫺, leading to a whole body metabolic acidosis. In individuals with defective NBCe1, plasma pH (7.08 –
7.23) and plasma [HCO3⫺] (5.6 –15 mM) are both below the
normal range. A genetic defect specific to NBCe1-A “Q29X”
(FIGURE 25 AND TABLE 6) is predicted to produce no functional NBCe1-A transporter, but not to affect the production or activity of either the NBCe1-B or -C variants (412).
Two further nonsense and nine missense mutations have
also been identified in the SLC4A4 gene in individuals with
pRTA (see TABLE 6). The genetic linkage between defective
NBCe1 and pRTA is further strengthened by the demonstration of whole body acidosis in Slc4a4-null mice, which
die shortly after weaning (313), and in a transgenic mouse
model of pRTA that carries the human NBCe1/W516X
pRTA-associated mutation (602). It is likely that at least
some of the nonrenal sequelae associated with pRTA, such
as retarded growth and mental retardation, may in part be
secondary to the whole-body metabolic acidosis.
breeding age (313) and the fertility of humans with NBCe1associated pRTA remains unreported.
MARK D. PARKER AND WALTER F. BORON
D) THE SLC4A5 GENE. The human NBCe2 gene maps to chromosomal locus 2p13(FIGURE 26A and Ref. 767) and has at least
31 exons that encompass ⬃127 kb of genomic DNA (FIGURE
26B). Reports that the 5=-UTR of NBCe2 includes sequence
from exons shared with its neighboring gene DCTN1 (770),
which encodes the p150GLUED subunit of dynactin, appear to
have been premature. The presently assigned SLC4A5 and
DCTN1 gene boundaries are separated by ⬃15 kb (NCBI
human genome assembly 37 version 1) and at least two promoters for SLC4A5 transcription are located within this intergenic region (916). The initiator codon for all presently known
variants of NBCe2 is located in exon 6 of SLC4A5. One
SLC4A5 promoter regions is located upstream of exon 1, but
transcripts most often exclude exon 1 and start at exon 2, and
promotes robust expression of a reporter gene in a humanlung and a mouse-myoblastoma cell line, but not in a human
embryonic kidney cell line. The second SLC4A5 promoter is
upstream of exon 5 and promotes robust expression of a reporter gene in the lung and kidney cell lines, but not in the
myoblastoma cell line (916).
a/NBC4a and NBCe2-c/NBC4c are predicted to encode a
functional transporter, NBCe2-c being the more abundant of
the two. GenBank protein accession numbers for the variants
discussed in this section are provided in Appendix IV.
I) Sources of variation in coding sequence among NBCe2
variants. Unusually for an NCBT, the Nt and Ct sequences
of NBCe2 are not known to be variant, although in silico
analysis suggests the existence of yet-to-be-reported gene
products. As there are only two validated variants of
NBCe2, there is only one validated source of variation that
distinguishes NBCe2-a from NBCe2-c.
E) STRUCTURAL FEATURES AND VARIANTS OF NBCe2. Of the six
originally reported splice variants of NBCe2 (NBC4a-f)
(768, 770, 835, 1009, 1056), the cDNA sequences of only
four (NBC4a-d) match the human genome. Only NBCe2-
A
52
For example, baboon (XP_003908879), gibbon (XP_003268733),
and orangutan (XP_003775944).
Locus 2p13
20 kb
MTHFD2
B
C
DCTN1
Gene structure
P1
12
SLC4A5
10 kb
P2
3
5
27
6
Transcript variation
P1
P2
M
NBCe2-a
1
2
3
4
5
NBCe2-c
1
2
3
4
5
6
26
6
26
27
28
29
30
*
31
32
28
29
30
*
31
32
M
FIGURE 26. SLC4A5 gene structure and NBCe2 transcript variants. Scale diagrams showing the human
SLC4A5 gene locus together with the position of neighboring genes (A), the position of promoters (P1, P2, and
P3), and the position of exons within SLC4A5 (B). Transcript variants are represented, not to scale, as
numbered boxes joined by a horizontal line (C). Each numbered box represents the inclusion of that exon in the
mature transcript. “//” denotes that both transcripts include exons 6 –26. Exons that include the initiator ATG
codon (“M”) and termination codon (“*”) are marked for each transcript. Colored exons, or parts of exons,
correspond to the protein regions that each encodes, which are identically colored in FIGURE 27. Uncolored
exons, or parts of exons, denote untranslated 5= and 3= sequence. Exons that are connected with a dashed line
are predicted, but not demonstrated, to be included in the mRNA. Not shown are NBC4b and NBC4d that are
unlikely to encode stable/functional transporters, or NBC4e and NBC4f that are cloning artifacts (see text).
874
32
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A) Extension between putative TMs 11 and 13. Transcripts that encode NBCe2-a include exon 27 (FIGURE
26C). The inclusion of the novel exon is predicted to
lengthen, by the 16-amino acid sequence “MGTGGSEFKIQKKLTP,” the predicted extended structure between putative TMs 11 and 13, which includes an intracellular loop (FIGURE 27). This extension, also predicted to be
included in NBCe2 variants from various primates,52 includes
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
Nt
TMD
100 aa
1–5
6–9
Ct
10–14
1,137
NBCe2-a
16
1,121
NBCe2-c
FIGURE 27. NBCe2 protein variants. Scale diagram of protein variants that are encoded by the transcripts
represented in FIGURE 26C. Horizontal bars represent protein sequence laid out from Nt to Ct. Vertical bars
represent position of ␣-helical TMs. Protein cassettes are labeled with a number denoting their size in amino
acids and colored to denote their genetic origin as shown in FIGURE 26C. A color-matched protein sequence
alignment of the variants is provided in Appendix V.
II) Cloned NBCe2 variants that are demonstrated or likely
to exhibit NCBT activity. A) NBCe2-a/NBC4a (NCBT activity untested). NBCe2-a is the longest NBCe2 variant, as it
includes the 16-amino acid splice cassette mentioned above
(FIGURE 27). We regard NBCe2-a as likely to exhibit NCBT
activity because we have no evidence that the 16-amino acid
insertion would disrupt function.
B) NBCe2-c/NBC4c (NCBT activity demonstrated). This
remains the only variant that has been functionally characterized (835, 1009). NBCe2-c lacks the 16-amino acid insertion found in NBCe2-a (FIGURE 27) and is therefore
structurally most similar to other NCBTs within its TMD.
III) Predicted NBCe2 variants. Based on our in silico analysis, the alternative splicing of NBCe2 RNA has the potential to generate a novel protein variant (i.e., not NBC4a–f).
Excision of exon 30 would produce an NBCe2 variant in
which the last 39 amino acid of NBCe2-c are replaced with
an alternative 77-amino acid Ct appendage that, like the
61-amino acid Ct appendage of NBCe1-C, is predicted to
terminate with the class I PDZ-binding domain motif
“ETTL.” However, such transcripts have yet to be amplified from mammalian cDNA.
C) NBC4e and NBC4f (probable cloning artifacts). The
nucleotide sequences of NBC4e and NBC4f (1056) poorly
match the human genome and contain frame-shifts and
nonsense mutations (producing premature termination)
that distinguish them as being artifactual. At least as expressed in oocytes, NBC4e, which has a truncated Ct, was
reported to mediate a substantial pHi recovery from a CO2/
HCO3⫺–induced acid-load. In separate experiments on the
same cell, Vm was unperturbed by the application of CO2/
HCO3⫺ (1056). Taken at face value, these data suggest that
the peculiarities of the NBC4e construct compromise its
electrogenicity. DIDS sensitivity, Na⫹ dependence, and
HCO3⫺ dependence of the pHi recovery mechanism were
not examined in these experiments.
F) DISTRIBUTION OF NBCe2. The major organ most often associated with NBCe2 expression is the liver, although
NBCe2 is expressed in many other organs. The distribution of NBCe2 in specific organ systems is discussed below. The distribution of NBCe2 is summarized and compared with that of other NCBTs in TABLE 5.
IV) Other NBCe2 variants. A) NBC4b (potentially legitimate transcript, NCBT activity unlikely). NBC4b is identical to NBCe2-a except for the presence of a 16-nt exon
(768) that produces a frame-shift, causing the last 8 amino
acids of putative TM14 as well as the entirety of the 83
amino acids in the Ct to be replaced by 28 novel amino
acids. Especially with the changes to TM14, it is not clear
that the protein would be stable or functional.
I) Central nervous system. A) Blood-brain barrier and elsewhere. RT-PCR analysis reveals the presence of NBCe2
transcripts in human brain (214, 835), specifically in the
choroid plexus epithelium (CPE), hippocampus, cerebrum,
and cerebellum (214) and in the hippocampi of mice (73).
Western blotting and immunohistochemical studies localize
NBCe2 protein to the apical membranes of rat and mouse
CPE (113, 470), see cartoon in FIGURE 28. Immunogold
staining confirms the presence of NBCe2 protein in the
membranes of apical microvilli of mouse CPE (113). Interestingly, human CPE is not labeled by existing anti-NBCe2
antibodies (113), despite the presence of NBCe2 transcripts
in this choroid-plexus preparation.
B) NBC4d (potentially legitimate transcript, NCBT activity
unlikely). NBC4d (770) lacks sequence encoding TMs
11–13 and is thus unlikely to encode a functional transporter.
II) Sensory organs. A) Eye. Analysis of EST abundance
suggests that NBCe2 is expressed in the human and mouse
eye (Appendix VI). In the retina, NBCe2 protein is located
in the outer plexiform layer (470).
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a consensus PKA phosphorylation site. The functional consequences of the splicing of this cassette are unknown.
MARK D. PARKER AND WALTER F. BORON
CSF
Tight junction
Interstitial space
H2O
2 K+
Na-K pump
3
Cl–
HCO3–
Na+
H+
K+
KCC4
Na+
HCO3–
K+
Cl–
++
cAMP
H+
NHE
CA
NBCe2
3 HCO3–
NBCn1
HCO3–
CO2
H2O
Na+
Cl–
NBCn2
HCO3–
Na+
Na+
K+
2
Cl–
Cl–
NDCBE
–
2 HCO3
Choroid plexus epithelium
FIGURE 28. Role of NCBTs in the choroid plexus. The secretion of cerebrospinal fluid by the choroid plexus
(CP) and intracellular pH regulation in CP epithelia is achieved by a combination of NCBTs. NBCe2 is the major
⫺
NCBT that is responsible for HCO3
secretion across the apical membrane. NBCn1, NDCBE, and NBCn2 have
⫺
all been detected in the basolateral membrane of CP epithelia, mediating HCO3
influx. NBCn1 has also been
detected in the apical membrane of the CP in some strains of mice. Note that NDCBE is not present in the CP
of adults. Reported, but not shown, is the presence of NBCe1 in the basolateral membrane.
III) Peripheral nervous system. A) Trigeminal ganglion.
NBCe2 transcripts are detected by RT-PCR in preparations
of rat trigeminal ganglion neurons (408).
IV) Respiratory system. A) Lungs. Northern blot analysis
reveals the presence of NBCe2 transcripts in a human lung
preparation (767, 768). Western blotting and immunohistochemical studies localize NBCe2 protein to the basolateral membranes of a human airway epithelial cell line (515).
V) Circulatory system. A) Heart. NBCe2 transcripts have been
detected in heart preparations from humans (767, 768), mice
(31), and rats (1056). Indeed, the archetypa human NBC4a
transcript was cloned from heart cDNA (768). Analysis by
qPCR reveals NBCe2 transcripts in mouse ventricles at a similar abundance to other cardiac HCO3⫺ transporters such as
AE3 and NBCe1 (31). A preliminary immunocytochemical
study on rat ventricular myocytes suggests that NBCe1 protein
is more abundant than NBCe2 in these cells (311).
876
VI) Musculoskeletal system. A) Muscles. RT-PCR reveals
the presence of NBCe2 transcripts in a human muscle preparation (835). NBCe2 protein is detected in rat and human
muscle homogenates (518). In rat muscle, NBCe2 is predominantly localized to the sarcolemma with an additional
presence in T tubules (518).
VII) Upper digestive system. A) Stomach. Northern blot
analysis reveals the presence of NBCe2 cRNA in a human
stomach preparation (767, 768).
VIII) Lower digestive system. A) Widespread, abundant in
liver. The liver appears to be one of the major sites of expression of NBCe2 transcripts (767, 768, 1056). A western
blot and immunohistochemical study have localized
NBCe2 protein to the basolateral (i.e., sinusoidal) membrane of rat hepatocytes (FIGURE 29) and to the apical membrane of cholangiocytes in rat intrahepatic bile ducts (8).
Northern blots reveal the presence of NBCe2 transcripts in
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Na+
Na+
NKCC1
AE2
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
H+
Interstitial space
NHE1
++
NBCe2
K+
Na+
Na+
cAMP
2 HCO3–
Hepatocyte
H+
HCO3–
Cl–
CAII
HCO3–
AE2
CO2
H2O
CFTR
Cl–
2 K+
Na-K pump
3 Na+
⫺
FIGURE 29. Role of NBCe2 in hepatocytes. NBCe2 mediates HCO3
influx across the basolateral membrane
⫺
of hepatocytes. This activity regulates intracellular pH and supports AE2-mediated HCO3
secretion into the bile
ducts.
human small intestines (767, 768), a distribution that was
determined by RT-PCR to correspond to NBCe2 expression in at least ileum, jejunum, and duodenum (214). Elsewhere, NBCe2 transcripts have also been detected in preparations of human pancreas (835) and proximal and distal
colon of rodents (512, 1056).53 However, in mouse duodenum, jejunum, ileum, and colon, the abundance of NBCe2
mRNA is trivial compared with the abundance of NBCe1 or
NBCn1 mRNAs (180). Accordingly, the duodena of
NBCe2-null mice exhibit no detectable HCO3⫺ secretion
defects (180).
53
It has been noted by Odgaard et al. (694) that the NBCe2specific primers reported by Xu et al. are not derived from NBCe2
sequence. This appears to have been an errant description of the
primer sequences in the paper rather than in the design of the actual
primers used by Xu et al. that were GCCAGCTATGCATGAAATTG
(sense) and ATGGGTCCTGTGCTGCTGAG (antisense; J. Xu and M.
Soleimani, personal communication).
IX) Lymphatic and immune systems. A) Spleen and leukocytes. NBCe2 transcripts are have been detected in preparations of human spleen (767, 768) and peripheral blood
leukocytes (835).
X) Endocrine system. A) Thyroid. In situ hybridization experiments reveal the presence of NBCe1 transcripts in the
thyroid glands of 1-day-old and adult mice (341).54 Analysis of the abundance of ESTs suggests that NBCe2 is
expressed in the human thyroid and parathyroid glands
(Appendix VI).
XI) Urinary system. A) Kidney. NBCe2 transcripts have
been detected in human kidney preparations (767, 768),
54
Despite the abundance of NBCe2 transcripts in the mouse thyroid, the morphology and serum T4 abundance were normal in
NBCe2-null mice (341).
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Canaliculus
MARK D. PARKER AND WALTER F. BORON
corresponding to the presence of NBCe2 transcripts in both
the kidney cortex and medulla (214, 341, 1056). Within the
medulla, NBCe2 transcripts have been detected in the outer
medullary segments of the TAL of rats (1056), and in
IMCD of humans (986) and mice (341). Immunohistochemical studies suggest an apical localization of NBCe2
protein in outer medullary collecting duct cells of humans
(214), and in uroepithelial cells of the renal pelvis of rats (8).
XII) Reproductive system. A) Male. NBCe2 transcripts have
been detected in preparations of human testis (767, 768, 835)
and in mouse testis, epididymis, and vas deferens (599). The
archetypa clones of NBC4b (767, 768), NBC4c (835), and
NBC4d (835) originate from human testicular cDNA.
C) Placenta. NBCe2 transcripts have been detected by
northern blot of human placenta RNA (767, 768).
G) PHYSIOLOGICAL ROLES OF NBCe2. Despite the broad distribution
of NBCe2 throughout the body, few physiological roles have
been ascribed to NBCe2 action. Aside from its roles in the
choroid plexus and liver, described below, NBCe2 likely contributes to pHi regulation in all of the cell types in which it is
located. Further physiological roles are suggested by the signs
exhibited by NBCe2-null mice, although primary and secondary effects of NBCe2 loss have yet to be distinguished.
I) Central nervous system. A) Support of CSF secretion. In
the choroid plexus epithelium, which is basically a backwards proximal tubule, the apical membrane faces the CSF.
Thus apical NBCe2 (113), which appears to operate with a
1:3 stoichiometry (645), would be in a position to mediate
the apical step (i.e., HCO3⫺ efflux) of HCO3⫺ secretion into
the CSF (FIGURE 28). Other NCBTs such as NBCn2, and in
some instances NBCn1 and NDCBE, are present at the
basolateral (blood-side) membrane of CPE, and presumably mediate the basolateral (i.e., HCO3⫺ uptake) step of
HCO3⫺ secretion. The role of NBCe2 in proper CSF secretion is supported by the exhibition of defective CSF secretion in NBCe2-gene-trapped mice, although the CPE in
these mice exhibit other defects. Furthermore, ventricle size
is normal in a different strain of NBCe2-null mice (341).
II) Peripheral nervous system. A) Potential contribution to
neuronal excitability. NBCe2 in cultured rat trigeminal
ganglion neurons could contribute towards countering the
excitability-dampening effects of intracellular acidification,
although NBCe1-B/C appears to be the dominant DIDSsensitive Na/HCO3 transporter in these cells (408).
III) Lower digestive system. A) pHi regulation in hepatocytes. The basolateral location of hepatocellular NBCe2
(FIGURE 29) is consistent with the previously demonstrated
878
B) [Na⫹]i regulation in hepatocytes. The addition of CO2/
HCO3⫺ to primary cultures of rat hepatocytes causes a substantial increase in hepatocellular [Na⫹]i. The increase in
[Na⫹]i accompanying NBCe2-mediated HCO3⫺ influx has a
substantial stimulatory effect on the activity of the Na-K
pump (288). Moreover, in perfused livers, the addition of
CO2/HCO3⫺ increases O2 consumption. These data are consistent with the hypothesis that a Na/HCO3 cotransporter,
most likely NBCe2, mediates a substantial Na⫹ influx,
which in turn increases the demand on the Na pump, and
thus on oxidative metabolism.
C) Choliangiocyte viability. The application of the NCBT
inhibitor S3705 inhibits the growth of and promotes apoptosis in cholangiocarcinoma cells (247), perhaps in part by
inhibition of cholangiocyte-expressed NBCe2.
D) Potential contribution to transepithelial HCO3– transport in cholangiocytes. Na/HCO3 cotransport, most likely
mediated by NBCe2, may constitute a basolateral step (i.e.,
HCO3⫺ uptake) in secretion of HCO3⫺ into the hepatic bile
canaliculi (60, 88). However, in cholangiocytes lining the
bile ducts of rats, NBCe2 apparently has an apical distribution (8). Considering that a major role of the bile duct is to
secrete HCO3⫺ into the lumen, and that the accepted major
pathway for apical HCO3⫺ exit from rat cholangiocytes is
Cl-HCO3 exchange mediated by AE2 (46, 59, 631, 902,
987), the role of an apical NBCe2 in these cells is unclear.
The cholangiocytes of mice express NBCe1 (987). In these
animals, NBCe1 may compensate for AE2 insufficiency
(987). It is possible that NBCe2 plays a similar support role
in the cholangiocytes of rats.
IV) Urinary system. A) Possible role in HCO3– reabsorption. Because NBCe2-null mice exhibit signs of urinary
HCO3⫺ wasting, it has been suggested that NBCe2 might
normally contribute towards renal HCO3⫺ reabsorption
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B) Female. NBCe2 transcripts have been detected by RTPCR of mouse ovary, uterus, and vagina (599).
presence of an electrogenic NCBT activity in the basolateral
membranes of hepatocytes (291, 792). The influx of Na⫹
and HCO3⫺, presumably in a 1:2 stoichiometry, mediated
by the transporter under basal conditions in vivo (288, 291)
plays a major role in regulating hepatocellular pHi (266,
289, 326) and, as discerned by studies of perfused rat liver,
is the primary mechanism of pHi regulation in the intact
liver under physiological conditions (266). The maintenance of hepatocyte pHi within a narrow range is crucial for
the functioning of the diverse cellular processes such as
gluconeogenesis, biotransformation of xenobiotics, and mitogenesis (reviewed in Ref. 287). NBCe2 is cooperatively
regulated by the activity of a pH-dependent K⫹ conductance pathway (gK), such that 1) a decrease in pHi causes a
downregulation of gK and depolarizes the plasma membrane (presumably reflecting the decrease in gK) and 2) the
depolarization stimulates NBCe2 activity and causes a compensatory increase in pHi (290).
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
(341). However, as noted below, this phenotype could reflect enhanced HCO3⫺ secretion.
H) CAUSES OF NBCe2 UPREGULATION.
To date, only two groups
have reported a maneuver that upregulates NBCe2.
I) General. Increased transcript abundance in response to
HDAC and methyltransferase inhibitors. NBCe2 transcript
levels are increased in an adenocarcinoma cell line after
treatment with inhibitors of histone deacetylase and DNA
methyltransferase, evidence for the specific regulation of
NBCe2 gene expression by epigenetic factors (478).
I) CAUSES OF NBCe2 DOWNREGULATION.
I) Central nervous system. A) Decreased transcript abundance in brain of a mouse
model of drug-responsive depression. Mice that become
indifferent to rewards after exposure to chronic mild stress
are a model for anhedonia, a symptom of depression. A
subset of those mice in which such behavior is induced are
responsive to treatment with the antidepressant escitalopram. NBCe2 transcripts are twofold lower in the hippocampi of mice that are responsive to escitalopram treatment compared with NBCe2 transcript levels in the hippocampi of nonresponsive mice (73). The physiological
relevance of this finding is unclear, although the finding is
consistent with a possible link between NBCe2 and drug
resistance.
J) CONSEQUENCES OF NBCe2 DYSFUNCTION.
Despite its abundance in the liver, and presumed importance for hepatic
function, NBCe2-null mice have no reported hepatic phenotype. In an early report, NBCe2 was considered as a
candidate gene for the neurodegenerative and metabolic
disease Alström syndrome (767), which maps to the same
genetic locus as NBCe2 (i.e., 2p13). However, subsequent
work has shown that mutations in the ALMS1 gene on
2p13 cause this syndrome (197, 373).
I) Central nervous system. A) Defective CSF secretion in
mice with a disrupted Slc4a5 gene.55 As expected by com-
55
As the authors of this study note, the Slc4a5 gene of these mice
is disrupted in the third extracellular loop, and it cannot be discounted that the phenotypes observed in these mice are due to the
expression of misfolded Slc4a5 product in Slc4a5-expressing cells.
Thus these mice may be a better model of the effects of mutations
that cause NBCe2 to misfold, rather than a model for drawing
inferences about the physiological roles of NBCe2.
B) Decreased neuronal excitability in mice with a disrupted Slc4a5 gene. Slc4a5– gene-trapped mice have a
reduced sensitivity to the proconvulsive drug pentylenetetrazol, a finding that the authors of that study interpret as either a consequence of the reduced intracranial
pressure of these mice or of the increased [K⫹]/decreased
[HCO3⫺] that is characteristic of the CSF of these mice
(470). As NBCe2 has not been demonstrated to be expressed in neurons or glia, it is unlikely that defective pHi
regulation in these cells could explain the decreased neuronal excitability, as is thought to underlie a similar resistance in NBCn2-null mice.
C) Retinal abnormalities and detachment in mice with a
disrupted Slc4a5 gene. Among the diverse morphological
abnormalities in Slc4a5– gene-trapped mice, these mice
have impaired vision and detached retinas (470). The authors of that study suggest that at least some of these signs
may be related to the decreased intracranial pressure in
these mice.
II) Circulatory system. A) Blood pressure-related traits.
Genetic analysis links single nucleotide polymorphisms
(SNPs) in the SLC4A5 gene locus with hypertension and
other blood pressure-related traits (61, 404, 619, 915,
953, 954), including peripheral artery disease (472). One
study reports that the contribution of a particular
SLC4A5 SNP to elevated systolic blood pressure in African-American women is greater in individuals with dark
versus medium skin color (955). A preliminary report
suggests that (SNPs) in the SLC4A5 gene locus are also
associated with the relative thickness of the left ventricular wall in hypertensive African-Americans (924).
NBCe2-null-mice exhibit elevated blood pressure compared with wild-type littermates (341), although the expression of other hypertension-linked genes (e.g., Slc4a7; see p.
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II) Lower digestive system. A) Increased transcript abundance in colon of mice treated with probiotics. Probiotic
treatment of mice is a model used to investigate the molecular mechanisms that underlie the pathogenesis of inflammatory bowel disorder, ulcerative colitis, and the health
benefits associated with probiotic treatment. NBCe2 transcript abundance is doubled in the mouse colon 20 days
after a probiotic treatment (512).
parison with mice that lack NBCn2, another NCBT that is
expressed in CPE and that contributes to CSF secretion, one
strain of NBCe2-deficient mice exhibits defects in CSF secretion and thus a decreased brain ventricle volume and
intracranial pressure (470). Unexpectedly however, these
Slc4a5– gene-trapped mice also exhibit in their CPE, among
other defects, a partial redistribution of NBCn2 into the
apical membrane, a partial redistribution of the Na/K pump
␣1 subunit into the basolateral membrane, and a complete
loss of the Na pump ␤2 subunit (470). Because all of these
changes are predicted to exacerbate the CSF secretion defect, we cannot ascribe the decreased CSF secretion solely to
the loss of NBCe2 activity per se. CSF composition is also
perturbed in these mice inasmuch as it is deficient in HCO3⫺
(but not Na⫹) but contains an overabundance of K⫹ (470).
In a second strain of NBCe2-deficient mouse, no decrease in
brain ventricle size was observed, although the presence of
compensatory mechanisms was not examined in these mice
(341).
MARK D. PARKER AND WALTER F. BORON
113) is upregulated in these mice (341). The molecular basis
for the observed hypertension in NBCe2-null mice is unknown.
B) Polyuria. NBCe2-null mice exhibit polyuria and polydipsia, although urine osmolality is normal (341).
B. Mammalian Electroneutral NCBTs:
NBCn1, NDCBE, and NBCn2
Three of the five mammalian NCBTs perform electroneutral
Na⫹-coupled HCO3⫺ transport: NBCn1, NDCBE, and
NBCn2. These three transporters are encoded by a group
of three closely related Slc4 genes (Slc4a7, -a8, and -a10)
that is distinct from two gene groups that encode electrogenic NCBTs or AEs. The three electroneutral transporters appear to have somewhat overlapping distributions,
all being abundantly expressed in the CNS where they
contribute to enhancing neuronal excitability. As we
shall see, the three transporters differ most in their molecular actions. The physiological relevance of these differences is unknown, but these transporters all have the
ability to regulate pHi without affecting or being influenced by Vm.
1. NBCn1 (Slc4a7)
A) SUMMARY. The electroneutral Na/HCO3 cotransporter
NBCn1 (encoded by the Slc4a7 gene) is unique among
NCBTs in its low sensitivity to blockade by DIDS as well as
in having a HCO3⫺-independent conductance. NBCn1 is
unique among electroneutral NCBTs because it does not
transport Cl⫺. NBCn1 has the greatest known multiplicity
of products for an Slc4 family member (at least 12 known
variants, -A through -L) and is present in many organs/
organ systems throughout the body. In common with
NBCe1-B/C, NDCBE, and NBCn2, NBCn1 is stimulated by
the soluble protein IRBIT. NBCn1 is notably abundant in
1) the central nervous system, where NBCn1 contributes
towards neuronal excitability; 2) the eye and the ear;
3) secretory epithelia, where NBCn1 contributes towards
transepithelial HCO3⫺/fluid movement (e.g., in the intes-
880
B) NOMENCLATURE OF Slc4a7 PRODUCTS. The first report of what
we now know as NBCn1 was a partial cDNA, called SBC2
or hNBC2, from a human retinal cDNA library (420). The
cDNA was interpreted as the product of a novel gene, which
was assigned the name SLC4A6. Subsequent to this study
was a report of a full-length NBCn1 cDNA, called mNBC3,
from human skeletal muscle (765). Unfortunately, because
of differences between the SBC2/hNBC2 and mNBC3
cDNA sequences,56 mNBC3 was interpreted as the product
of a novel gene, which was assigned the name SLC4A7
(765).57 By the time NBC2 and NBC3 had been rationalized
as alternative products of the same gene (139, 189), the
designation SLC4A7 had already been introduced to the
literature. As a consequence, the designation SLC4A6,
which had never been mentioned in the literature, was withdrawn. Following the characterization of a rat Slc4a7 product as an electroneutral Na/HCO3 transporter, the gene
product was renamed NBCn1 (189). The confusion caused
by this changeable nomenclature was fortunately minimal.
Few papers refer to NBC2 or SBC2 (344, 426, 427, 457,
485, 814, 989) and fewer still refer to NBCn1, NBC2,
SBC2, and NBC3 as though they are the products of distinct
genes (344, 426, 457, 485, 530). We further note that
NBC2 has been used on one occasion to refer to NBCe1-B
(485) and XNBC2 to refer to a Xenopus Slc4a11 product
(1102). Due to the withdrawal of the name “NBC2” and
because the term “NBC3” is degenerate (i.e., it has been
used to refer to both Slc4a7 and Slc4a8 products), we consider NBCn1 as the preferred nomenclature for Slc4a7
products.
C) MOLECULAR ACTION OF NBCn1. NBCn1 is an electroneutral
Na/HCO3 cotransporter with an associated HCO3⫺-independent conductance (FIGURE 30; 30; Ref. 189), conclusions supported by the following observations.
I) NBCn1 is an electroneutral NCBT with poor DIDSsensitivity. In Xenopus oocytes subjected to a CO2/
HCO3⫺-induced acid load, rat NBCn1 mediates a pHi
recovery that is electroneutral, insensitive to 5-(N-ethyl-
56
In addition to numerous artifacts in the hNBC2/SBC clone, the
inclusion in mNBC3 versus hNBC2/SBC2 of a different complement
of splice cassettes also misled investigators to believe that these two
cDNAs were transcribed from different genes.
57
This report was concurrent with a report of a partial product of
truly novel gene (Slc4a8), which was also then named NBC3 (35).
This product has since been renamed NDCBE.
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III) Urinary system. A) Metabolic acidosis. NBCe2-null
mice exhibit a compensated metabolic acidosis that is
evidenced by normal blood pH, reduced plasma CO2 and
[HCO3⫺], and elevated urinary HCO3⫺ excretion (341).
These observations are consistent for a role for NBCe2 in
HCO3⫺ reabsorption. However, the NBCe2-null mice
also exhibit an increased abundance of pendrin (341),
which secretes HCO3⫺ in the collecting duct (reviewed in
Ref. 1016). Thus the urinary phenotype might not be
solely related to loss of NBCe2 function. Another, untested, possibility is that the acidosis is secondary to disrupted hepatobiliary interactions (e.g., reduced glutamine synthesis).
tines); and 4) the kidney, where NBCn1 promotes NH4⫹
excretion. In keeping with its presence in the eye and ear,
mice that lack NBCn1 are both blind and deaf. Multiple
studies report genetic linkage between the SLC4A7/NEK10
gene locus and breast cancer in humans. Although the genetic basis underlying the linkage has not been elucidated,
the association between pHi regulation and tumor viability
is clear.
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
NBCn1
Na+
HCO3–
2 Na+ CO32–
Na+
A
Na+
CO32–
Na+
B
NaCO3–
Na+
C
HCO3–
Na+
D
HCO3–
FIGURE 30. Molecular action of NBCn1. Possible molecular mechanisms by which an electroneutral NCBT
⫺
⫺
could operate with an apparent Na⫹:HCO3
stoichiometry of 1:1. NBCn1 also exhibits a HCO3
-independent
conductance that is represented by the red dashed arrow. Note that we have not included any models that are
based on CO3/H cotransport, such as those represented for NBCe1 in FIGURE 16.
II) NBCn1 operates independently of Cl⫺. In Xenopus oocytes,
the HCO3⫺ efflux mediated by NBCn1, when operating in “reverse” (i.e., initiated by the removal of extracellular Na⫹), does
not depend on extracellular Cl⫺ (189). Nor does the HCO3⫺ influx mediated by NBCn1, when operating in the “forward” direction, result in the net efflux of Cl⫺ from the cell (719). Thus
NBCn1 is not a Na⫹-driven Cl-HCO3 exchanger.
III) NBCn1 has an associated cation leak. After several days
incubation, Xenopus oocytes expressing rat NBCn1 are unusually depolarized and loaded with nearly 40 mM [Na⫹]i,
more than six times the [Na⫹]i of H2O-injected control
oocytes from the same batch (189). Furthermore, NBCn1expressing oocytes, even in the nominal absence of CO2/
HCO3⫺, are substantially hyperpolarized by the removal of
extracellular Na⫹, indicating the presence of an associated
58
The first report of NBCn1 expression in oocytes characterized
the protein as being DIDS-insensitive, EIPA-sensitive, and capable of
substantial Na/OH cotransport or Na-H exchange (765). Subsequent work published with members of the same laboratory reports
that HEK-293-expressed NBCn1 does not have these qualities
(711). Thus the authors conclude that the EIPA sensitivity and
Na/OH cotransport are a feature of NBCn1 expression in oocytes.
It is therefore likely that the initial report was confounded by endogenous NHE activity, and possible that none of the reported acid-base
transport in fact represented NBCn1. In the meantime, an apical
EIPA-sensitive Na-base cotransport activity in ␣-intercalated cells
from the rabbit collecting duct (from the inner stripe of the outer
medullary) was attributed to NBCn1 in two studies (774, 1083),
guided by the original report of EIPA sensitivity of NBCn1 and the
apical distribution of NBCn1 suggested by the use of the “anti-NBC3”
antibody discussed in Appendix VII. Finally, in aortic smooth muscle
of rats (a site of NBCn1 expression), the authors report both an
EIPA-sensitive NCBT activity and a distinct SITS-sensitive, and unusually EIPA-sensitive, NDCBE-like activity (592).
Na⫹-conductance pathway (189), that is also a feature of
NBCn1 expressed in HEK 293 cells (201). About 50% of the
current through the conductive pathway is carried by Na⫹
(189, 201), and Na⫹ conduction is estimated to be responsible
for 1/300 of the total Na⫹ movement though the transporter
(201). The reversal potential of the conductance varies in a less
than Nernstian manner with respect to extracellular [Na⫹],
which indicates that the conductance cannot be explained by a
simple Na⫹-channel model (201). It is not clear what carries
the remainder of the current. Indeed, the Na⫹-independent
component of the current remains even in the absence of all
extracellular ions except Mg2⫹, Cl⫺, and HEPES, perhaps indicative of an anion-efflux component. The magnitude of the
conductance exhibited by membranes of NBCn1-expressing
oocytes is not reduced in cells preincubated in Cl⫺-free media,
although the Cl⫺ depletion of these cells was not demonstrated
(201). The Na⫹-conductive pathway neither depends on nor carries HCO3⫺ and is paradoxically stimulated by DIDS exposure
(189). A preliminary report shows that both the HCO3⫺transport and Na⫹-conductive elements of NBCn1 function are
upregulated by coexpression of NBCn1 with the NCBT binding
partner IRBIT (722), further evidence that the two components
may be mediated by the same protein. Finally, a study of NBCe1/
NBCn1 chimeras indicates that the Na⫹ conductance requires
elements in the back half of the TMD (i.e., TM6-TM14, inclusive: subdomain 8 in FIGURE 15) of NBCn1 (193).
D) THE SLC4A7 GENE.
The human SLC4A7 gene was originally
mapped to chromosome 3p22 (766), although more recent
genomic assembly suggests that 3p24.1 is a more accurate
assignment.59 SLC4A7 occupies at least 27 exons spread
over ⬃100 kb (FIGURE 31A). The upstream neighbor of
SLC4A7 is the T-box region (TBR) gene EOMES, aka TBR2,
which encodes the neuronal transcription factor eomesodermin (495). The SLC4A10 gene, which encodes a second electroneutral Na/HCO3 cotransporter, also has a TBR gene as its
59
A 67-nt sequence with 87% identity to a portion of SLC4A7
(exon 10, encoding sequence in the Nt) is found on chromosome
1p36, 13 kb upstream of the RhD gene. There is currently no
evidence to suggest that this sequence is ever transcribed, or is part
of an miRNA sequence.
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N-isopropyl)amiloride (EIPA),58 and dependent on both
Na⫹ and HCO3⫺, consistent with the activity of an electroneutral NBC (189). Human NBCn1 also mediates a
Na⫹-dependent pHi recovery when overexpressed in
HEK-293 cells (711). Unusually for an NCBT, NBCn1
cotransport activity is not greatly sensitive to DIDS; in
oocytes, 500 ␮M inhibits only 25% of the NBCn1-mediated pHi recovery (189), whereas the Ki for DIDS of the
related transporter NBCe1 is ⬃40 ␮M (612).
MARK D. PARKER AND WALTER F. BORON
A
Locus 3p24.1
20 kb
SLC4A7
NEK10
EOMES/TBR2
B
Gene structure
P1
P2
1
2
C
10 kb
3
7 8
26
27
Transcript variation
P2
M
3
4
5
6
7
8
9
25
*
27
1
3
4
5
6
7
8
9
25
*
27
1
3
4
5
6
7a
8
9
25
26
*
27
1
3
4
5
6
7
8
9
25
26
*
27
1
3
4
5
6
7
9
25
NBCn1-A
2
M
NBCn1-B
M
NBCn1-C
M
NBCn1-D
M
NBCn1-E
*
27
FIGURE 31. SLC4A7 gene structure and NBCn1 transcript variants. Scale diagrams showing the human
SLC4A7 gene locus together with the position of neighboring genes (A), the position of promoters (P1 and P2),
and the position of exons within SLC4A7 (B). Transcript variants NBCn1-A to NBCn1-E, which among themselves display the diversity of NBCn1-A to NBCn1-E, are represented, not to scale, as numbered boxes joined
by a horizontal line (C). Each numbered box represents the inclusion of that exon in the mature transcript. “//”
denotes that all transcripts include exons 9 –25. Exons that include the initiator ATG codon (“M”) and
termination codon (“*”) are marked for each transcript. Sequence that is derived from part of a larger exon
sequence are labeled with an “a” (e.g., exon 7a is a subdivision of exon 7). Colored exons, or parts of exons,
correspond to the protein regions that each encodes, which are identically colored in FIGURE 32. Uncolored
exons, or parts of exons, denote untranslated 5= and 3= sequence. Exons that are connected with a dashed line
are predicted, but not demonstrated, to be included in the mRNA.
upstream neighbor (FIGURE 39A), suggesting a longstanding
association between the two gene families, and one that predates the duplication of this gene region.
E) STRUCTURAL FEATURES AND VARIANTS OF NBCn1. Slc4a7 products that encode full-length transporters are currently
named NBCn1-A through -L and are the most diverse in
terms of number of reported and predicted variant transcripts. Here we describe the nature of the variant features
(i.e., alternative promoters, splice cassettes), followed by a
description of NBCn1-A through -L. A diagrammatic representation of the sources of transcript variation is provided
in FIGURE 31C. A representation of each NBCn1 protein
variant is provided in FIGURE 32.
I) Sources of variation in coding sequence among NBCn1
variants. Alternative promoter choice and Nt (“MERF”- versus “MEAD”-). Mammalian NBCn1 transcription can initiate
882
at either exon 1 or exon 2, presumably dictated by a choice
of alternative promoter regions (FIGURE 31B). The result is
variant protein products whose Nt begins either with an 11amino acid or a 16-amino acid protein cassette. In humans,
exon 2, which encodes the 11-amino sequence (beginning with
the sequence MERF), is located 4 kb upstream of exon 3 in the
SLC4A7 gene, whereas exon 1, which encodes the 16-amino
acid sequence (beginning with the sequence MEAD), is 32 kb
upstream of exon 3. The consequences of this promoter choice
for NBCn1 function and/or regulation are unclear.
The full-length NBCn1 protein variants that begin with “MERF”
are NBCn1-A, -F, -J, -K, and -L, whereas the variants that begin
with “MEAD” are NBCn1-B, -C, -D, -E, G, -H, and -I.
A) Variation in the length of “MEAD”-encoding exon 1
(NBCn1-X=). In some variants, the 3= boundary of exon 1 is
extended by the use of an alternative splice site such that the
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P1
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
III
6–9
10–14
124
1,214
124
1,219
124
PDZ
NBCn1-B
1–5
PDZ
11
Ct
PDZ
NBCn1-A
TMD
C
as
se
tte
100 aa
C
as
se
tte
I
C
as
se
tte
II
Nt
1,242
13
16
NBCn1-C
36
124
NBCn1-E
NBCn1-F
NBCn1-G
NBCn1-H
124
NBCn1-I
NBCn1-J
NBCn1-K
124
NBCn1-L
1,255
1,095
1,090
1,131
1,206
1,118
1,126
1,201
1,113
FIGURE 32. NBCn1 protein variants. Scale diagram of protein variants that are encoded by the transcripts represented in FIGURE 31C. Horizontal bars represent protein sequence laid out from Nt to Ct. Vertical bars represent
position of ␣-helical TMs. Protein cassettes are labeled with a number denoting their size in amino acids and colored to
denote their genetic origin as shown in FIGURE 31C. All NBCn1 variants are presumed to include an
autoinhibitory domain and IRBIT-binding determinants in their Nt. All NBCn1 variants terminate with a PDZ
binding motif. A color-matched protein sequence alignment of the variants is provided in Appendix V.
16-amino acid sequence is lengthened at its carboxy-terminal end by the 4-amino acid sequence “VTSR”. The terminology for such variants has not been settled. Some have
been designated either with a prime (e.g., NBCn1-D= is
identical to NBCn1-D except for the inclusion of “VTSR”;
Appendix IV and Appendix V) and others with a lowercase
“a” and “b” (e.g., NBCn1-Hb is identical to NBC1-Ha
except for the inclusion of “VTSR”). The consequences of
the inclusion of “VTSR” are unknown.
B) Cassette I (aka “cassette A”). A 13-amino acid “cassette
I” (originally termed “cassette A” in Ref. 189)60 is encoded
by a 3= extension of exon 7 (FIGURE 31C). The protein
60
Analysis of the Slc4a7 gene structure leads us to deduce that
cassette I encompasses the 13-amino acid sequence “GKKHSD
PHLLERN” and not, as originally deduced in Ref. 189, the 14-amino
acid sequence “GKKHSDPHLLERNG.”
sequence encoded by cassette I is located in the Nt loop
subdomain (FIGURE 15) and is absent from some variants of
NBCn1 (FIGURE 32). The consequences of this splice for
NBCn1 function and/or regulation are unclear.
Several studies have used RT-PCR to address the spatial distribution of splice cassette I, as well as II and III (discussed
below). The most exhaustive to date is presented in Reference
213. In preparations from various organs/tissues of adult mice,
NBCn1 splicing generally does not appear to favor the omission or inclusion of cassette I (55, 213). However, a preference
for cassette I inclusion seems to exist in kidney cortex, submandibular gland, parotid gland, and liver. Conversely, a preference for cassette I exclusion appears to exist in the lung.⬎
The full-length NBCn1 protein variants that include cassette I are NBCn1-A, -B, -D, -E, -F, -G, and -J. Variants
lacking cassette I are NBCn1-C, -H, -I, -K, and -L. Among
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PDZ PDZ PDZ PDZ PDZ PDZ PDZ PDZ PDZ
NBCn1-D
MARK D. PARKER AND WALTER F. BORON
those variants lacking cassette I, at least NBCn1-H is functional.
Comparisons of Xenopus-oocyte– expressed NBCn1 variants, with or without cassette II (specifically NBCn1-B versus NBCn1-E), indicate that NBCn1 ⫹ cassette II accumulates in the plasma membrane more slowly than NBCn1 ⫺
cassette II (1078). However, after 72 h of expression, both
variants accumulate to a similar extent in the oocyte membrane, and both mediate a Na⫹ conductance of equivalent
magnitude (1078). Some differences are evident between
the two variants at low [Na⫹]o: 1) NBCn1 ⫹ cassette II
appears to have a lower Na⫹ affinity, and 2) the variants
exhibit differences in the appearance of a conductive leak of
an as-yet-unidentified nature (1078).
The reduced functional expression of NBCn1 ⫹ cassette II
is also indirectly evident in an opossum kidney cell line,
inasmuch as the endogenous Na pump in these cells is less
active in cells coexpressing NBCn1 ⫹ cassette II than in cells
coexpressing NBCn1 ⫺ cassette II, presumably because the
less active variant of NBCn1 imposes a lesser Na⫹ load on
the cells (1078).
Thus cassette II appears to be inhibitory in at least certain
contexts, perhaps because it serves as a binding site for
calcineurin or other proteins with an inhibitory effect.
In mice (55, 213) and in rats (200), most NBCn1 transcripts
lack cassette II, as assessed by PCR across a region including
cassette II. However, the inclusion of cassette II appears to
61
“Exon 7” is a misnomer. Although cassette II is encoded by the
seventh exon of individual NBCn1 transcripts, cassette II is encoded
by exon 8 of the Slc4a7 gene (see FIGURE 31C).
884
The full-length NBCn1 protein variants that include cassette II are NBCn1-A, -B, -C, -D, -H, and -K. Variants
lacking cassette II are NBCn1-E, -F, -G, -I, -J, and -L.
Variants that include both cassettes I and II are NBCn1-A,
-B, and -D. The only two variants that lack both cassettes I
and II are NBCn1-I and -L.
D) Cassette III (aka “cassette B”). The final published source
of NBCn1 variation lies in the Ct of the protein (FIGURE 32).
NBCn1 variants are unique among NCBTs in that the sequence of the extreme Ct is invariant: all variants terminate
with the PDZ-binding domain ligand -ETSL. Instead, Ct variation among NBCn1 forms is achieved by the optional inclusion of a 36-amino acid “cassette III” (originally termed “cassette B” in Ref. 189). As far as other NCBTs are concerned,
cassette III–like sequence is also present in the Ct of NDCBE-A
and NDCBE-C, and inclusion of cassette III–like sequence is
obligatory in the Ct of all NBCn2 variants (see extended domain alignment in Appendix V).
Experiments on oocytes expressing NBCn1 variants indicate that cassette III stimulates overall functional expression in the absence (but not in the presence) of cassette II
(compare NBCn1-G versus -E and -D versus -B).
In preparations from various organs/tissues of adult mice,
NBCn1 splicing generally does not appear to favor the omission or inclusion of cassette III (55, 213). However, in rodents,
a preference for cassette III inclusion seems to exist in kidney
cortex (213); renal mTAL (694); submandibular gland (213),
specifically the acini, see Reference 615; sublingual gland
(213); pylorus (213); colon (213); pancreas (213); lung (213);
cerebrum (213) and certain other areas of the brain (755); and
epididymis (213). On the other hand, a preference for cassette
III exclusion appears to exist in cardiac ventricles (213) and in
the ducts of submandibular glands (615).
The full-length NBCn1 protein variants that include cassette III are NBCn1-C, -D, -G, I-, -J, and -L. Variants lacking cassette III are NBCn1-A, -B, -E, -F, -H, and -K.
Of the 12 confirmed transcripts, only NBCn1-D includes all
three cassettes I, II, and III.
II) Cloned NBCn1 variants that are demonstrated or likely to
exhibit NCBT activity. The known NBCn1 variants are listed
below along with their features and their demonstrated locations. Information about clones that have not yet been reported in a full manuscript is derived from their GenBank
entries (accession numbers are provided in Appendix IV).
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C) Cassette II (aka “exon 7”).61 Abutting cassette I, and
also within the Nt loop subdomain (FIGURE 15), is the 124amino acid cassette II (123 amino acids in rodents due to the
lack of an Ala residue close to the Ct end of the cassette),
encompassing the entirety of exon-8 – encoded sequence
(FIGURES 31C AND 32). Cassette II sequence is unlike that
encoded by any other mammalian gene, but contains a
number of consensus PKA and PKC phosphorylation sites
and, as an extension of the Nt loop subdomain, is likely
accessible to a number of cytosolic binding partners. A preliminary report demonstrates that an isolated cassette II is
able to interact with calcineurin-A␤ in vitro (715). Indeed,
cassette II includes a motif “PTVVIH” that is similar to a
consensus calcineurin-binding motif (45). Moreover, perturbation of this sequence disrupts the in vitro interaction
between isolated cassette II and calcineurin (715). The
physiological relevance of this interaction remains untested,
although perhaps pertinent is the observation that NBCn1
protein abundance is decreased in the renal medulla of rats
treated with the calcineurin inhibitor FK506 (655).
be favored in rodent aorta (55, 200), rat heart (fetal ⬎
adult; interventricular septum ⬎ ventricles ⬎ auricles ⬎
atria ⬎ AV node; Ref. 200), human heart and muscle (200,
765), and in fetal rat hippocampal neurons (201).
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
A) NBCn1-A (NCBT activity demonstrated). NBCn1-A is the
archetypal human NBCn1 clone (765). NBCn1-A initiates
with the “MERF” Nt, includes cassette I and II, but omits
cassette III. A full-length NBCn1-A clone has been isolated
from a human skeletal muscle cDNA preparation (765).
B) NBCn1-B (NCBT activity demonstrated). NBCn1-B is
the archetypal rat NBCn1 clone (189). NBCn1-B initiates
with the “MEAD” Nt, includes cassette I and II, but omits
cassette III. A full-length NBCn1-B clone has been isolated
from rat aorta (189) and embryonic rat hippocampal neuron cDNA preparations (201).
D) NBCn1-D and D= (NCBT activity demonstrated).
NBCn1-D initiates with the “MEAD” Nt sequence, and
includes cassettes I, II, and III. NBCn1-D= is the most complete “MEAD”-initiated NBCn1 clone as it initiates with
the extended 20-amino acid “MEAD” Nt sequence and also
includes all three splice cassettes. Full-length NBCn1-D has
been isolated from rat aorta cDNA preparations (189).
Full-length NBCn1-D= has been isolated from human liver
cDNA preparations.
E) NBCn1-E (NCBT activity demonstrated). NBCn1-E initiates with the “MEAD” Nt, includes cassette I, but omits
cassettes II and III. Full-length NBCn1-E clones have been
isolated from human skeletal muscle, adult-rat hippocampal neurons (201), and mouse reproductive tract cDNA
preparations.
F) NBCn1-F (NCBT activity untested). NBCn1-F initiates
with the “MERF” Nt, includes cassette I, but omits cassettes II and III. Full-length NBCn1-F clones have been isolated from human kidney cDNA preparations.
G) NBCn1-G (NCBT activity demonstrated). NBCn1-G initiates with the “MEAD” Nt, includes cassettes I and III, but
omits cassette II. Full-length NBCn1-G clones have been isolated from human skeletal muscle cDNA preparations.
H) NBCn1-H and H= (NCBT activity demonstrated).
NBCn1-H initiates with the “MEAD” Nt, includes cassette
II, but omits cassettes I and III. NBCn1-H= is the same but
initiates with the extended 20-amino acid “MEAD” Nt sequence. Full-length NBCn1-H and -H= clones have both
I) NBCn1-I (NCBT activity untested). NBCn1-I initiates
with the “MEAD” Nt, includes cassette III, but omits
cassettes I and II. Full-length NBCn1-I clones have been
isolated from mouse reproductive tract cDNA preparations.
J) NBCn1-J (NCBT activity untested). NBCn1-J initiates
with the “MERF” Nt, includes cassettes I and III, but omits
cassette II. Full-length NBCn1-I clones have been isolated
from mouse ovary and testis cDNA preparations.
K) NBCn1-K (NCBT activity untested). NBCn1-K initiates
with the “MERF” Nt, includes cassette II, but omits cassettes I and III. Full-length NBCn1-I clones have
been isolated from mouse skeletal muscle cDNA preparations.
L) NBCn1-L (NCBT activity untested). NBCn1-L initiates
with the “MEAD” Nt, includes cassette III, but omits cassettes I and II. Full-length NBCn1-L clones have been isolated from mouse reproductive tract cDNA preparations.
III) Predicted NBCn1 variants. The choice of three alternative first exons and the omission or inclusion of any of the
three splice cassettes I, II, or III could, together, produce as
many as 24 variants, although presently only 15 of the 24
have been cloned as full-length cDNAs.62 No pattern of
association between promoter/cassette usages has emerged
that suggests any of the “missing” combinations63 are unfavored; thus it is likely that their existence will be documented in due course.
IV) Other NBCn1 variants. A) Unusual variants that
represent only the isolated Nt. Six unusual cDNA species
from brain, heart, and skeletal muscle cDNA are identical to full-length NBCn1 transcripts except for the omission of exon 13 (GenBank DNA accessions nos.
FJ178574, FJ178575, FJ178576, GU354307, GU354309,
and GU354310).64 If translated, each of these cDNAs is
predicted to produce a soluble protein that would include
62
In a personal communication, Drs. Liming Chen and Ying Liu
report to us the existence of a new promoter and a new cassette
that could increase the number of possible variants to 64.
63
Considering only “MEAD” versus “MERF” and the three cassettes, the “missing” combinations are MEAD/-I/-II/-III, MERF/⫹I/
⫹II/⫹III, MERF/-I/-II/⫹III, MERF/-I/⫹II/⫹III, and MERF/-I/-II/-III.
64
If these clones included exon 13, FJ178574 would encode
NBCn1-H, FJ178575 would encode NBCn1-G, FJ178576 would
encode NBCn1-E, GU354307 would encode NBCn1-C, GU354309
would encode NBCn1-E=, and GU354310 would encode NBCn1-G=.
Because some of these full-length NBCn1 clones differ only in the
protein sequence of their Ct, the accession pair FJ178574/
GU354307 is predicted to encode identical isolated-Nt polypeptides
as are the pairs FJ178575/FJ178576 and GU354309/
GU354310.
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C) NBCn1-C and C= (NCBT activities untested). NBCn1-C
initiates with the “MEAD” Nt sequence, includes cassette
II, but omits cassettes I and III. NBCn1-C= is the same but
initiates with the extended 20-amino acid “MEAD” Nt sequence. A full-length NBCn1-C clone has been isolated
from rat aorta cDNA preparations (189). A full-length
NBCn1-C= clone has been isolated from human skeletal
muscle cDNA preparations.
been isolated from human skeletal muscle cDNA preparations.
MARK D. PARKER AND WALTER F. BORON
almost the entire “MEAD” Nt (i.e., the sequence encoded
by exons 1 and 3–12, per normal) plus two residues
“VQ” followed by a termination codon (encoded by an
out-of-frame exon 14). The protein would be truncated
at a point 25 amino acids upstream of TM1, and thus
would precisely terminate in the region that, in AE1, is
predicted to be an unstructured linker that join the Nt to
the TMD. The physiological relevance of these clones
remains obscure, and the cognate protein has yet to be
identified. It is possible that the premature termination
codons included in these unusual mRNAs would make
them targets for nonsense-mediated decay (170). These
clones are reminiscent of isolated Nt variants of NDCBE
and NBCn2.
F) DISTRIBUTION OF NBCn1. The distribution of NBCn1 is
broad, as summarized in TABLE 5. Its location in specific
organ systems is discussed below. Note that the tissue distributions of the alternate promoters and three splice cassettes were discussed above in section VB5 on p. 882.
I) Central nervous system. A) Brain. At the level of subregions of the central nervous system, NBCn1 transcripts
(755) and protein (174, 709) are widespread, being present in the cerebral cortex, hippocampus, subcortex, cerebellum, and olfactory bulb of rodents. NBCn1 immunoreactivity in wild-type rats and ␤-galactosidase staining
of heterozygous mice with a lacZ insertion in the Slc4a7
gene shows that the highest level of NBCn1 promoter
activity, at the tissue level, is in the pyramidal cell layers
of the hippocampus, appearing equally robust in the
CA1, CA2, and CA3 regions, and in the granule cells
layer of the dentate gyrus (91, 709). In these “lacZ” mice,
NBCn1 promoter activity is also evident in certain regions of the cortex and the dentate nucleus of the cerebellum (91).
At the cellular level, NBCn1 expression is detected in hippocampal neurons of embryonic rats (201) and adult mice
(91). In primary cultures of hippocampal neurons from embryonic rats, most cells, both GABAergic and non-GABAergic, express NBCn1 transcripts (201). NBCn1-like activity is detected in locus coeruleus neurons (479), although the presence of NBCn1 transcripts and protein in these cells has yet
to be demonstrated.
At the subcellular level in embryonic rat neurons, NBCn1
protein is detected in the plasma membrane of the cell soma
as well as in the dendrites, with a punctuate distribution
that is consistent with a presence in the dendritic spines
(201, 709).
B) Choroid plexus. NBCn1 protein has a basolateral presence (see FIGURE 28) in the choroid plexus of rats (213, 709,
755), some strains of mice (755), and humans (756).
NBCn1 also has a small apical presence in the choroid
plexus of human ventricle IV (756) and is predominantly
apically expressed in some strains of mice (470, 755).
NBCn1 distribution has been the focus of much investigation, revealing some discrepancies between studies. That is
to say, certain NBCn1-directed polyclonal antibodies suggest a different transporter distribution than others. Some
studies report that the localization of NBCn1 in a tissue
may vary among species (485) and even among mouse
strains (216). Data concerning NBCn1 protein distribution
come from the use of the three types of antibodies, those
that are generated against: 1) epitopes in the Ct of rat
NBCn1 (1014), 2) epitopes in the Ct of human NBCn1
(“anti-NBC3”; Ref. 774), and 3) an epitope in the Nt of rat
NBCn1 (213). The studies using antibodies 1 and 3 reinforce each other and are confirmed by ␤-galactosidase staining in mice heterozygous for the insertion of lacZ into the
Slc4a7 gene (91). It is therefore the results of these studies
that are cited here for NBCn1 protein localization.
II) Sensory organs. A) Eye. NBCn1 promoter activity is
evident in the photoreceptor and ganglion cells of mouse
retina (91) and NBCn1 transcripts have been detected in
Northern blots of rabbit eyes (814). Microarray analysis
demonstrates that cultured mouse keratocytes express
NBCn1 transcripts (see Gene Expression Omnibus database entry GDS85765 that accompanies Ref. 162). See Appendix VII for subretinal distribution based on the “antiNBC3” antibody.
The distribution of NBCn1 as reported by antibody 2, the
“anti-NBC3” antibody, is unusual in many respects (dis-
65
Gene Expression Omnibus Entry at (http://www.ncbi.nlm.nih.
gov/sites/GDSbrowser?acc⫽GDS857).
886
B) Ear. See Appendix VII for distribution of NBCn1 within
the inner ear according to the “anti-NBC3” antibody.
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B) SBC2/hNBC2 (probable cloning artifact). The SBC2/
hNBC2 cDNA sequence (GenBank protein accession no.
BAA25898) was the first reported SLC4A7 product (420).
It differs from subsequently reported SLC4A7 products in
three respects: 1) hNBC2 lacks exons 0 – 4 of verified
SLC4A7 transcripts. 2) The 5=-UTR and the initial portion
of the purported open reading frame of hNBC2 is derived
from the exons 5 and 6 of the SLC4A7 gene, but this sequence has an inverted orientation with respect to the remainder of the transcript. 3) The 3= end of the hNBC2
transcript is not predicted by the genomic sequence, presumably due to low fidelity of the amplified cDNA: because
of this the ORF contains three missense mutations and
reaches a premature stop, 18 amino acid short of the Ct end
of verified Slc4a7 products.
cussed in Ref. 334) and is considered separately in Appendix VII (see section V).
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
III) Peripheral nervous system. A) Trigeminal ganglion. In
rats, NBCn1 transcripts are detected by RT-PCR in trigeminal ganglion preparations (408).
B) Osteoblasts. A proteomic study reveals NBCn1 protein
to be present in the hydroxyapatite-releasing microvesicles
that bud from the apical membranes of osteoblasts (969).
IV) Respiratory system. A) Trachea and lung. NBCn1 transcripts and protein are present in preparations of rat lung
(189, 213), and NBCn1 transcripts are present in the Calu-3
airway cell line (see “NBC2” in Ref. 515). NBCn1 promoter activity is evident in nonvascular smooth muscle cells
of mouse trachea (91).
C) Osteoclasts. Osteoclasts express NBCn1 protein (112,
797), specifically in the ruffled membrane that faces the bone
resorption lacuna (see cartoon in FIGURE 33 and ref. 797).67
B) Vasculature. As noted in the previous paragraph,
NBCn1 is present in cardiac endothelial cells. In vascular
smooth muscle, NBCn1 cDNA has been amplified from
pulmonary artery and aorta (189), and NBCn1 protein is
detected in the portal vein, and in the hepatic, mesenteric,
and intrarenal cortical arteries of rats. NBCn1 protein is
also present in rat skeletal muscle vasculature. In the arteries, it is the endothelial cells and the smooth muscle cells of
the tunica media that are NBCn1 positive (213). LacZ/␤galactosidase staining provides evidence for NBCn1 promoter activity in mouse cerebral arteries and veins, mesenteric arteries, and renal arteries of mice (91). In the vascular
smooth muscle of a mouse mesenteric small artery, immunogold staining detects NBCn1 expression in the sarcolemmal membrane (90).
VI) Musculoskeletal system. A) Skeletal muscle. The archetypal human NBCn1 clone was amplified from skeletal
muscle cDNA. Within skeletal muscle of rats, NBCn1 protein localizes to vasculature as well as to the vicinity of
neuromuscular junctions. However, NBCn1 does not colocalize with ␣-bungarotoxin, suggesting that NBCn1 is present in motor neuron terminals or sarcolemmal areas that
lack the nicotinic acetylcholine receptor (213).
66
The antibody in this study detected only NH2 termini beginning
with “MEAD” (see green cassette in FIGURE 32).
B) Salivary gland. In human, but not rat, parotid and submandibular salivary glands, NBCn1 protein is enriched in
the basolateral membranes of the striated duct cell (FIGURE
21B). However, NBCn1 is not detected in acinar cells (334),
which are a site of basolateral NBCe1 expression (FIGURE
21A). NBCn1 is expressed in the basolateral membranes of
an immortalized cell line from rat parotid acini (740). The
evidence for the presence of NBCn1 in the apical membrane
of salivary gland duct cells is indirect, being based on Co-IP
of NBCn1 and CFTR in isolated tissues, rather than immunohistochemical data (711).
See Appendix VII for distribution of NBCn1 within the
salivary glands according to the “anti-NBC3” antibody.
C) Stomach. Results of qPCR show that rabbit NBCn1
transcripts are expressed in gastric mucosa, most prominently in the chief cells and mucous cells, with lesser expression in parietal cells (814). LacZ/␤-galactosidase staining is
negative in mouse gastric mucosa (91).
VIII) Lower digestive system. A) Intestines. In rabbits,
NBCn1 transcripts are more abundant in the duodenal and
ileal mucosa than in either gastric or colonic mucosa (427).
In mice, NBCn1 transcripts are detected in duodenal and
colonic epithelia (55, 180, 213). In mice, anti-NBCn1 antibodies localize NBCn1 protein to the basolateral membrane
of the enterocytes of duodenal villi (see FIGURE 22 as well as
Refs. 180, 213, and 753). The presence of NBCn1 in the
enterocytes of mouse duodenal villi, but not of crypts, is
confirmed by lacZ/␤-galactosidase staining of NBCn1/lacZ
transgenic mice (91). In the colons of NBCn1/lacZ mice,
␤-galactosidase staining reveals the presence of Slc4a7
products in villar epithelial cells, but not in crypt epithelial
cells (91). In colonic crypts, qPCR indicates a low level of
NBCn1 transcript expression that is swamped by an ⬃80fold greater abundance of NBCe1-B transcripts (1087).
67
The osteoclast membrane has a ruffled border (facing the resorption lacuna) and a free surface (not facing the lacuna). Although
these domains are sometimes considered equivalent to the apical
and basolateral membranes of epithelia, the free surface is composed of subdomains that contain both apical and basolateral markers of classic epithelia, whereas the ruffled border could be considered as a “giant extracellular lysosome” (662, 991).
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V) Circulatory system. A) Heart. In human heart, NBCn1
cDNAs have been amplified from tissue dissected from
aorta, apex, atria, auricles, ventricles, interventricular septum, and atrioventricular node (200). In rat heart, NBCn1
protein66 is found in myocardial capillaries, the endothelium and vasa vasorum of aorta, and in the endothelia of
atria and ventricles. In ventricular endothelia, immunogold
staining reveals NBCn1 protein in both the luminal and
abluminal (i.e., basal) membranes (213). ␤-Galactosidase
staining of mice with a lacZ insertion in Slc4a7 provides
evidence for NBCn1 promoter activity in the aorta and in
cardiac myocytes of the atria, but not the ventricles (91). A
preliminary study reports a diffuse NBCn1 immunoreactivity in rat ventricular myocytes (311).
VII) Upper digestive system. A) Enamel organ. NBCn1 immunoreactivity is reported in the papillary cell layer (456)
of the enamel organ (FIGURE 20).
MARK D. PARKER AND WALTER F. BORON
HCO3–
Ca2+
Osteoclast
NCX
AE2
3 Na+
Cl–
HCO3–
CO2
CAII
H+
H2O
H+
Na+
++
NHE
cAMP
Ca2+
HCO3– Na+
H-pump
NCX
NBCn1
Sealing
zone
Bone matrix
3 Na+
H+
CaCO3
Resorption lacuna
FIGURE 33. Role of NBCn1 in osteoclasts. Intracellular carbonic anhydrase generates H⫹ that are secreted
by the H-pump into the resorption lacuna to dissolve bone minerals. H⫹ secretion is supported by AE2 in the
⫺
is absorbed across the lacunar membrane by NBCn1 and across
contra-lacunar membrane. Liberated HCO3
the contra-lacunar membrane by AE2. Liberated Ca2⫹ is absorbed by the combined actions of NCX and TRPV5
channels (580, 994).
LacZ/␤-galactosidase staining of NBCn1/lacZ mice reveals
that some of the NBCn1 transcripts detected by qPCR in the
duodenum and colon, and the majority of the NBCn1 transcripts detected in the jejunum and ileum (180), represent
NBCn1 expression in the nonvascular smooth muscle cell
layers, rather than the epithelium (91).
B) Liver. Slc4a7 products have been amplified from cDNA
preparations of rat and mouse liver (189, 213). See Appendix VII for hepatic distribution of NBCn1 according to the
“anti-NBC3” antibody.
C) Pancreas. The detection of NBCn1 transcripts in preparation of mouse pancreas are reported as unpublished data
by Xuo and Muallem (711). Furthermore, ESTs appear to
be common in preparations from mouse pancreas (Appendix VI).
See Appendix VII for pancreatic distribution of NBCn1
according to the “anti-NBC3” antibody.
IX) Lymphatic and immune systems. A) Spleen and macrophages. Slc4a7 products have been amplified from cDNA
preparations of macrophages (630) and spleen (189, 213).
888
X) Endocrine system. A) Thyroid. According to an NCBIcurated database of ESTs, the human thyroid gland is a site
of NBCn1 transcription (Appendix VI).
XI) Urinary system. A) Kidney. NBCn1 transcripts are amplified from cortical preparations from rabbits (427), as
well as the inner stripe of the outer medulla (754), IMCD
(986), and mTAL (694) preparations from rats. AntiNBCn1 antibodies detect an 180-kDa protein in western
blots of rat preparations of the inner medulla as well as the
inner and outer stripes of the outer medulla (213, 1014). An
anti-NBCn1-Nt antibody, but not an anti-NBCn1-Ct antibody, detects NBCn1 protein in the renal cortex (213),
where NBCn1 expression appears to be less than in the medulla (213). LacZ/␤-galactosidase staining indicates that
NBCn1 expression in mouse cortex may predominantly represent vascular expression in the afferent arterioles and renal
corpuscles (91). No evidence of NBCn1 promoter activity is
detected in cortical collecting ducts (CCDs, Ref. 91).
At the cellular level, anti-NBCn1 antibodies detect a basolaterally located protein in mTAL epithelial cells (334, 431,
491, 530, 754, 797, 1014, 1026), intercalated cells in the
inner stripe of the outer medulla (1014), ␣-intercalated cells
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TRPV5
Ca2+
⫺
TRANSPORTERS
Na⫹-COUPLED HCO3
in the IMCD (694, 1014), and renal papilla epithelial cells
(754) of rats. Only an anti-NBCn1-Nt antibody detects
NBCn1 protein at the basolateral membrane of a subset of
outer medullary collecting ducts (OMCD) intercalated cells
(213). This presence of NBCn1 in rat mTAL, IMCD, and
OMCD matches the pattern of Slc4a7 promoter activity
disclosed by lacZ/␤-galactosidase staining (91). In these
mice, renal Slc4a7 promoter activity is particularly robust
in the epithelium lining the renal pelvis.
See Appendix VII for renal distribution of NBCn1 according to the “anti-NBC3” antibody.
B) Bladder. Slc4a7 promoter activity has been detected in
nonvascular smooth muscle cells from mouse bladder (91).
See Appendix VII for distribution of NBCn1 in the epididymis according to the “anti-NBC3” antibody.
B) Female. NBCn1 transcripts have been detected by RTPCR of mouse ovary, uterus, and vagina (599), and ESTs
are abundant in mouse mammary gland preparations
(Appendix VI). NBCn1 protein is present in the lobular
acini of the human breast (182). Slc4a7 promoter activity
has been detected in the myometrium of the uterus of
mice (91).
G) PHYSIOLOGICAL ROLES OF NBCn1.
We have seen that NBCn1
has a broad distribution throughout the body and likely
supports HCO3⫺ secretion across a number of epithelia and
contributes to pHi regulation in all of the cell types in which
it is located. Further physiological roles are suggested by
characteristics exhibited by NBCn1-null mice, although
primary versus secondary effects of NBCn1 loss have yet to
be distinguished.
I) General. A) pHi regulation. DIDS-insensitive (or poorly
DIDS-sensitive) Na/HCO3 cotransport, a strong indicator
of NBCn1 activity, contributes to pHi regulation in many
tissues, including the choroid plexus (113), duodenum
(427), renal mTAL (530, 694) and IMCD (754), as well as
ureter (13). NBCn1 is also strongly implicated as a contributor to pHi regulation of mouse vascular smooth muscle
cells, although, as discussed below, overall Na/HCO3 transport in these cells is partly sensitive to DIDS (90). As discussed in footnote 54, NBCn1 is unlikely to be responsible
for the DIDS-insensitive, EIPA-sensitive Na/base transport
that has been detected at the apical membrane of OMCD ␣
intercalated cells.
B) Potential contribution to CSF secretion. In choroid
plexus epithelia (FIGURE 28), the basolateral presence of
NBCn1 protein (213, 755, 756) parallels the distribution of
NBCn2. In light of the prominent role played by NBCn2 in
CSF secretion (429), it has been suggested that the role
played by NBCn1 in CSF formation may be less significant.
Indeed, in some strains of mice, NBCn1 in the CPE is predominantly at the apical membrane (216, 470), where the
protein would not be in a position to contribute to CSF
secretion. Moreover, in NBCn2 and NBCe2 knockout
mice, endogenous NBCn1 does not compensate for defective CSF secretion (216, 470).
III) Peripheral nervous system. A) Potential contribution to
neuronal excitability. Although NBCn1 transcripts are
present in neurons cultured from trigeminal ganglions of
rats, the NCBT activity in these cells is fully blocked by
DIDS, an observation that is inconsistent with the relative
DIDS insensitivity of NBCn1 in oocytes (see Ref. 408).
IV) Circulatory system. A) Tone and contractility of vascular smooth muscle. In the vascular smooth muscle cells of
mice, Na⫹-dependent HCO3⫺ transport makes a major contribution to the recovery of pHi from an acid-load (90). The
presence of CO2/HCO3⫺ in the extracellular fluid contributes to enhanced myogenic tone and the ability to maintain
contractile ability during sustained agonist exposure, presumably due to transporter-mediated HCO3⫺ uptake. Two
pieces of data speak to the importance of NBCn1 in mediating this HCO3⫺ uptake. 1) At the transcript level, NBCn1
is the only Na⫹-dependent Slc4 family member detectable in mesenteric, coronary, and cerebral arteries, and
2) siRNA-directed knockdown of NBCn1 (to ⬃50% normal levels) reduces both the steady-state pHi of these cells
and the rate at which their pHi recovers from an acidload (90). One usual aspect of these studies is that the pHi
recovery in mesenteric arteries is unusually DIDS sensitive (90) whereas, as mentioned above, NBCn1 activity is
relatively DIDS insensitive in other tissues and heterologous expression systems.
V) Musculoskeletal system. A) Osteoclast survival and
function. Treatment of osteoclasts with CSF-1 results in an
increase in pHi via a mechanism that depends on Na⫹ and
HCO3⫺, but that is not sensitive to DIDS or EIPA—the hallmarks of NBCn1 activity. Because treatment of osteoclasts
with CO2/HCO3⫺ reduces apoptosis in osteoclasts, an effect
further promoted by the addition of CSF-1, it has been
proposed that NBCn1, by raising pHi and/or [HCO3⫺]i, inhibits caspase activity and thereby promotes osteoclast sur-
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XII) Reproductive system. A) Male. NBCn1 transcripts
have been detected by RT-PCR of mouse testis, epididymis,
and vas deferens (599) as well as by Northern blot of human
testis (420). NBCn1 protein (213) has been detected in
preparations of rat epididymis (213).
II) Central nervous system. A) Potential contribution to
neuronal excitability. Being widely expressed in neurons
throughout the brain, NBCn1 likely contributes towards
control of neuronal excitability, as has been demonstrated
for other NCBTs, such as NBCe1 (FIGURE 24).
MARK D. PARKER AND WALTER F. BORON
vival (112). More recently, NBCn1 has been suggested to
play a direct role in reabsorbing the HCO3⫺ liberated from
the hydroxyapatite matrix during bone remodeling (797).
This observation is supported by the presence of NBCn1
protein in the ruffled membrane that faces the resorption
space (lacuna, see FIGURE 33) and the decreased bone absorptive capabilities of osteoclasts when NBCn1 abundance
is reduced in these cells by shRNA (797).
that maintains sperm in a quiescent state (771). On the
other hand, as discussed above and in Appendix VII, the
anti-NBC3 antibody has not been a reliable tool. Independent verification of this apical polarity of NBCn1 distribution is presently lacking. If NBCn1 were instead basolaterally disposed, it could contribute towards HCO3⫺ secretion,
and thence fertility, as proposed for NBCe1 (p. 73).
H) CAUSES OF NBCn1 UPREGULATION. NBCn1 transcript and pro-
VII) Lower digestive system. A) Transepithelial HCO3⫺ secretion in intestines. NBCn1 is present in the basolateral
membranes of epithelia in the lower digestive system (FIGURE 22), enabling the transporter to contribute to the basolateral step of transepithelial HCO3⫺ secretion into the gut
lumen and thereby protect the mucosa from gastric acid.
The importance of NBCn1 for this process is demonstrated
by a substantial reduction in the basal and forskolin-stimulated rates of HCO3⫺ secretion by the duodena of NBCn1null mice (180).
VIII) Urinary system. A) Enhancement of renal NH4⫹ excretion. The renal medullary thick ascending limb is a major
site of NH4⫹ reabsorption, which occurs as NH4⫹ enters the
cell across the apical membrane via NKCC2 and the renal
outer medullary K⫹ channel (ROMK) and then sheds a
proton—thereby acidifying the cell—to form NH3 (FIGURE
34). This NH3 exits across the basolateral membrane and
then enters the medullary collecting duct, where it is
trapped as NH4⫹ which appears in the urine and thereby
plays a major role in urinary acid secretion (316). One
would expect that NBCn1, present at the basolateral
membrane of mTAL cells, would tend to neutralize pHi
during NH4⫹ reabsorption. Indeed, NBCn1 protein is
upregulated during metabolic acidosis and downregulated during metabolic alkalosis. Furthermore, the influx
of ammonium and methylammonium in NBCn1-expressing Xenopus oocytes is stimulated in the joint presence of
Na⫹ and HCO3⫺ (556, 557). However, in the absence of
an acid load, NBCn1-knockout mice lack an obvious
renal phenotype (93).
IX) Reproductive system. A) Possible role in HCO3⫺ reabsorption and/or secretion in the epididymis. An apical distribution of NBCn1 protein in certain cells along the rat
epididymis is indicated by the use of the “anti-NBC3” antibody discussed in Appendix VII. At the apical membrane,
NBCn1 would be positioned to reabsorb HCO3⫺ from the
epididymal fluid, contributing to the luminal acidification
890
tein abundance are typically increased by maneuvers that
elicit an acidosis, reflecting a general pattern of increased
abundance of acid extruders (e.g., NBCe1 and NHE1) and
decreased abundance of acid loaders (e.g., AE3) under these
conditions.
I) Central nervous system. A) Increased transcript and protein abundance in brain in response to acidosis. In primary
cultures of rat hippocampal neurons, lowering extracellular
pH below 6.8, a maneuver that presumably lowers pHi to
some extent, results in an increase in NBCn1 protein levels
(202). The abundance of NBCn1 transcripts and protein in
the brain is increased in a rat model of chronic metabolic
acidosis (709).
B) Increased protein abundance in response to hypercapnia.
Chronic hypercapnia generally increases NBCn1 protein
abundance in the neonatal, but not adult, mouse cerebral
cortex (463), which may help to counter the acidifying effects of hypercapnia.
II) Circulatory system. A) Increased transcript abundance
and transporter activity in heart during pressure-overload
hypertrophy. Hypertrophy of ventricles in rats with constricted aortas is accompanied by an increase in ventricular
NBCe1 and NBCn1 transcript abundance (1071) and an
increase in HCO3⫺-dependent acid extrusion in myocytes
isolated from the hypertrophic ventricles. The presence of
NBCn1 protein has yet to be demonstrated in ventricular
myocytes. Indeed, the authors do not exclude the possibility
that the ventricular NBCn1 transcripts may originate from
nonmyocytes. Nevertheless, they suggest that NBCn1 contributes to an increased intracellular Na⫹ load in hypertrophic myocytes, which would tend to reverse the Na-Ca
exchanger, and thereby promote arrhythmia and reperfusion injury (1071).
B) Increased protein abundance in response to hypercapnia.
Chronic hypercapnia generally increases NBCn1 protein
abundance in the neonatal, but not adult, mouse heart (463).
C) Upregulation of NBCn1-like activity in cardiac myocytes by ANG II. In cat cardiac myocytes, 10⫺7 M ANG II
stimulates HOE64268-insensitive pHi recovery from an
acid-load (presumed to represent the sum of NBCe1 and
68
An NHE1 blocker.
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VI) Upper digestive system. A) Transepithelial HCO3⫺ secretion in salivary gland. The concerted action of NBCe1 and
NBCn1 in the basolateral membranes of striated duct epithelia
could support transepithelial HCO3⫺ secretion into the saliva
⫺
(FIGURE 21B). A suggested role for NBCn1 in apical HCO3
salvage in salivary gland duct cells lacks evidence that NBCn1
resides in the apical membrane of these cells.
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
Lumen
Tight junction
Interstitial fluid
Na+
NH4
ROMK
NH3
Na+
H+
NHE1
NH3
NH4+
NKCC2
2
Cl–
H+
H2O
CO2
NBCn1
HCO3–
Na+
Cl–
H+
HCO3–
cAMP
AE2
mTAL epithelial cell
FIGURE 34. Role of NBCn1 in the renal medulla. To avoid absorption of ammonia into the blood, NH4
traveling along the nephron bypasses the renal cortex by passing through the medullary interstitium to the
⫹
collecting tubules. NH4
enters thick ascending limb epithelia via K⫹ channels (ROMK) and the Na/K/Cl
cotransporter. NH3 is absorbed across the basolateral membrane into the medullary interstitium, perhaps via
⫺
that enters the cell via NBCn1. The basolateral
a channel. Residual H⫹ is extruded by NHE and titrated by HCO3
Na-pump has been omitted for clarity.
NBCn1 action, Ref. 223). However, this same dose of ANG
II inhibits a DIA that is blocked by S085969 (presumed to
represent isolated NBCe1 action, Ref. 224). Therefore, the
authors of the study conclude that the stimulatory effect of
ANG II upon NCBT activity in cat cardiac myocytes represents activation of NBCn1, rather than of NBCe1. However, although some electroneutral NCBT activity is evident
in cardiac myocytes (1072), compelling evidence for
NBCn1 expression in cardiac myocytes, as opposed to endothelia, is presently lacking.
A pharmacological dissection of the pathway of NCBT activation in cat cardiac myocytes by De Giusti and co-workers led the authors of the study to propose that the stimulatory effect of ANG II upon NBCn1 involves activation of
NADPH oxidase, generation of reactive oxygen species
(ROS), ROS-induced release of mitochondrial ROS, and
stimulation of the extracellular-signal regulated kinase
(ERK) signaling pathway (223, 224).
69
An NCBT inhibitor of undemonstrated specificity.
III) Urinary system. A) Increased transcript and protein
abundance in kidney in response to acidosis. Multiple reports demonstrate that chronic metabolic acidosis upregulates NBCn1. NBCn1 transcript abundance is increased in
the rodent kidney by the oral administration of NH4Cl
(207, 688),70 oral administration of HCl (207), or by the
acidosis that accompanies hyperkalemia (664). Compared
with wild-type controls, NBCe2-null mice exhibit a slightly
greater abundance of NBCn1 transcripts and a compensated metabolic acidosis (341). At the level of NBCn1 protein, the acidosis that follows oral administration of NH4⫹
increases the levels of NBCn1 protein (530, 664), consistent
with the increase of NBCn1-like activity in the isolated
mTAL of these animals (694), as discussed above. NBCn1
protein levels in the mTAL also increase during the acidosis
that accompanies Li⫹-induced nephrogenic diabetes insipidus (491) and hyperkalemia (431). Finally, NBCn1 protein
70
One preliminary report found that rat renal NBCn1 transcript
abundance was not increased by NH4Cl feeding, but that NBCn1
protein levels were increased (664).
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Na+
H-pump H+
NHE3
++
cAMP
+
MARK D. PARKER AND WALTER F. BORON
abundance in the ST-1 mTAL cell line rises following a 24-h
exposure to a medium that is acidic (pH 6.8) or that contains 10 mM NH4Cl (557).
B) Increased protein abundance in response to hypercapnia.
Chronic hypercapnia generally increases NBCn1 protein
abundance in the neonatal, but not adult, mouse kidney (463).
I) CAUSES OF NBCn1 DOWNREGULATION.
Maneuvers that downregulate NBCn1 have only been reported in the brain and
kidney.
B) Apparent lack of decreased protein abundance in response to alkalosis. Note that in cultured rat hippocampal
neurons, raising extracellular solution to pH 8.3 does not
significantly change NBCn1 protein levels, although it
might be noted that the high pHo does not cause these
neurons to acquire a substantially higher pHi (202).
II) Urinary system. A) Decrease in protein abundance in
response to ureteral obstruction. In the renal mTAL of rats
in which both ureters are occluded for 24 h by tying with a
silk ligature, NBCn1, together with NKCC2 (previous
topic), protein abundance is substantially decreased four
days after ligature release (1024).
In rats in which only one ureter is obstructed within 48 h of
birth, NBCn1 protein abundance is unchanged after 7 wk in
the continuously obstructed kidney but is increased ⬃30%
in the contralateral unobstructed kidney (1025), a pattern
consistent with a compensation to the metabolic acidosis
that accompanies ureteral obstruction. After 14 wk of continuous unilateral obstruction, both kidneys exhibit a
⬃20% decrease in NBCn1 protein abundance (1025), a
pattern consistent with a contribution to the metabolic acidosis.
B) Apparently decreased protein abundance in pendrin
knockouts. Pendrin/Slc26a4 is a Cl-HCO3 exchanger that
mediates the secretion of HCO3⫺ across the apical membrane of renal ␤- and non-␣/non-␤-intercalated cells (819).
The physiological importance of pendrin is underlined by
the observation that perfused collecting ducts from pendrin-null mice absorb rather than secrete HCO3⫺ (819). One
immunohistochemical study suggests that NBCn1 protein
892
C) Decreased protein abundance in response to FK506 administration. NBCn1 protein abundance falls by ⬃20%
during and following the renal tubular acidosis that accompanies administration of the calcineurin inhibitor FK506
(655). Inasmuch as acidosis per se appears to increase
NBCn1 protein abundance, this seemingly counterintuitive
observation in FK506-treated mice probably reflects an effect of calcineurin blockade in these animals.
D) Decreased protein abundance in response to alkalosis.
Hypercalcemia caused by infusion of parathyroid hormone
(PTH) inhibits acid secretion by the proximal tubule but
causes a mild, paradoxical metabolic alkalosis and decreased
urine pH. The paradox is at least in part due to an increased
expression of the B1 subunit of the V-type H⫹ pump in the
inner medullary collecting duct (1026). The challenge also
causes a reduction in ammonium excretion. Indeed, rats
treated with PTH have a ⬃60% decrease in NBCn1 protein
abundance in the basolateral membranes of their mTAL and
IMCD epithelia (1026). The downregulation of NBCn1 expression in the mTAL would presumably reduce urinary
NH4⫹ (i.e., acid) excretion by the mechanism above. Furosemide-induced alkalosis also reduces NBCn1 protein abundance in the renal medulla of rats (754).
J) CONSEQUENCES OF NBCn1 DYSFUNCTION.
Much of what we
know about the pathology of NBCn1 dysfunction comes
from NBCn1-null mice that, exhibiting hearing and vision
loss, are a potential model of human Usher 2B syndrome.
Human genetic studies have linked the SLC4A7 locus with
substance abuse, neuropathy, lead accumulation, and
breast cancer. These studies are considered below.
I) Central nervous system. A) Neuroprotection from glutamate cytotoxicity in a model of stroke-induced epilepsy.
Ischemic injury, such as might follow a stroke, causes the
release of glutamate (134) and can be associated with lowering of extracellular magnesium levels (reviewed in Ref.
635). Both glutamate addition (920) and magnesium depletion (42, 933)71 induce seizure-like activity in hippocampal
neurons and are models used to study the etiology of strokeinduced epilepsy. Furthermore, glutamate can cause longterm changes in neuronal excitability and cell death (920).
71
By analogy to the inhibitory effect of intracellular Mg2⫹ on
NBCe1-B, one might expect Mg2⫹ depletion to activate other AIDincluding NCBTs, such as NBCn1, which could lower the seizure
threshold of hippocampal.
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I) Central nervous system. A) Decreased protein abundance
in brain in response to hypoxia. As is usually the case for
NDCBE and NBCn2, NBCn1 protein levels generally fall in
response to chronic continuous hypoxia in the hippocampus, cerebral cortex, subcortex, and cerebellum of neonatal
and adult mice (174). One possibility for these effects is that
the hypoxia downregulates a range of energy-requiring systems, including NCBTs. Another is that the hypoxia triggers hyperventilation and thus respiratory alkalosis, which
indirectly causes a downregulation of NCBTs.
levels are decreased in the cortical collecting ducts of pendrin-knockout mice, particularly in those cells that usually
express pendrin (492). However, as discussed above and in
Appendix VII, the anti-NBC3 antibody used in this study
yields results that conflict with those obtained using other
methods of detecting NBCn1 protein.
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
In cultures of mouse hippocampal neurons, NBCn1 knockdown is neuroprotective inasmuch as fewer NBCn1-null
than wild-type neurons die when glutamate is applied in the
nominal absence of extracellular Mg2⫹ (202). The neuroprotective role of NBCn1 knockdown in these experiments
is consistent with the hypothesis that a reduction in pHi
reduces neuronal excitability and is also in accordance with
the higher seizure threshold of brain slices from NDCBE
and NBCn2 knockout mice (429, 889), the enhanced neuronal survival in NHE1-null mice following ischemic injury
(614), and the proposed enhancement of neuronal excitability by NBCe1.
C) Genetic linkage with propensity towards substance abuse.
A chromosomal locus associated with a high degree of allelic
variation in substance abusers includes the SLC4A7 gene.
Ishiguro and co-workers (423) studied the association of addictive behavior with the frequency of occurrence of 22 singlenucleotide polymorphisms (SNPs) in the SLC4A7 gene region. Of these 22 SNPs, 12 occurred with a significantly
increased frequency in genomic DNA samples from substance abusers compared with control samples. Of these 12
SNPs, 5 are located in exons but only one, designated
rs3755652, changes the predicted coding sequence of the
NBCn1 protein, producing a Glu to Lys mutation midway
through splice cassette II. The effect of any of these SNPs on
the functional expression of NBCn1 activity is untested.
Ishiguro et al. note that far more than 22 SNPs may need to
be examined in the gene region,72 and they do not exclude
the importance of SNPs in neighboring genes. Furthermore,
SLC4A7 was not sequenced in its entirety for mutations.
Thus the role, if any, of NBCn1 in the etiology of addictive
72
Indeed, as of May 2012, the number of allelic SLC4A7 variations
in the NCBI SNP database stands at 1449, 85 of which are located
in exons and 46 of which alter the predicted NBCn1 coding sequence.
II) Sensory organs. A) Vision and hearing loss: a potential
model of Usher syndrome. One strain of NBCn1 knockout
mice develop blindness and auditory impairment due to the
degeneration of photoreceptors in the retina (93) and of
hair cells in the inner and outer ear (93, 607). The signs
manifested in the knockout mouse are similar to those of
Usher syndrome 2B, a disease once linked to the human
chromosomal locus 3p23–3p24.2 (383), which is close to
the location of the human SLC4A7 gene. However, the
original assignment of an Usher locus at 3p23–24 has since
been retracted (384). Ironically, molecular evidence gathered in the interim suggests that NBCn1 could be part of the
Usher protein network (790), disruption of which is considered to be the molecular basis of Usher syndrome (789).
In the absence of a clear demonstration that mutations in
human SLC4A7 gene are linked to Usher syndrome 2B, the
Slc4a7-null mouse remains only a potential model of the
human disease. In a screen of 172 individuals with Usher
syndrome, no mutations localized within the exons that
encode the NBCn1-A product (549). This observation does
not exclude the possibility that disease-associated mutations could be located in the promoter, introns, or exons
included in NBCn1 variants besides NBCn1-A (e.g., those
that include cassette III). However, the majority of the 172
individuals in the study exhibited mutations in known Usherassociated genes (549).
B) Genetic linkage with central cornea thickness in mice.
Strains of mice with thicker corneas tend to exhibit, in their
corneas, greater abundance of certain transcripts, including
those encoded by Slc4a7 (601). Corneal thickness in the
studied mouse strains is mostly determined by the number
of lamellae in the corneal stroma, an observation presumed
to be due to altered keratocyte function (601). The role of
NBCn1 in this likely complex phenotype, has not been determined.
III) Peripheral nervous system. A) Possible role in hereditary sensory neuropathy. The human SLC4A7 gene locus
falls within the boundaries of a region (3p24) that has been
linked to a mild sensory neuropathy associated with a
chronic cough and gastroesophageal reflux (505). In their
2004 study, Kok et al. (504) sequenced the coding exons of
SLC4A7 from genomic DNA amplified from the white
blood cells of at least one affected individual and found no
nonsynonymous mutations. On these grounds alone, the
authors exclude SLC4A7 as a candidate gene for the neuropathy. However, this study does not identify nonsynonymous mutations in the coding exons of any other genes in
this candidate region and does not consider the possibility
that causal mutations may be located in regulatory regions
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B) Possible contribution to enhanced NMDA-associated
neurotoxicity in acidosis. NMDA, an agonist for the
NMDA class of ionotropic glutamate receptors, causes cell
death through excitotoxicity. As judged by caspase-3 activation, NMDA-induced neuronal death is greater in acidotic rats than in wild-type rats (709). Although upregulation of NBCn1 in acidotic rats normally counters intracellular acidosis, NBCn1 is not upregulated by acidosis in
NMDA-treated rats (709). The authors suggest that the
lack of enhanced NBCn1 activity might render neurons
more susceptible to acid injury in NMDA-treated rats, resulting in increased cell death (709). This hypothesis is consistent with the anti-apoptotic effect of NBCn1, apparently
mediated by a rise in pHi, in CSF1-stimulated osteoclasts.
An alternative explanation provided by those authors is
that NMDA is killing the cells before they have a change to
upregulate NBCn1 (709). It is unknown how other neuronally expressed NCBTs are affected by NMDA treatment in
this model.
behaviors is unknown. However, the association seems reasonable, given the potential contribution of NBCn1 toward
control of neuronal excitability.
MARK D. PARKER AND WALTER F. BORON
of the SLC4A7 gene or in additional genes outside of 3p24.
Therefore, it is premature to exclude a role for SLC4A7 in
the physiopathology of this syndrome.
NBCn1-null mice are mildly hypertensive at rest, an observation that accords with the finding that isolated, precontracted arteries from these mice exhibit a reduced ability to
relax in response to acetylcholine application (92). Underlying this phenotype is reduced pHi in vascular cells. Moreover, endothelial nitric oxide synthase (eNOS) is inhibited
by acidosis in endothelial cells, whereas rho-kinase signaling is inhibited in vascular smooth muscle cells, rendering
contraction of isolated arteries less sensitive to Ca2⫹ (92).
B) Suggested linkage with blood lead accumulation. A genetic linkage study suggests a quantitative trait locus for
erythrocyte lead accumulation, with a linkage peak near the
gene locus of 62 genes or putative genes, including human
SLC4A7 (1033, 1034). NBCn1 is the only transporter encoded by any of these 62 genes, leading the authors to
conclude that NBCn1 affects lead transport. However, this
conclusion must be regarded with caution because the authors present no evidence that 1) erythrocytes express
NBCn1,73 2) NBCn1 mediates lead transport, or 3) mutations in SLC4A7 affect lead transport in any cell type. On a
related note, the author of one study on red blood cells
reports evidence consistent with AE1-mediated transport of
PbCO3⫺ (886).
V) Lower digestive system. A) Potential role in susceptibility to duodenal ulcers. Helicobacter pylori markedly inhibits the ability of the duodenal epithelium to increase HCO3⫺
secretion in response to the appropriate stimuli (984), leading to duodenal ulceration (reviewed in Ref. 639). Because
NBCn1 action supports duodenal HCO3⫺ secretion, NBCn1
defects could increase susceptibility to duodenal ulceration.
VI) Reproductive system. A) Linkage to breast cancer. An
association between NBCn1 and cancer was first broached
In the first study that linked cancer with SLC4A7, Chen and
co-workers (182) identified NBCn1 as a tyrosine kinase
substrate expressed in the lobular acini of the breast. The
authors make two observations that link NBCn1 to cancer.
1) In the MCF10AT cell line model of breast cancer progression, NBCn1 tyrosine phosphorylation was increased
threefold in premalignant and low-grade-lesion-like cells
but was decreased twofold in high-grade-lesion-like cells
(182). However, the effect of phosphorylation events on the
functional expression of NBCn1 activity in tumor cells is
untested.
2) NBCn1 protein abundance is decreased in MCF10AT
cells and in most of the cancerous breast-tissue samples
examined in their study. However, the downregulation of
NBCn1 as a contributory factor in breast cancer seems
counterintuitive: NBCn1 expression would help cancer
cells to maintain a normal pHi in the acidic environment of
a tumor and would enhance local extracellular acidity.
Other studies provide evidence that cancer is associated
with upregulation of NBCn1. The breast-cancer cell line
MCF-7, when overexpressing a truncated ErbB2 receptor,
exhibits enhanced acid-extruding capability in part by increasing the abundance of NBCn1 protein in the plasma
membrane (546). A report by Wong et al. (1041) demonstrates the importance of NCBT activity, including a DIDSinsensitive component, to pHi regulation in two human and
one murine breast-cancer cell lines. Note that both groups
discount a significant role for NBCn1 action in cancer cell
migration (547, 849), although in theory NCBT activity
could contribute to a regulated volume increase.
In summary, the role of NBCn1 in the etiology of cancer
progression is presently speculative, although NBCn1
abundance and phosphorylation may be useful markers for
cancer screening (182).
2. NDCBE (Slc4a8)
73
SLC4A7 products are not among the 340 red cell proteins
identified in a proteomic study by Pasini and co-workers (727), nor
among the 751 proteins reported in a review of the human red cell
proteome (329).
894
The electroneutral Na⫹-driven Cl-HCO3 exchanger NDCBE (encoded by the Slc4a8 gene) exchanges Na⫹
and two HCO3⫺ equivalents for Cl⫺, a function shared by
A) SUMMARY.
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IV) Circulatory system. A) Genetic linkage with hypertension. A genome-wide association study (GWAS) links a single nucleotide polymorphism, rs13082711, in the SLC4A7
gene locus with slightly elevated systolic and diastolic blood
pressure in individuals of European and African ancestry
(961). Inasmuch as NBCn1 is a Na⫹ transporter that is
expressed in the vasculature and kidney, the protein has the
potential to influence blood pressure. However, the SNP is
located ⬃10 kb upstream of any known transcriptional
start site for NBCn1, and the effect of this SNP, or of yet to
be discovered SNPs in the linked region, upon NBCn1 expression has yet to be established.
in a 2003 review by Izumi et al. (426). Since then, 10 studies
have been published concerning the link between susceptibility to breast cancer and a genetic locus marked by an
SNP, rs4973768, that is located in the long terminal exon
that encodes the 3=-UTR of NBCn1 (12, 44, 148, 182, 364,
605, 648, 670, 733, 917). It is important to note that it is
the genetic locus marked by this SNP, rather than the SNP
itself, that is linked to breast cancer susceptibility. Thus it is
possible, as noted by the authors of these studies, that dysregulation of the neighboring NEK10 gene, a UV-stimulated kinase that is independently associated with cancers
(656), could underlie the genetic susceptibility.
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
NBCn2 under certain conditions. NDCBE has four known
variants (-A through -D). In common with several other
NCBTs, NDCBE-B is stimulated by the soluble protein IRBIT.
Although present in many organs, NDCBE is notably abundant in the brain. In neurons, NDCBE-mediated HCO3⫺ influx
enhances neuronal excitability, a role corroborated by a study
of NDCBE-null mice. To date, no human pathologies have
been linked to NDCBE dysfunction.
C) MOLECULAR ACTION OF NDCBE.
When expressed in Xenopus
oocytes, human NDCBE mediates electroneutral codependent Na⫹ and HCO3⫺ influx (i.e., Na⫹ influx requires
HCO3⫺ and vice versa), accompanied by a Na⫹ and HCO3⫺dependent Cl⫺ efflux (337). When NDCBE operates in the
“reverse” direction, that is to say, mediating the coefflux of
Na⫹ and HCO3⫺ from the oocyte, the transport process has
an absolute dependence on extracellular Cl⫺ (337). Thus
NDCBE is a Na⫹-driven Cl-HCO3 exchanger (FIGURE
2⫺
35A). A preliminary study suggests that it is CO3 and not
⫺
HCO3 that is the transported base (Fig. 35, B and C; Ref.
335). An NCBT activity attributed to NDCBE in mouse
IMCD cells is poorly selective for Na⫹ over Li⫹ (35). The
approximate stoichiometry of transport is estimated to be
1Na⫹:2HCO3⫺, which would require the net countertransport of 1 Cl⫺ for electroneutrality (337). Inasmuch as 1) the
estimated unidirectional efflux of 36Cl is manyfold greater
than the estimated fluxes of Na⫹ and HCO3⫺ (337) and 2)
the NDCBE-mediated efflux of Cl⫺ has a trans-side Cl⫺
dependence (719), it seems likely that the net movement of
chloride by NDCBE is accompanied by a much larger component of futile Cl-Cl self-exchange (FIGURE 35D; Ref.
74
This partial human clone (AF107099) is identical along its length
to subsequently cloned human SLC4A8 products. A full-length
mouse Slc4a8 product (now called NDCBE-A) was subsequently
cloned in its entirety by a group that included the same authors
(1029).
75
GenBank nucleotide accession number AF069512.
I) Assignment of transport activity to NDCBE. It is straightforward to overexpress NDCBE in a cell such as a Xenopus
oocyte and convincingly demonstrate NDCBE activity. However, because of the vagaries of Cl⫺ transport, it can be a
challenge to demonstrate that transport activity is indeed due
to NDCBE in a setting where the transporter coexists with
other NCBTs, Cl-HCO3 exchangers (including those from the
Slc26 family), Na-H exchangers, and Cl⫺ channels. Thus reports of NDCBE activity, hereafter referred to as NDCBE-like
activity, can rarely be taken at face value. To illustrate, we will
note the potential difficulties in using three common approaches to test for the presence of NDCBE activity.
A) 36Cl fluxes. An NDCBE should mediate an efflux of 36Cl
that requires extracellular Na⫹ and HCO3⫺, and that is
blocked by DIDS. As discussed in a later section, the human
electroneutral Na/HCO3 cotransporter NBCn2, as expressed in oocytes, does not normally mediate net Cl⫺ transport, but is nonetheless capable of futile Cl-Cl exchange (detected as 36Cl efflux) that requires HCO3⫺ but not Na⫹, and
that is blocked by DIDS. It is not known whether the closely
related NBCn1 can also mediate futile Cl-Cl exchange. Thus a
convincing demonstration of NDCBE activity requires evidence of net Cl⫺ efflux, either from a direct and quantitative
comparison 36Cl influx and 36Cl efflux, or from surface-[Cl⫺]
transients as discussed below. Further complicating matters, in
the absence of extracellular Cl⫺, NBCn2 appears capable of
Na⫹-driven Cl-HCO3 exchange. Even the red cell anion exchanger AE1 has been reported to be capable of exchanging
Cl⫺ for either the NaCO3 or the LiCO3⫺ ion pairs under certain
conditions (303, 304).
B) Net Cl⫺ fluxes. An NDCBE should mediate a net efflux
of Cl⫺. However, in cells that express a Cl⫺ channel plus
NBCe1 or NBCe2, the coupled influx of Na⫹, HCO3⫺, and
net negative charge would hyperpolarize the cell and thus
drive the net efflux of Cl⫺ through the Cl⫺ channel. Voltage-clamp experiments could test this possibility.
C) Washout of intracellular Cl⫺. The net influx of Na⫹ and
HCO3⫺ mediated by an NDCBE should require intracellular
Cl⫺. Unfortunately, it is notoriously difficult to wash Cl⫺
out of cells (115, 850, 851). Moreover, human NDCBE appears to require extracellular Cl⫺ for NDCBE activity (719).
Finally, as noted above, NBCn2 appears to act as a Na⫹driven Cl-HCO3 exchanger in the absence of extracellular
Cl⫺. Thus removing extracellular Cl⫺ for the purpose of Cl⫺
washout could have unintended consequences for key NCBTs.
Thus a demonstration of a pHi recovery from an acid load,
together with dependence on Na⫹ and HCO3⫺ but blockade
by DIDS, is only the beginning of a physiological assignment of NDCBE. The next critical step is to demonstrate net
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B) NOMENCLATURE OF SLC4A8 PRODUCTS. Following the provisional assignment of NBC1 (now called NBCe1) to refer to
Slc4a4 products, and the provisional assignment of NBC2
(now called NBCn1) to refer to Slc4a7 products, two
groups simultaneously reported the cloning of two different
Slc4 products to which they inadvertently assigned the degenerate name NBC3. We now appreciate that the “NBC3”
reported by Pushkin et al. (765) is an Slc4a7 product (139),
whereas the “NBC-3” reported Amlal et al. (35) is a partial
human Slc4a8 product.74 In fact, the report of the partial
sequence postdated the depositing of a full-length human
SLC4A8 product by Grichtchenko et al.75 With the physiological characterization of the SLC4A8 product as a Na⫹driven Cl-HCO3 exchanger, the product was renamed
NDCBE1 (337). With the assumption that the SLC4A8
gene encodes the sole human Na⫹-driven Cl-HCO3 exchanger, we propose to drop the numerical suffix, and refer
to the transporter as NDCBE.
337). Moreover, this Cl-Cl self-exchange has an absolute
requirement for extracellular Na⫹ and HCO3⫺ (337, 719).
MARK D. PARKER AND WALTER F. BORON
Na+
CO32–
NDCBE
Na+
HCO3–
HCO3–
A
Na+
CO32–
B
Cl–
(n) Cl–
NaCO3–
C
Cl–
D
Cl–
(n+1) Cl–
FIGURE 35. Molecular action of NDCBE. Possible molecular mechanisms by which NDCBE could operate in
⫺
an electroneutral mode to exchange Na⫹ and HCO3
equivalents for Cl⫺. Note that we have not considered any
2⫺
/H⫹ cotransport. In the original characterization of NDCBE action, Cl⫺ flux was
models that are based on CO3
estimated to be sixfold greater than Na⫹ flux.
D) THE SLC4A8 GENE. The human SLC4A8 gene maps to 12q13
(337, 673), locus 12q13.13 in version 36.3 of the NCBI human
genome map, and includes at least 28 exons spread over 124 kb
(FIGURE 36A; Ref. 717). SLC4A8 is located between
GALTNT6 (that encodes UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase) and
SCN8A. We discuss alternative splicing of SLC4A8 premRNA in the following section. The intrinsic promoter
activity of a virally derived long-terminal repeat sequence
LTR129, present normally in intron 5 of the human
SLC4A8 gene, is activated in testicular cancer. The transcript promoted by the LTR is a short antisense-SLC4A8
intronic sequence. However, this antisense pre-mRNA
does not appear to affect NDCBE transcript abundance
in seminoma versus normal testicular parenchyma (143).
However, when overexpressed in a testicular carcinoma
cell line, the antisense sequence does have the capability
to reduce NDCBE transcript abundance, acting at the
level of pre-mRNA (327).
E) STRUCTURAL FEATURES AND VARIANTS OF NDCBE. The human
SLC4A8 gene has the capacity to encode at least five variant
products named A–E (FIGURES 36, B and C, and 37). Evidence for other minor variants has also been reported, including NDCBE-D=, which has an alternative 5=-UTR.
NDCBE-A and NDCBE-B share a common Nt, but
NDCBE-A has a longer and different Ct. NDCBE-C and
NDCBE-D are identical to “A” and “B,” respectively, but
their Nt are truncated by 54 amino acid. NDCBE-E has
a longer and different Nt appendage compared with
NDCBE-B. Protein variants A–D exhibit NCBT function
896
when expressed in Xenopus oocytes; NDCBE-E ought to be
functional but has not been tested. The choice of Nt has no
obvious bearing on basal functional expression of NDCBE.
However, variants with the shorter Ct (i.e., NDCBE-B and
-D) show reduced functional expression compared with
variants with the longer Ct (i.e., NDCBE-A and -C). A
comparison of the functional expression of NDCBE-A and
NDCBE-B with an artificial construct that includes neither the
17-amino acid nor the 66-amino acid sequence (FIGURE 37)
demonstrates that the 17-amino acid sequence is inhibitory
to the functional expression of NDCBE. The 66-amino acid
sequence has no effect on the basal functional expression of
NDCBE (717).
I) Sources of variation in coding sequence among NDCBE
variants. Known NDCBE variants differ only in the length
of their Nt and the choice of one of two Ct appendages.
Unique among NCBTs, transcripts that encode each Ct include mutually exclusive 3=-UTR regions.
A) Alternative promoter choice and truncated Nt. Some
variants of NDCBE are truncated by 54 amino acid in their
Nt as a result of alternative promoter choice (717). The
SLC4A8 gene appears to have two promoters (FIGURE 36C).
One promoter (P1) is located just upstream of exon 1 and
promotes transcription from exon 1. Transcription initiated at exon 1 produces pre-mRNAs that can be processed
to form either NDCBE-C, NDCBE-D, or NDCBE-E. Because exons 2– 4 are omitted from NDCBE-C/D transcripts
and because neither exon 1 nor exon 5 contains an initiator
Met, translation of NDCBE-C/D is predicted to begin with
an initiator Met located within exon 6 (that encodes internal Met55 of NDCBE-A/B). Thus in NDCBE-C/D the first
54 amino acids of NDCBE-A/B are absent. The consequence of the loss of this Nt sequence are currently unclear,
but preliminary data suggest that unlike NDCBE-B (722),
NDCBE-D may not be sensitive to stimulation by IRBIT
due to the loss of sequence homologous to that which contains IRBIT binding determinants in NBCe1-B (Parker and
Boron, unpublished data). Translation of NDCBE-E is predicted to begin with an initiator Met located within exon 3.
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Cl⫺ efflux under conditions in which Vm does not change or
in a cell verified to be devoid of electrogenic NBCs or Cl⫺
channels. Finally, it is advisable to demonstrate the presence
of NDCBE protein at the plasma membrane, and show that
knock-down of the protein eliminates the hypothesized
NDCBE activity. With these caveats in mind, we summarize
the reports of Na⫹-dependent Cl-HCO3 exchange activity
and indicate, where possible, which reports are strongly
linked to the accompanying presence of NDCBE itself and
which are unlikely to involve NDCBE.
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
A
Locus 12q13
20 kb
GALNT6
B
SLC4A8
SCN8A
Gene structure
10kb
P2
P1
1
C
2-3
4
5
25
6
28
Transcript variation
P2
M
4
NDCBE-A
5
6
7
24
5
6
7
24
6
7
24
M
4
NDCBE-B
25
a
26
27
*
28
26
27
*
28
*
25
M
25
a
NDCBE-C
1
5
NDCBE-D
1
5
6
7
24
*
25
NDCBE-D
1
5
6
7
24
*
25
M
M
2
3
FIGURE 36. SLC4A8 gene structure and NDCBE transcript variants. Scale diagrams showing the human
SLC4A8 gene locus together with the position of neighboring genes (A), the position of promoters (P1 and P2),
and the position of exons within SLC4A8 (B). Transcript variants are represented, not to scale, as numbered
boxes joined by a horizontal line (C). Each numbered box represents the inclusion of that exon in the mature
transcript. “//” denotes that all five transcripts include exons 7–24. Exons that include the initiator ATG codon
(“M”) and termination codon (“*”) are marked for each transcript. Sequences that are derived from part of a
larger exon sequence are labeled with an “a” (e.g., exon 25a is a subdivision of exon 25). Colored exons, or
parts of exons, correspond to the protein regions that each encodes, which are identically colored in FIGURE
37. Uncolored exons, or parts of exons, denote untranslated 5= and 3= sequence. Exons that are connected
with a dashed line are predicted, but not demonstrated, to be included in the mRNA.
A second promoter (P2) is situated upstream of exon 4 and
promotes transcription of NDCBE from exon 4, which encodes an initiator Met. Transcription from this promoter
produces pre-mRNAs that can be processed to form either
NDCBE-A or NDCBE-B.
B) Alternative Ct and 3=-UTR. NDCBE variants can have
either a 17-amino acid or a 66-amino acid sequence appended to the ⬃30 amino acid that is common to the Ct
of all NDCBE variants. A 66-amino acid Ct appendage is
produced when exons 25a-28 are spliced together, making exon 28 the terminal exon (FIGURE 36C). The 66amino acid appendage (encoded by exons 26 –28) is common to NDCBE-A and NDCBE-C. The role of the 66amino acid Ct is presently unknown; this 66-amino acid
Ct can be removed without any apparent consequence to
functional expression of the transporter in Xenopus
oocytes (717).
An alternative mRNA is produced when the splice machinery does not recognize the exon-25a/intron-25 splice
boundary and a polyadenylation signal located ⬃3 kb
downstream of the start of exon 25 is used to produce a
mature, polyadenylated mRNA. This “long” version of
exon 25 is defined as a composite terminal exon (268). The
17-amino acid appendage (encoded by exon 25) is common
to NDCBE-B, -D, and -E and constitutes an autoinhibitory
domain, inasmuch as a mutant NDCBE that lacks the 17amino acid sequence has a greater functional expression
than an NDCBE that includes the 17-amino acid sequence
(717).
The 3=-UTR of NDCBE-B/D/E (comprised of exon 25 sequence) is different from and shorter than that of NDCBEA/C (comprised of exon 28 sequence), probably accounting
for the two groups of transcript sizes (9.5–12 kb and 4.4 –
6.3 kb) observed in Northern blots (35, 337, 717). This
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P1
MARK D. PARKER AND WALTER F. BORON
Nt
TMD
1–5
100 aa
6–9
Ct
10–14
NDCBE-A
66
16
1,093
AID
1,044
NDCBE-B
17
16
NDCBE-C
∆54
NDCBE-D
∆54
66
1,040
991
17
1,071
43
17
FIGURE 37. NDCBE protein variants. Scale diagram of protein variants that are encoded by the transcripts
represented in FIGURE 36C. Horizontal bars represent protein sequence laid out from Nt to Ct. Vertical bars
represent position of ␣-helical TMs. Protein cassettes are labeled with a number denoting their size in amino
acids and colored to denote their genetic origin as shown in FIGURE 36C. A color-matched protein sequence
alignment of the variants is provided in Appendix V.
choice of alternative 3=-UTRs is a mechanism of variation
that is unique among SLC4 products.
NDCBE-C has been cloned from human brain, heart, and
kidney cDNA preparations (717).
C) Cloned NDCBE variants that are demonstrated or likely
to exhibit NCBT activity. There are five NDCBE protein
variants, the features of which are described below and
depicted in FIGURE 37. GenBank protein accession numbers
for the variants discussed in this section are provided in
Appendix IV.
4) NDCBE-D and -D= (NCBT activity demonstrated).
NDCBE-D is identical to NDCBE-B, except that it does
not include the first 54 amino acids of the NDCBE-B Nt.
NDCBE-D includes the autoinhibitory 17-amino acid Ct
appendage. NDCBE-D protein is the shortest of the four
variants. Full-length NDCBE-A has been cloned from
human brain and kidney cDNA preparations (717).
NDCBE-D= is identical to NDCBE-D, except for a 5= extension to exon 6, which extends the 5=-UTR and is specifically
expressed in the heart. The relevance of the 5=-UTR extension is presently unclear (717).
1) NDCBE-A (NCBT activity demonstrated). Human
NDCBE-A is the counterpart of the archetypal mouse
NDCBE variant that was reported in Reference 1029.
NDCBE-A includes the full-length Nt and the 66-amino
acid Ct appendage. NDCBE-A protein is the longest of the
four variants. Full-length NDCBE-A has been cloned from a
mouse renal cell line (1029) and human brain (717) cDNA
preparations.
2) NDCBE-B (NCBT activity demonstrated). NDCBE-B is
the archetypal human NDCBE clone reported in Reference 337. NDCBE-B includes the full-length Nt and the
autoinhibitory 17-amino acid Ct appendage. As a consequence, NDCBE-B has a lower per-molecule activity than
NDCBE-A when expressed in Xenopus oocytes. Fulllength NDCBE-B has been cloned from human brain
cDNA preparations (337, 717).
3) NDCBE-C (NCBT activity demonstrated). NDCBE-C is
identical to NDCBE-A, except that it does not include the
first 54 amino acids of the NDCBE-A Nt. NDCBE-C includes the 66-amino acid Ct appendage. Full-length
898
5) NDCBE-E (NCBT activity untested). A singleton cDNA
from brain (GenBank DNA accession no. AB018282) that
appears to represent a full-length mRNA would, if translated, produce a protein product in which the 16 amino
acids encoded by exon 4 of NDCBE-A/B are replaced by 43
amino acids encoded by exon 3. Such modification of the Nt
appendage is unlikely to eliminate NCBT activity and thus
NDCBE-E is likely to be functional.
D) Predicted NDCBE variants. 1) Partial clones from human
cDNA. A number of other human NDCBE cDNA sequences
that have been deposited in GenBank have a structure similar
to NDCBE-C/D, in that as their transcription begins at a position that is upstream of, but omits, exon 2. If a full-length
NDCBE-C/D clone was modified to include any of these partial sequences (e.g., GenBank DNA accession nos. CN286464
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NDCBE-E
⫺
TRANSPORTERS
Na⫹-COUPLED HCO3
and DB090766), the altered sequence of the Nt appendage
ought not eliminate NCBT function.
E) Other NDCBE variants. 1) An unusual variant that represents only the isolated Nt. A singleton NDCBE cDNA amplified from thymus (GenBank DNA accession no. AK128321)
includes exons 4 –11, and exon 12, which becomes a composite terminal exon that includes intron 12 sequence and a polyadenylation signal therein. Such a transcript would encode
residues 1–314 of NDCBE-C/D plus 15 novel amino acids
encoded by the exon 12 extension, followed by a termination
codon: that is, most of the soluble Nt of NDCBE-C/D. It is not
clear whether this protein product, truncated within the Nt,
would be stable. This cDNA is reminiscent of isolated Nt
variants of NBCn1 and NBCn2.
2) Putative variants cloned from rodent cDNA (potentially
legitimate transcripts, NCBT activity unlikely). Mouse
Slc4a8 encodes NDCBE-A, but it is unknown whether the
gene has the capability to encode orthologs of NDCBE-B,
-C, or -D. The rat Slc4a8 gene has not been demonstrated to
produce orthologs of any of the four human variants. The
three reported transcripts from rat kidney are named
“NDCBE1-A,” “NDCBE1-B,” and “NDCBE1-C” and appear to be unique to rat and are not the same as the human
NDCBE-A/B/C variants. In fact, none of the three rat
clones is predicted to encode a functional transporter.
Rat “NDCBE1-A” lacks putative TM6 and part of putative
TM7. Rat “NDCBE1-B” lacks 25 amino acids in the cytoplasmic Nt close to TM1; at least for NBCe1, this sequence
is necessary for functional expression of NCBT activity
(575). Rat “NDCBE1-C” lacks both of the regions missing
from “NDCBE1-A” and “-B”. The rat Slc4a8 gene does
have the potential to encode a complete ortholog of the
human NDCBE-A variant, but cDNA representing such a
transcript has yet to be cloned.
F) DISTRIBUTION OF NDCBE. NDCBE expression is particularly
abundant in brain, specifically in neurons, although
NDCBE transcripts are also abundant in the testis and are
expressed to a lesser extent in many other organs. The distribution of NDCBE in specific organ systems is discussed
below. The distribution of NDCBE is summarized and compared with that of other NCBTs in TABLE 4.
I) Central nervous system. A) Brain. In Northern blots and
RT-PCR analysis of mouse and human RNA preparations,
At the regional level, NDCBE-A/B transcripts are present in
RNA preparations from amygdala, caudate nucleus, cerebellum, cerebral cortex, corpus callosum, hippocampus,
medulla, substantia nigra, and thalamus (337, 1029). RTPCR amplifies NDCBE, as well as NBCe1 and NBCn1,
transcripts from basal ganglion, occipital cortex, hypothalamus, and frontal lobe (327). The widespread distribution of NDCBE throughout the CNS of rats and mice is
confirmed by the use of anti-NDCBE antibodies (553, 889).
The use of a pan-NDCBE antibody further extends the distribution of NDCBE to the entorhinnal cortex, midbrain,
striatum, pons, thalamus, and olfactory bulb of rats (553).
An anti-NDCBE-A/C antibody does not detect NDCBE in
the corpus callosum of mice (889).
At the cellular level, and in brain slices, anti-NDCBE-A/B
antibodies detect NDCBE in hippocampal pyramidal neurons of human (214), rat, and mouse (176; decreasing in
abundance from CA1 to CA3) and in cerebellar Purkinje
cells of rat (214) and mouse (176). In mouse brain slices,
NDCBE-A/B is further detected in cerebellar granule cells,
white matter, substantia nigra, and neurons of the brain
stem (176) as well as in unipolar brush cells and cartwheel
cells (interneurons) in the dorsal cochlear nucleus and in
unipolar brush cells in the cerebellum (489).
NDCBE-A/B is mainly expressed in neurons, as evidenced
by staining of brain slices, freshly dissociated neurons, and
cultured neurons, and does not have a substantial astrocytic
presence (176, 889). However, a Cl⫺-dependent NCBT activity detected in cultured rat cerebellar astrocytes (500)
may be attributable to an alternative Slc4a8, or even an
Slc4a10, gene-product. At the subcellular level, NDCBE is
abundant throughout the cell body, and to a lesser extent
the processes, of hippocampal pyramidal neurons of rats
(553). NDCBE-A/C immunoreactivity is predominantly localized to the presynaptic nerve endings of glutamatergic
neurons where it is colocalized with glutamate transporters
(136, 889), with only a marginal presence in GABAergic
neurons (136, 889).
B) Spinal cord. Northern blots detect the presence of
NDCBE transcripts in human spinal cord preparations
(35), a result not duplicated using an NDCBE-A/B specific
probe (337) perhaps indicating that NDCBE-C/D are prevalent here. Indeed, an antibody that should recognize all
NDCBE variants detects NDCBE protein in protein preparations from rat spinal cord (553).
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A polyadenylation signal has been identified in intron 5
of SLC4A8 (see Supplemental Table 1 of Ref. 970). The
existence of a transcript composed of exon 4 and a composite terminal exon 5 has not been demonstrated, but
such an mRNA could, for example, encode amino acid
residues 1– 44 of NDCBE-B plus the 7-amino acid sequence “GKNCHAV” followed by a termination codon.
The function, if any, of such a polypeptide is unknown.
of those organs tested, NDCBE transcripts are particularly
abundant in the brain (35, 214, 337, 673, 684, 1029). In
Northern blots of human brain shown in Reference 337,
probed with oligonucleotide specific for NDCBE-A/B, a
⬃12 kb transcript (likely NDCBE-A) predominates over a
⬃6.3 kb transcript (likely NDCBE-B).
MARK D. PARKER AND WALTER F. BORON
C) Choroid plexus. RT-PCR amplifies NDCBE, as well as
NBCe1 and NBCn1, cDNAs from human choroid plexus
preparations (214) but not from adult mouse or rat CPE
(755). NDCBE-A/B protein is however, expressed at the
basolateral membrane of choroid plexus epithelia in fetal,
but not adult, rats (176).
II) Sensory organs. We are not aware of any reports of
NDCBE expression in the eye, ear, or olfactory system.
However, NDCBE-like activity has been reported in mammalian lens cells (33, 265).
IV) Respiratory system. A) Trachea. Northern blots of
RNA preparations reveal the presence of NDCBE transcripts in the human trachea (35).
B) Lung. Northern blots of RNA preparations reveal the
presence of NDCBE transcripts in the lungs of mice (1029)
and humans (35). NDCBE transcripts are also detected in a
Calu-3 human airway epithelia cell line (515).
V) Circulatory system. A) Heart. NDCBE-C and NDCBE-D=
transcripts can be amplified from human heart cDNA, and
the significant presence of NDCBE transcripts in mouse
ventricle preparations has been confirmed by qPCR (31).
B) Vasculature. NDCBE cDNAs have been amplified from
preparations of mouse aorta (571).
VI) Musculoskeletal system. A) Skeletal muscle. Northern
blots of RNA preparations reveal the presence of NDCBE
transcripts in human skeletal muscle (35).
VII) Upper digestive system. A) Stomach. Northern blots of
RNA preparations reveal the presence of NDCBE transcripts in the human stomach (35).
X) Endocrine system. A) Widespread. Northern blots of
RNA preparations reveal the presence of NDCBE transcripts in the thyroid glands of mice (571) and in the thyroid
and adrenal glands of humans (35).
XI) Urinary system. A) Kidney. Northern blots of RNA
preparations reveal the presence of NDCBE transcripts in
the kidneys of mice (1029) and humans (35). In rat kidney,
Northern blotting reveals that NDCBE transcripts are enriched in the medulla compared with the cortex (1029), a
result confirmed by RT-PCR from human RNA preparations (214). In rat kidney, NDCBE transcripts predominate
in the inner medulla (986, 1029), with lower abundance in
outer medulla (1029). RT-PCR also detects NDCBE transcripts in a mouse IMCD-3 cell line from the inner medullary collecting duct (1029).
Immunocytochemistry using an antibody directed against
an epitope common to NDCBE-A and -B has not demonstrated the presence of NDCBE protein in any renal structure except endothelial cells (214), whereas an antibody
directed against an epitope common to NDCBE-A and -C
exhibits strong immunoreactivity in mouse renal cortical
preparations, including isolated CCD preparations (571).
Thus it is possible that renal epithelia predominantly express NDCBE-C and/or -D, or novel NDCBE variants.
VIII) Lower digestive system. A) Widespread. Northern
blots of RNA preparations reveal the presence of NDCBE
transcripts in the human pancreas and liver (35). RT-PCR
analysis confirms the presence of NDCBE transcripts in the
pancreas and extends this distribution to include the human
duodenum, ileum, and colon (214).
XII) Reproductive system. A) Male. Northern blots of RNA
preparations reveal the presence of NDCBE transcripts in the
human testis (337). RT-PCR analysis confirms the presence of
NDCBE transcripts in rat testis (214) and extends the distribution, in mice, to testis, epididymis, and vas deferens (599).
IX) Lymphatic and immune systems. A) Widespread.
Northern blots of RNA preparations reveal the presence of
NDCBE transcripts in the bone marrow and lymph nodes of
humans (35). PCR analysis extends this distribution to include a T-cell-derived cell line (983).
B) Female. NDCBE transcripts have been detected by RTPCR of mouse oocytes, ovary, uterus, and vagina (275,
599). According to an NCBI-curated database of ESTs,
NDCBE transcripts may be abundant in human mammary
gland preparations (Appendix VI).
900
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III) Peripheral nervous system. A) Possible presence in
trigeminal ganglion neurons. Although a preliminary
study suggested the presence of NDCBE transcripts in
trigeminal ganglion neurons (407), a later single-cell PCR
study by the same authors was negative for NDCBE in
these cells (408).
An NDCBE-like activity has been described in rat lymphocytes. Stakisaitis et al. (903) report that Na⫹-dependent ClHCO3 exchange is responsible for the net Cl⫺ efflux, measured as a fall in [Cl⫺]i, observed when cells are bathed in a
solution lacking Cl⫺ (903). However, this result is not consistent with the phenotype of human NDCBE heterologously
expressed in Xenopus oocytes: human NDCBE mediates a
36
Cl efflux only in the presence of extracellular Cl⫺ (719). The
apparent discrepancy between the lymphocyte and oocyte
data could represent systematic differences between rat versus
human NDCBE, or between native lymphocytes versus heterologous expression in oocytes. However, it is possible that the
Cl⫺ efflux observed by Stakisaitis et al. in rat lymphocytes is
mediated by NBCn2. Expression of NDCBE and/or NBCn2 in
lymphocytes has, to our knowledge, never been formally demonstrated.
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
C) Placenta. Northern blots of RNA preparations reveal the
presence of NDCBE transcripts in the human placenta (35).
G) PHYSIOLOGICAL ROLES OF NDCBE.
NDCBE likely contributes
to pHi regulation in all of the cell types in which it is expressed. In neurons, pHi, and therefore NDCBE action,
influences neuronal excitability. In epithelia, NDCBE has
been suggested to contribute to HCO3⫺ secretion and Cl⫺
reabsorption.
II) Central nervous system. A) Enhancement of neuronal excitability. In 1992, Church (194) found that the switch from a
CO2/HCO3⫺-free HEPES buffer to a CO2/HCO3⫺ buffer is associated with enhanced neuronal excitability in CA1 neurons
of rat hippocampal slices. He suggested that excitability increases because CO2/HCO3⫺ causes pHi to rise. Later Bevensee
et al. (78) demonstrated that CA1 neurons in fact exist in two
resting pHi states, those with a relatively low and those with a
relatively high pHi (78). In those with a relatively low initial
pHi in a CO2/HCO3⫺-free buffer, the switch to CO2/HCO3⫺
causes a net increase in steady-state pHi (78, 121). This elevated pHi is most likely maintained at least in part by NDCBE
and NBCn1, which are robustly expressed in pyramidal neurons from this region. In CA1 neurons with a relatively high
initial pHi, the switch to CO2/HCO3⫺ has no effect on steadystate pHi or causes it to fall (78, 121). The most straightforward explanation of Church’s data is that he was mainly
working with low-pHi neurons.
Three studies on genetically altered mice support Church’s
hypothesis: the NDCBE- and NBCn2-null mice (which lack
a single acid extruder) exhibit signs of reduced neuronal
excitability, whereas the AE3-null mouse (which lacks a
single acid loader) has a reduced seizure threshold (378).
The link between neuronal pHi regulation and excitability
is reviewed in References 76, 186, 187, and 898.
B) Role in central nervous system plasticity. The switch of
glycine evoked responses of cartwheel cells (glycinergic interneurons) in the dorsal cochlear nucleus of mice from
excitatory to inhibitory follows the lowering of [Cl⫺]i,
which shifts ECl from a value more positive to a value more
negative than Vm (see review in Ref. 34). The shift in ECl is
C) Potential contribution to CSF secretion. The basolateral
presence of NDCBE protein in the choroid plexus epithelium of fetal, but not adult, rats suggests that NDCBE contributes to CSF secretion in early developmental stages
(176). In adult rats, the basolateral step of transepithelial
HCO3⫺ transport across the CPE is likely mediated by
NBCn2 and, to a lesser extent, by NBCn1. Recall that
NDCBE transcripts are present in the choroid plexus of the
adult human, where it is possible that NDCBE plays a functional role.
III) Urinary system. A) Unproven role in Cl⫺ reabsorption
in the PT. Na⫹-dependent Cl-HCO3 exchange has been
proposed to contribute to the basolateral step of Cl⫺ reabsorption by the renal proximal tubule (29, 419, 833). However, this hypothesized Na⫹-dependent Cl-HCO3 exchange
activity has not been demonstrated to be directly coupled to
Na⫹ flux in the proximal tubule, and is difficult to isolate
experimentally due to the much larger HCO3⫺ flux mediated
by the electrogenic Na/HCO3 cotransporter NBCe1 in the
same basolateral membrane (29, 419, 677, 833). Three observations speak directly to the issue of whether NDCBE
contributes to proximal-tubule Cl– reabsorption: 1) antiNDCBE immunoreactivity has not been observed in the
PT;76 2) although removing peritubular Na⫹ does indeed
reduce Cl⫺ efflux across the basolateral membrane of the
proximal tubule, removing peritubular Cl⫺ does not lead to
a change in intracellular Na⫹ activity; and 3) the 1:1 Cl⫺:
HCO3⫺ exchange stoichiometry estimated for the activity
(506) is different from the expected 1:2 coupling ratio for
NDCBE activity (337, 719).
The molecular identity of the transporter(s) responsible for
this basolateral Na⫹-dependent Cl-HCO3 exchange phenomenon have yet to be determined.77 NDCBE is also suggested to contribute to NaCl reabsorption in the collecting
ducts of Na⫹-depleted mice.
76
Immunohistochemistry using an antibody specific to NDCBE-A
and -B does not detect NDCBE protein in the proximal tubule (J.
Praetorius, personal communication), although this observation
does not preclude the presence of NDCBE-C or -D.
77
The difficulty of resolving NDCBE and NDCBE-like activities are
discussed on p. 895.
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I) General. A) pHi regulation. Investigators have proposed
that NDCBE-like activity plays a key role in pHi regulation
in many mammalian cell types, including pyramidal neurons from the CA1 region of hippocampi (850, 889), aortic endothelial cells (280), fibroblasts (157, 531), cultured vascular smooth muscle cells (459, 775), cultured
lens cells (33), esophageal epithelia (973), glomerular
mesangial cells (114, 115), thyrocytes (477), intrahepatic
bile duct cells (354, 913), lymphocytes (786), a monocyte-lymphoma cell line (541), macrophages (949), and
mouse oocytes (275). Note that the presence of NDCBE
mRNA or protein is not documented for all of these cells types.
subsequent to cellular acidification which follows rapid
spiking events (489). The shift requires CO2/HCO3⫺ and is
blocked by H2DIDS (489). Immunohistochemistry reveals
that these cells express NDCBE and thus, taken together,
these data are consistent with the hypothesis that NDCBE
contributes to the manifestation of inhibitory signaling
(489). Lowering of [Cl⫺]i by an NDCBE-like activity has
also been implicated in the development of inhibitory
GABA-evoked responses during central nervous system
maturation. A similar role is shared by the NDCBE-like
SLC4 homolog ABTS-1 in nematodes.
MARK D. PARKER AND WALTER F. BORON
II) Central nervous system. A) Increased protein abundance
in some brain regions in response to metabolic acidosis. At
the level of the whole brain, NDCBE protein abundance is
unperturbed by metabolic acidosis in rats (553). However,
at the regional level, the protein abundance in the hippocampal CA3 region, traditionally a region of lower
NDCBE expression than other regions of the hippocampus,
is 2.5-fold more abundant in acidotic than control rats
(553). NDCBE abundance is also increased in some populations of cortical neurons (553). The presumed increase in
acid extrusion in these cells would tend to counter decreases
in pHi causes by acidosis.
B) Lack of increased protein abundance in response to hypercapnia. Different from the response of NBCn1 in the
brain, but similar to the response of NBCn2, NDCBE protein abundance is not increased in the brain of mice exposed
to chronic hypercapnia (463).
III) Lymphatic and immune systems. A) Increased transcript abundance in a model of systemic lupus erythematosus. Systemic lupus erythematosus (SLE) is an autoimmune
disease causing inflammation in multiple organs. Two mutants of the T-cell receptor ␨ chain have been linked to SLE,
and overexpression of these unstable mutants in murine
T-cells is associated with an eightfold increase in NDCBE
transcripts in these cells (983). The cause and effect of this
upregulation remains to be studied, although it has been
noted that apoptosis of thymocytes, T-cell precursors, is
increased by cellular alkalinization, yet is inhibited by stilbene derivatives, consistent with a proapoptotic action of
902
NDCBE (980). Note that this proapoptotic effect of
NDCBE contrasts with the antiapoptotic effect of NBCn1
in osteoclasts.
IV) Urinary system. A) Increased transcript abundance and
activity in a renal cell line in response to metabolic acidosis.
In a mouse collecting duct cell line, metabolic acidosis increases NDCBE transcript abundance and induces a robust
DIDS-sensitive, Na⫹- and HCO3⫺-dependent pHi recovery
attributed to NDCBE. These results are consistent with a
protective role for NDCBE during metabolic acidosis (35).
B) Increased NDCBE-like activity in CCD of alkali-loaded
rabbits. Although the renal CCD normally reabsorbs
HCO3⫺, the CCD in alkali-loaded animals secretes HCO3⫺
and thereby tends to restore a normal (i.e., less alkaline)
blood pH (636). In alkali-loaded rabbits, the blood-to-lumen movement of HCO3⫺ across the basolateral membrane
of CCD ␤-intercalated cells involves a stilbene-sensitive,
Na⫹ and Cl⫺-dependent mechanism consistent with the activity of NDCBE (271). However, the molecular identity of
the transporter(s) responsible for this activity is presently
unknown.
C) Increased NDCBE-like activity in the CCD of Na-deficient mice. Feeding mice a Na⫹-restricted diet leads to the
upregulation of a novel thiazide-sensitive NaCl reabsorption pathway in cortical collecting ducts, contributing to an
increase in Na⫹ reabsorption (959). Leviel and co-workers
make three observations consistent with a contribution of
NDCBE to the NaCl-reabsorption pathway (571):
1) NDCBE protein is expressed in mouse CCD preparations, 2) an apical thiazide-sensitive NDCBE-like activity
is upregulated in the intercalated cells of Na⫹-depleted
mice, and 3) the CCDs of NDCBE-null mice that have been
fed a Na⫹-restricted diet are unable to reabsorb NaCl
(571). According to the authors’ model, the parallel action
of apical pendrin would recycle HCO3⫺ out of the cell and
mediate the requisite uptake of Cl⫺ (571). Not demonstrated are stilbene sensitivity of the NaCl reabsorption
pathway and the presence of NDCBE protein in the apical
membranes of CCD intercalated cells.
I) CAUSES OF NDCBE DOWNREGULATION. I) Central nervous system. A) Decreased protein abundance in brain in response
to hypoxia. Chronic continuous hypoxia (CCH) decreases
the amount of NDCBE protein in the cortex, subcortex,
hippocampus, and cerebellum of adult rat brains, but generally not in neonates (175). CCH also reduces the abundance of NBCn1 and NBCn2.
II) Reproductive system. A) Decreased transcript abundance in testes of feminized mice. NDCBE transcripts are
abundant in testis, but virtually absent in the testes of feminized mice, that is to say, mice with a disrupted androgen
receptor, or those whose testes cannot descend due to sur-
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H) CAUSES OF NDCBE UPREGULATION. I) General. A) Increased
NDCBE-like activity in response to cell shrinkage. In Chinese hamster ovary cells, a rise in pHi upon exposure to
hypertonic medium is dependent on extracellular Na⫹, Cl⫺,
and HCO3⫺, consistent with the activity of NDCBE (793). It
is unlikely that NHE1 contributes inasmuch as the pHi
increase is insensitive to amiloride. A question that arises is
whether Na⫹-driven Cl-HCO3 exchange would contribute
to a net increase in intracellular osmotically active particles,
and thereby to a regulatory volume increase. If the nonHCO3⫺ buffering power of the cell were infinite (so that pHi
did not change), then the Na⫹-driven Cl-HCO3 exchanger
would be osmotically silent (219): the uptake of 1 Na⫹
would be balanced by the efflux of 1 Cl⫺, and the equivalent
uptake of 2 HCO3⫺ would have no net effect as intracellular
buffers released H⫹ to titrate the HCO3⫺ to CO2 ⫹ H2O,
which would exit the cell. However, at finite non-HCO3⫺
buffering powers, Na⫹-driven Cl-HCO3 exchange activity
would cause pHi to rise, leading to a rise in [HCO3⫺]i, more
so for lower buffering powers, which would in principle
contribute to cell swelling. Finally, to the extent that a rise
in pHi stimulates Cl-HCO3 exchange, the net effect would
be the intracellular accumulation of NaCl, which would
contribute to cell swelling.
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
gical intervention prior to puberty (693). The authors suggest that downregulation of this and other transporter activities may perturb the composition of seminiferous fluid,
inhibiting germ cell maturation and contributing to the feminized phenotype.
J) CONSEQUENCES OF NDCBE DYSFUNCTION.
NDCBE null-mice
exhibit reduced neuronal excitability and renal Na⫹-reabsorption defects. Human pathologies that are linked to
NDCBE dysfunction have yet to be described.
1) In the pyramidal layer of the CA1 region of hippocampal
slices prepared from NDCBE-null mice, the frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs)
is twofold less than in preparations from wild-type mice (889).
A reduction in mEPSC frequency can be mimicked in wildtype preparations by simultaneously lowering both pHo and
pHi. Moreover, an increase in mEPSC frequency occurs in
preparations from NDCBE-null mice with an increase in either
pHo and pHi together or pHi alone. These results are consistent with the idea that the reduced excitability in NDCBE-null
mice results from a low pHi (889).
2) In the CA1 region, the amplitude of population spikes
evoked by stimulating Schaffer collaterals is lower in
NDCBE-null than in wild-type mice (889). Wild-type mice
subjected to a second round of stimulation exhibited population spikes with a 1.5-fold greater amplitude than in the
first round, whereas NDCBE-null mice exhibited population spikes with a 2-fold greater amplitude than in the first
round, consistent with a greater presynaptic plasticity in the
mutant mice (889).
3) During repetitive stimulation, the time constant for the
release of vesicle contents from boutons of hippocampal
slices was greater (i.e., the release was slower) in NDCBEnull versus wild-type mice (889).
4) NDCBE-null mice have an increased latency until onset
of seizures/ictal activity in response to interperitoneal administration of the proconvulsive substances pentylenetetrazol and pilocarpine and to hyperthermia (889).
II) Urinary system. A) Defective regulation of NaCl reabsorption. NDCBE-null mice fed a Na⫹-deficient diet are
unable to upregulate a thiazide-sensitive NaCl reabsorption
pathway that would tend to enhance Na⫹ retention (see
Ref. 571). Thus, under conditions of Na⫹ restriction,
NDCBE dysfunction might be expected to be associated
with volume depletion.
A) SUMMARY.
The electroneutral Na/HCO3 cotransporter
NBCn2 (encoded by the SLC4A10 gene) cotransports Na⫹
and HCO3⫺ with accompanying futile cycles of Cl-Cl exchange. NBCn2 appears to undergo a mode switch into a
Na⫹-driven Cl-HCO3 exchanger (“NCBE”) under certain
assay conditions when extracellular Cl⫺ is unavailable.
Some investigators report that mouse and rat Slc4a10 products act in NCBE mode even under physiological conditions. In common with several other NCBTs, NBCn2 is
stimulated by the soluble protein IRBIT. NBCn2 is present
in many organs but is notably abundant in the central nervous system, where its action is predicted to enhance neuronal excitability. Such a role is corroborated by a study of
NBCn2-null mice. Genetic disruption of the SLC4A10 gene
locus in humans is linked with autism and epilepsy.
B) NOMENCLATURE OF Slc4a10 PRODUCTS.
Slc4a10 products were
initially termed NCBE following a report that mouse
Slc4a10 functions as a Na⫹-driven chloride/bicarbonate exchanger in both Xenopus oocytes and HEK-293 cells
(1021). However, under near-physiological conditions, the
isotopic Cl⫺ efflux associated with human SLC4A10 activity in Xenopus oocytes does not require extracellular
Na⫹ and does not represent a net movement Cl⫺ but
rather Cl-Cl exchange. Thus,the human SLC4A10 product normally functions as an electroneutral Na/HCO3
cotransporter (FIGURE 38A) that the authors propose to
rename NBCn2 (719), an acronym that we will use in
following sections in place of NCBE.
C) MOLECULAR ACTION OF NBCN2. Four functional studies demonstrate that NBCn2, whether it is human NBCn2 heterologously expressed in Xenopus oocytes, or mouse or rat
NBCn2 in mammalian cells, mediates a Na⫹-dependent
HCO3⫺ uptake that can be blocked by DIDS (212, 317, 719,
1021). One of these studies further demonstrates that the
NBCn2
Na+
HCO3–
Cl–
Na+
HCO3–
HCO3–
A
B
Cl–
Cl–
FIGURE 38. Molecular action of NBCn2. Possible molecular
mechanisms by which NBCn2 could operate in an electroneutral
⫺
with accompanying futile cycles
mode to cotransport Na⫹ and HCO3
⫺
of HCO3
-dependent Cl-Cl self-exchange (A). In the absence of extracellular Cl⫺, NBCn2 performs Na⫹-driven Cl-HCO3 exchange (B). The
Slc4a10 gene product from mice and rats is reported to act like
NDCBE (FIGURE 35) even in the presence of extracellular Cl⫺. Note
2⫺
or
that we have not considered any models that are based on CO3
H⫹ cotransport.
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I) Central nervous system. A) Reduced network excitability
and increased presynaptic plasticity in NDCBE-null mice.
As discussed, the action of NDCBE is hypothesized to enhance neuronal excitability. Four observations demonstrate
that neuronal excitability is reduced in NDCBE-null mice.
3. NBCn2/NCBE (Slc4a10)
MARK D. PARKER AND WALTER F. BORON
I) A study of mouse Slc4a10 expressed in Xenopus oocytes.
When expressed in Xenopus oocytes, mouse Slc4a10 mediates HCO3⫺-dependent isotopic influxes of Na⫹ and Cl⫺
and efflux of Cl⫺. This Cl⫺ efflux is maximal in the collective presence of extracellular Na⫹, Cl⫺, and HCO3⫺. In their
original description of “NCBE,” Wang and co-workers
(1021) interpreted these data as evidence for Na⫹-driven
Cl-HCO3 exchange. However, three additional observations in the same study are more consistent with Cl-Cl exchange in parallel with Na/HCO3 cotransport rather than a
classical model of Na⫹-driven Cl-HCO3 exchange activity.
1) The transporter mediates an influx (in addition to an
efflux) of 36Cl in the presence of extracellular Na⫹ and
HCO3⫺, 2) Cl⫺ influx does not require extracellular Na⫹ or
HCO3⫺, and 3) Cl⫺ efflux requires extracellular Cl⫺ (i.e.,
trans-side dependence).
II) A study of human SLC4A10 expressed in Xenopus
oocytes. To address the question of whether the transporter
mediates a net efflux of Cl⫺, Parker et al. (719) in a later
study expressed human NBCn2-B in oocytes and used a
Cl⫺-sensitive microelectrode to monitor [Cl⫺] on the extracellular surface ([Cl⫺]S) of the oocyte. Bulk extracellular
[Cl⫺] was maintained at 10 mM to enhance electrode sensitivity. The authors found that, when exposed to CO2/
HCO3⫺, oocytes expressing either AE1, human NDCBE, or
squid NDCBE exhibited a transient rise in [Cl⫺]S, indicating
a HCO3⫺-stimulated net efflux of Cl⫺. However, oocytes
expressing NBCn1 or NBCn2-B exhibited no [Cl⫺]S increase. Thus, under these conditions, SLC4A10 does not
mediate a net efflux of Cl⫺. The authors also found that the
NBCn2-mediated 36Cl efflux that is stimulated by application of HCO3⫺, is independent of the presence of extracellular Na⫹. Instead, the 36Cl fluxes must represent a futile
Cl-Cl exchange that accompanies the true physiological
function, the apparent 1:1 coupled influx of Na⫹ and
HCO3⫺ (FIGURE 38A).
904
Although the preceding study demonstrates that NBCn2 does
not normally mediate Na⫹-driven Cl-HCO3 exchange, an interesting observation is that human NBCn2-B appears to be
capable of Na⫹-driven Cl-HCO3 exchange under a particular
nonphysiological condition, namely, the absence of extracellular Cl⫺. Removing extracellular Cl⫺ reduces 36Cl efflux by
half (presumably by eliminating Cl-Cl exchange), and the remaining 36Cl efflux now requires both extracellular Na⫹ and
HCO3⫺ (719). Moreover, in the absence of extracellular Cl⫺,
NBCn2 mediates a robust, pHi recovery. Thus it appears that,
with no extracellular Cl⫺ to participate in Cl-Cl exchange, the
transporter is now obligated to engage in Na⫹-driven ClHCO3 exchange (FIGURE 38B).
III) Studies of rodent Slc4a10 expressed in mammalian cells.
In the case of mouse NBCn2-B heterologously expressed in
HEK-293 cells (1021) or mouse NBCn2-A and rat NBCn2C/D heterologously expressed in 3T3 cells (212, 317), removing extracellular Cl⫺ blocks HCO3⫺ influx. The most straightforward explanation for these data is that removing extracellular Cl⫺ switches the activity of Slc4a10 from electroneutral
Na/HCO3 cotransport to Na⫹-driven Cl-HCO3 exchange, as
predicted by Parker et al. (719), but that the concomitant
depletion of intracellular Cl⫺ eliminates this activity. On the
other hand, if extracellular Cl⫺ removal fails to deplete intracellular Cl⫺ over the time period examined, then an alternative explanation is that the rodent transporter expressed in mammalian cells behaves differently than the
human transporter expressed in oocytes.
A study of rodent-Slc4a10-transfected 3T3 cells (212) includes four novel observations that are provided as evidence
that Na⫹-driven Cl-HCO3 exchange is the normal mode of
action for Slc4a10.
1) Slc4a10-transfected cells alkalinize at a faster rate than
nontransfected cells in response to the acute removal of
extracellular Cl⫺ as if, in the absence of Cl⫺, the driving
force for Cl⫺ efflux and thence Na/HCO3 influx is increased
(212). This observation agrees with the findings in Reference 719, namely, that NBCn2 can act as a Na⫹-driven
Cl-HCO3 exchanger in the absence of bath Cl⫺. The 3T3cell data are complicated by the presence of substantial
endogenous anion exchange activity in these cells (212) that
would tend to enhance the rate of alkalinization upon Cl
removal in Slc4a10-transfected cells.
2) 36Cl efflux occurs at a greater rate from 3T3 cells transfected with rat Slc4a10 and bathed in a HCO3⫺-buffered
solution compared with similar cells bathed in a HEPESbuffered solution, but only in the presence of Na⫹ (212). In
this respect, the behavior of rat Slc4a10 in 3T3 cells appears
to differ from the behavior of human NBCn2. When expressed in oocytes, human NBCn2-B mediates a Cl⫺ efflux
that is independent of the presence of extracellular Na⫹
(719).
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transport mediated by human NBCn2 is electroneutral
(719). Three groups provide evidence that the Na⫹ -dependent HCO3⫺ influx mediated by NBCn2 is accompanied by
the efflux of 36Cl (212, 719, 1021). However, controversy
has arisen over whether this efflux represents a net movement of Cl⫺ under physiological conditions. Two groups of
investigators (212, 1021) interpret the Cl⫺ efflux data as
evidence that mouse and rat NBCn2 mediate Na⫹-driven
Cl-HCO3 exchange (like NDCBE) under physiological conditions (FIGURE 38B). A third group (719) provides evidence
that the 36Cl efflux that accompanies human NBCn2 action
represents futile cycles of Cl-Cl exchange under physiological conditions (FIGURE 38A) and that NBCn2 is a second
electroneutral Na/HCO3 cotransporter (the other being
NBCn1). However, NBCn2 does behave as a Na⫹-driven
Cl-HCO3 exchanger in the absence of extracellular Cl⫺
(719). The evidence pertaining to NBCn2-mediated Cl⫺
movement in these studies is considered below.
⫺
TRANSPORTERS
Na⫹-COUPLED HCO3
3) Cl⫺ efflux, but not Cl⫺ influx, is enhanced in the presence
of HCO3⫺, an observation offered as evidence that the Cl⫺
efflux mediated by mouse Slc4a10 represents a net efflux
(212). An alternative explanation is that the extent of 36Cl
influx over the 2-min duration of the influx assay is underestimated due to the simultaneous 36Cl efflux mediated by
NBCn2. With human NBCn2-B expressed in oocytes, 36Cl
efflux also is enhanced by HCO3⫺, which simply appears to
stimulate Cl-Cl exchange (719).
In summary, it is unknown whether human SLC4A10 and
rodent Slc4a10 exhibit true functional differences, or
whether the disparities between the studies are methodological in nature. Indeed, as human SLC4A10 is capable of
shifting between “NCBE” and “NBCn” modes, it is not
inconceivable that rodent Slc4a10 could behave differently
from human SLC4A10.
D) THE Slc4a10 GENE.
The human SLC4A10 gene occupies 27
exons over ⬃360 kb (FIGURE 39, A and B) on chromosome
2q24.2 (1081). A singleton EST (GenBank DNA accession no. BP229748) from fetal brain provides evidence
that the SLC4A10 locus may extend 150 kb further upstream than presently thought, filling the apparent gap
between SLC4A10 and its upstream neighbor TBR1.
TBR1 encodes a transcription factor that is expressed in
cortical neurons during development (133). The TBR1
paralog EOMES/TBR2 is the upstream neighbor of
SLC4A7 (FIGURE 31A), indicating a longstanding relationship between these two gene families. The downstream
neighbor of SLC4A10, DPP4, encodes the plasminogen
receptor dipeptidyl peptidase IV (328). DPP4 protein interacts with and enhances the activity of NHE3 in the
proximal tubule (324, 325) and also dampens stimulation of duodenal HCO3⫺ secretion by degrading glucagon-like peptide (418). It is unknown whether DPP4 influences the activity of NCBTs.
Intron 1 of SLC4A10 contains a binding site for the transcription factor and tumor-suppressor p53 (1031). SLC4A10 transcription is downregulated in a colon carcinoma cell line
by 5-fluorouracil induction of p53 expression (see supplemental data for Ref. 1031). It would be interesting to
E) STRUCTURAL FEATURES AND VARIANTS OF NBCn2.
Variation
among mammalian NBCn2 transcripts arises by alternative
splicing at any or all of the following three sites in Slc4a10
pre-mRNA (317).
I) Sources of variation in coding sequence among NBCn2
variants. The NBCn2 gene is only known to include a single
promoter. As depicted in FIGURE 39C and FIGURE 40, there
are two major sources of variation between NBCn2 transcripts: the optional inclusion of a 30-amino acid cassette in
the Nt and the choice of one of two Ct appendages (a
22-amino acid appendage that ends “-ETCL” or a 4-amino
acid appendage that ends “-SSPS”). A further, minor source
of variation arises due to the optional inclusion of a single
alanine residue due to an apparently degenerate splice
boundary. All four NBCn2 clones reported to date are predicted to include an autoinhibitory domain and IRBIT binding determinants in their Nt (718, 722).
A) Optional Ala. The 5= end of exon 7 which, at least in rat
NBCn2, contains a cryptic splice-site that, when utilized by
the splice machinery, shortens the transcript by three nts
(CAG) resulting in the loss of a single Ala residue from the
Nt of the transporter. Thus the Ala is optional in rats. In
humans, the Ala is always present.
B) Cassette A. NBCn2 transcripts can differ in the inclusion
or exclusion of exon 8, sometimes referred to as DNA cassette or insert “A,” the excision of which by splicing removes sequence that encodes a 30-amino acid protein “cassette A” (FIGURES 39C and 40). The inclusion of cassette A
is predicted to extend the Nt loop (FIGURE 15).
C) Choice of alternative Ct (“-SSPS” or “-ETCL”). Exon
26, sometimes referred to as DNA cassette or insert “B,”
encodes the 4-amino acid Ct appendage “-SSPS” (FIGURES
39C and 40). The excision by splicing of cassette B allows
translational read-through to an alternative downstream
termination codon in exon 27. Thus removal of cassette B
produces NBCn2 variants with a longer and different Ct
(21-amino acid of NBCn2-C/D replaces 4-amino acid of
NBCn2-A/B). The 21-amino acid Ct terminates with a
type I consensus PDZ-domain binding motif “ETCL”
(317).
In an astrocytic cell line, the inclusion of the 21-amino
acid Ct appendage in rat NBCn2 (rb2NCBE, see below)
results in increased colocalization of NBCn2 with the
actin cytoskeleton, compared with rb1NCBE, a variant
with the 4-amino acid Ct appendage (317). The cytoskeletal attachment of the 21-amino acid appendage is mediated via EBP50 and ezrin (562). Consistent with the hypothesis that an enhanced cytoskeletal interaction is im-
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4) A comparison of the rates of Na⫹ and HCO3⫺ influx into
Slc4a10-transfected cells, calculated from measurements of
fluorometric dyes, suggest that the Na:HCO3 cotransport
ratio is 1:2 for rodent Slc4a10. Thus the Cl⫺ efflux would
have to be net to maintain electroneutrality (212). However, there are potential risks in comparing rates obtained
by two different methods (Na⫹- versus pH-sensitive dyes)
as well as concerns that the Na dye (CoroNa) might not be
suitable for quantitative measurements at low [Na⫹] (see
Ref. 641). Cited as validation for the Slc4a10 stoichiometry
measurements, the stoichiometry of NBCn1 calculated by
this method was, as expected, 1:1.
know whether p53 in fact decreases the expression of
NBCn2.
MARK D. PARKER AND WALTER F. BORON
A
Locus 12q13
20 kb
SLC4A10
DPP4
TBR1
B
Gene structure
P
10 kb
1
C
27
8
Transcript variation
M
NBCn2-A
1
7
1
7
1
7
1
7
1
7
9
23
24
25
*
26
27
9
23
24
25
*
26
27
9
23
24
25
*
27
8
9
23
24
25
*
27
8
9
23
M
NBCn2-B
8
M
NBCn2-C
M
NBCn2-D
M
rb3NCBE
*
27
FIGURE 39. SLC4A10 gene structure and NBCn2 transcript variants. Scale diagrams showing the human
SLC4A10 gene locus together with the position of neighboring genes (A), the position of the promoters (P), and
the position of exons within SLC4A10 (B). Transcript variants are represented, not to scale, as numbered
boxes joined by a horizontal line (C). Each numbered box represents the inclusion of that exon in the mature
transcript. “//” denotes that all four transcripts include exons 1–7 and 9 –23. Exons that include the initiator
ATG codon (“M”) and termination codon (“*”) are marked for each transcript. Colored exons, or parts of exons,
correspond to the protein regions that each encodes, which are identically colored in FIGURE 40. Uncolored
exons, or parts of exons, denote untranslated 5= and 3= sequence. Exons that are connected with a dashed line
are predicted, but not demonstrated, to be included in the mRNA. Note that rb3NCBE has only been isolated
from rat cDNA.
portant for efficient trafficking of the transporter to the
plasma membrane, cytoskeletal disruption by cytochalasin B treatment of fibroblasts expressing rb2NCBE results in the loss of transporter activity from the plasma
membrane (562). Indeed, in a mouse fibroblast cell line,
rb2NCBE variant traffics more efficiently to the plasma
membrane than rb1NCBE (317). However, the opposite
is observed when NBCn2 variants are expressed in
MDCK cells (756) perhaps, the authors suggest, due to
the lack of an accessory protein.
II) Cloned NBCn2 variants that are demonstrated or likely
to exhibit NCBT activity. GenBank protein accession numbers for the variants discussed in this section are provided in
Appendix IV.
A) NBCn2-A (NCBT activity demonstrated). NBCn2-A
lacks the 30-amino acid cassette A and includes the 4-amino
906
acid Ct appendage that ends with “-SSPS.” It is orthologous
to the rat variant rb5NCBE. Full-length NBCn2-A transcripts have been isolated from a mouse pancreatic cell line
cDNA library (1021), and from rat hippocampus and
mouse brain cDNA preparations. In the brains of mice,
NBCn2-A appears to be the most abundant NBCn2 variant
in the subcortex (598).
B) NBCn2-B (NCBT activity demonstrated). NBCn2-B includes the 30-amino acid cassette A and the 4-amino acid Ct
appendage that ends with “-SSPS.” It is most orthologous to
the rat variant rb1NCBE, which does not include the optional Ala that is always present in humans. Full-length
NBCn2-B transcripts have been cloned from human kidney
cDNA (719) and from rat hippocampus and mouse brain
cDNA preparations. In the brains of mice, NBCn2-B appears to be the most abundant NBCn2 variant in the medulla (598).
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P
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
Ct
C
as
se
tte
100 aa
1–5
6–9
10–14
C
(e ass
xo e
n tte
26 B
)
TMD
A
Nt
1,088
NBCn2-A
3
1,118
NBCn2-B
30
3
PDZ
NBCn2-C
1,106
21
30
1,136
21
rb3NCBE
1,054
30
1 (exon 27)
FIGURE 40. NBCn2 protein variants. Scale diagram of protein variants that are encoded by the transcripts
represented in FIGURE 39C. Horizontal bars represent protein sequence laid out from Nt to Ct. Vertical bars
represent position of ␣-helical TMs. Protein cassettes are labeled with a number denoting their size in amino
acids and colored to denote their genetic origin as shown in FIGURE 39C. All NBCn2 variants are presumed
to include an autoinhibitory domain and IRBIT-binding determinants in their Nt. A color-matched protein
sequence alignment of the variants is provided in Appendix V.
C) NBCn2-C (NCBT activity untested). NBCn2-C lacks the
30-amino acid Nt cassette A and includes the 21-amino acid
Ct appendage that ends with “-ETCL.” The orthologous rat
variant is rb4NCBE. Full-length NBCn2-C transcripts have
been cloned from mouse brain cDNA preparations (598).
D) NBCn2-D (NCBT activity demonstrated). NBCn2-D
includes the 30-amino acid Nt cassette A and the 21-amino
acid Ct appendage that ends with “-ETCL.” The most similar rat variant is rb2NCBE, which does not include the
optional Ala that is always present in humans. Full-length
NBCn2-D transcripts have been cloned from mouse brain
cDNA (598).
E) rb3NCBE (NCBT activity untested). This variant cloned
from rat brain is similar to NBCn2-D except that, instead of
lacking exon 26, it lacks exons 24 –26. The now out-offrame exon 27 encodes a singleton His residue that is immediately followed by a termination codon. Thus, in
rb3NCBE, the most Ct 83 amino acids of NBCn2-D are
replaced by a single His. By comparison with an artificially
truncated version of human NDCBE that has a Ct of similar
length (717), rb3NCBE ought to be functional.
III) Predicted NBCn2 variants. A) Predicted variants with
an alternative Ct “-RS.” Although not yet demonstrated to
be included in a full-length transcript, a novel fragment
amplified from human brain cDNA includes a partial, outof-frame exon 26 created by the utilization of a cryptic
splice site with exon 26 (see supplemental material of Ref.
719). The internally spliced exon 26 includes the third base
position of a codon hung-over from exon 25 (the triplet in
the novel fragment now encodes an Arg rather than the
usual Ser) and a singleton Ser codon followed by a termination codon. Thus the fragment is predicted to be part of a
transcript that encodes a novel variant that terminates in a
protein kinase C consensus phosphorylation site “KRS.”
Such variants would be identical to NBCn2-A/B except
that, in the novel variants, the 2-amino acid “-RS” replaces
the 4-amino acid “-SSPS” in NBCn2-A/B.
IV) Other NBCn2 variants. A) An unusual variant that
represents only the isolated Nt. One variant, rb7NCBE
(GenBank DNA accession no. AY579377), which originates from rat brain, is identical to rb5NCBE at the transcript level save for the inclusion in rb7NCBE of sequence
derived from a cryptic exon between exons 11 and 12. The
novel sequence encodes 18 amino acids followed by a termination codon. Thus rb7NCBE encodes an isolated but
near-complete cytoplasmic Nt (equivalent to residues
1– 451 of human NBCn2-A) plus 18 novel residues. It is
possible that the premature termination codon included in
this mRNA would make it a target for nonsense-mediated
decay (170). rb7NCBE is reminiscent of isolated Nt variants of NBCn1 and NDCBE.
B) rb6NCBE (potentially legitimate transcript, NCBT activity unlikely). This variant (GenBank DNA accession
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PDZ
NBCn2-D
MARK D. PARKER AND WALTER F. BORON
As far as astrocytes are concerned, one group detected
NBCn2 transcripts in mouse cortical astrocytes (317),
whereas another did not detect NBCn2 protein in rat
hippocampal astrocytes (177). The difference may be explained by 1) phenotypic differences between astrocytes
isolated from different brain regions (56), 2) astrocytes
not maintaining substantial levels of NBCn2 protein despite the presence of mRNA, or 3) a species difference. As
to the splice variants expressed, an analysis of transcripts indicates that astrocytes, in distinction to the neurons discussed above, lack NBCn2-A and NBCn2-B messages, but instead are enriched in NBCn2-D transcripts
(317).
F) DISTRIBUTION OF NBCn2. NBCn2 is predominantly expressed
in the central nervous system. The distribution of NBCn2 in
the CNS and in other organ systems is discussed below and
compared with the distribution of other NCBTs in TABLE 5.
B) Choroid plexus and dura mater. In immunohistochemistry studies, the most striking anti-NBCn2 immunoreactivity is in the choroid plexus (see cartoon in FIGURE 28).
Antibodies raised against the Nt common to all NBCn2
variants, or to one or the other alternative NBCn2 Ct78 (i.e.,
short Ct versus long PDZ-binding-motif containing Ct) all
stain the basolateral membrane of the choroid plexus epithelium in human, mouse, and rat (113, 177, 216, 429, 755,
756). Immunogold staining of mouse choroid plexus epithelial cells from the third and fourth ventricles, using an
anti-Ct NBCn2 antibody, confirms the basolateral distribution and shows the protein to be especially abundant in
“highly folded membrane processes between neighboring
epithelial cells.” This study also confirms the cytosolic disposition of the Ct (755). An analysis of rodent cDNAs indicates that NBCn2-A may be the predominant transcript in
choroid plexus (755). NBCn2 transcripts are also present in
the dura mater (399).
I) Central nervous system. A) Brain. NBCn2 transcripts are
most abundant in and widely distributed throughout the
CNS (214, 317, 399, 428, 684, 719, 1021) in the forebrain
(frontal, temporal and occipital lobes and olfactory bulb),
the limbic system (in the hypothalamus, geniculate nucleus,
thalamic eminence, hippocampus, substantia nigra and in
the amygdala, caudate nucleus, and putamen of the corpus
striatum), and the hindbrain (cerebellum, medulla, spinal
cord).
In mouse brains, an antibody directed against an epitope
common to all known variants of NBCn2 has the highest
level of immunoreactivity in the cerebral cortex, cerebellum and hippocampus, and the least in subcortex (174).
This pattern is similar to the distribution of NBCn1 but
different from that of NDCBE, which is most abundant
in the subcortex compared with the other three tested
regions (175). Within CA1-CA3 regions of the hippocampus, NBCn2 also exhibits an expression pattern
complementary to that of NDCBE, inasmuch as NBCn2
expression is greatest in the CA3 region (429), whereas
NDCBE expression appears to be strongest in the CA1
and CA2 regions (176). A study of the developmental
expression of NBCn2 transcripts in rodent brains is presented in References 317 and 399. The abundance of
NBCn2-A through -D appears to vary among brain regions in mice (317, 598, 600). At the cellular level,
NBCn2-A and NBCn2-B transcripts are more abundant
than NBCn2-C and NBCn2-D transcripts in neurons
(317).
In prenatal rat hippocampal neurons, NBCn2 protein is
detected by immunocytochemistry in the soma of freshly
dissociated cells (177), as well as in the processes (177) and
the plasma membrane of the soma (177, 199) of cultured
cells (see cartoon in FIGURE 24A).
908
II) Sensory organs. A) Eye. In the retinas of mice, NBCn2
transcripts have been detected in the neuronal cell layer and
pigment epithelium (399). An NDCBE-like activity, which
could be mediated by NBCn2, has been reported in mammalian lens cells (33, 265).
B) Ear. NBCn2 transcripts have been detected in the cochlear ganglion (399).
III) Peripheral nervous system. A) We are not aware of any
reports of NBCn2 expression in the peripheral nervous system.
IV) Respiratory system. A) Lung. A mutation in the
SLC4A10 gene is associated with lung cancer, although the
expression of NBCn2 in healthy or cancerous lung tissue
has not been formally demonstrated.
78
The antibody raised against the short Ct of NBCn2-A/B has
been shown to cross-react with the long Ct of NBCn2-C/D (756),
although RT-PCR results predict that only NBCn2 variants with a
short Ct are expressed in mouse choroid plexus (755).
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no.AY579376), which originates from rat brain cDNA, is
identical to rb5NCBE at the transcript level (i.e., like
NBCn2-A, it omits cassette A and includes cassette B) except for the alternative splicing, at nonconsensus splice
sites, of exons 14 and 15. The effect is that the last twothirds of exon 14 are discarded, together with the first half
of exon 15. Furthermore, the remaining exon 15 sequence is
out of frame and encodes only seven amino acids followed
by a termination codon. The resulting rb6NCBE protein
product encodes the entire Nt and TM1–3 of NBCn2. However, the frame shift and premature termination at a point
within putative TM4 make it unlikely that this product is
functional or even stable.
⫺
TRANSPORTERS
Na⫹-COUPLED HCO3
V) Circulatory system. A) Heart. NBCn2 transcripts have
been detected in preparations of heart ventricles from
mice (31).
VI) Musculoskeletal system. A) Skeletal muscle. NBCn2
transcripts have been detected in human skeletal muscle
preparations (see supplemental data in Ref. 719). Transcripts including the PDZ-binding domain (i.e., NBCn2-C
and -D) appear to predominate over those lacking the PDZ
binding domain (i.e., NBCn2-A and -B; see supplemental
data of Ref. 719).
VII) Upper digestive system. A) Stomach. NBCn2 transcripts have been detected in preparations of human (214)
and mouse (399) stomach.
IX) Lymphatic and immune systems. A) Lymph node and
spleen. According to an NCBI-curated database of ESTs,
the human lymph node is a potential site of NBCn2 transcription (Appendix VI). NBCn2 transcripts have also been
detected in preparation of rat spleen (317).
X) Endocrine system. A) Pancreas. NBCn2-A was originally cloned from a mouse pancreatic beta-cell line (1021).
B) Pituitary gland. NBCn2 transcripts have been detected in
a preparation of rat pituitary glands (1021).
XI) Urinary system. A) Kidney. The archetypal NBCn2-B
variant was cloned from human kidney cDNA (719), and
transcripts have also been detected in rat kidney preparations (1021). In humans, NBCn2 transcripts are present at
least in the renal cortex (214).
XII) Reproductive system. A) Male. NBCn2 transcripts
have been detected in preparation of rat and mouse testes
(1021) and in preparations of mouse epididymis and vas
deferens (599).
B) Female. NBCn2 transcripts have been detected in preparations of ovary, uterus, and vagina of mice (599).
G) PHYSIOLOGICAL ROLES OF NBCn2.
At present, studies of
NBCn2-specific activity in situ are few in number. The major issues, to some extent common to all the NCBTs, are
that 1) a cell (particularly neurons and choroid plexus epithelia) may express more than one NCBT, 2) specific blockers are not available, 3) physiological dissections of NCBTs
are not straightforward because of the difficulty of performing a sufficiently wide range of assays on one cell, and
I) Central nervous system. A) Neuronal excitability. A comparison of average resting pHi values of cells in mouse brain
slices shows no significant different between wild-type and
NBCn2-knockout mice (429). Even so, the knockout of
NBCn2 substantially slows the HCO3⫺-dependent pHi recovery from an intracellular acid load in a mouse brain slice
from the hippocampal CA3 region and isolated cells from
the mouse choroid plexus (429). As discussed earlier in this
review, a faster recovery of pHi following neuronal firing
leads to a faster recovery of neuronal excitability.
B) CSF secretion. Basolateral NBCn2, along with other basolateral NCBTs (FIGURE 28), is suitably positioned to mediate the basolateral step in the transepithelial movement of
Na⫹ and HCO3⫺ from the blood into the CSF, thereby contributing to CSF secretion. This role appears to be confirmed by exhibition of CSF secretion defects in an NBCn2null mouse, although other transporters are perturbed in
the CPE of these mice.
C) Possible role in central nervous system maturation. The
detection of NBCn2 transcripts in the CNS of embryonic
mice led to the hypothesis that the expression of Slc4a10 is
a developmental switch in which the gene-product lowers
[Cl⫺]i and thereby shifts ECl from a value more positive to a
value more negative than Vm. Such a shift in ECl would
causes the GABA-evoked response to change from excitatory to inhibitory (399). Indeed, the probable Na⫹-driven
Cl-HCO3 exchanger ABTS-1 fulfills this role in nematodes.
However, the underlying premise of this hypothesis in mice
is that Slc4a10 mediates Na⫹-driven Cl-HCO3 exchange.
Inasmuch as human NBCn2 is unable to effect net Cl⫺
movements under physiological conditions (719), the original hypothesis is unlikely to be correct in humans. NDCBE
action could theoretically fulfill this role in humans, given
the correct temporal expression pattern.
II) Reproductive system. A) Possible role in sperm capacitation. In 1996, Zeng et al. (1090) reported that the recovery of pHi in sperm following an acid-load is stilbene-sensitive and requires Na⫹, Cl⫺, and HCO3⫺ (1090). Subsequently, Wang et al. (1021) speculated that NBCn2
contributes to the alkalinization of sperm required for their
capacitation. This speculation could be correct if 1) the
removal of external Cl⫺ converted the activity of NBCn2 to
Na⫹-driven Cl-HCO3 exchange, 2) the depletion of intracellular Cl⫺ blocked this Na⫹-driven Cl-HCO3 exchange
activity, and 3) the NDCBE-like activity is not mediated by
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VIII) Lower digestive system. A) Widespread. NBCn2 transcripts have been detected in preparations of human duodenum (214), rat ileum (1021), and human liver (719). In
the liver, NBCn2-A appears to be more abundant that
NBCn2-B-D (see supplemental data of Ref. 719).
4) evidence from knockout mice is complicated by dysregulation of other transporters. Ideally, one might use immunocytochemistry or single-cell PCR to verify that an identifiable cell has a single NCBT, which could be approached
with standard techniques for studying pHi regulation. Failing that, knockdown approaches are promising, although
one must remain wary of secondary effects.
MARK D. PARKER AND WALTER F. BORON
NDCBE. However, formal demonstration of NBCn2 expression in these cells is presently lacking.
H) CAUSES OF NBCn2 UPREGULATION.
To our knowledge there
are no reports of maneuvers that increase the transcript
abundance, protein abundance, or plasma membrane abundance of NBCn2. However, preliminary reports show that
the functional expression of NBCn2 is enhanced by coexpression with IRBIT (718, 722).
I) CAUSES OF NBCn2 DOWNREGULATION. We are not aware of any
reports of maneuvers that decrease NBCn2 transcript abundance. Two studies have reported maneuvers that downregulate NBCn2 at other levels.
II) Central nervous system. A) Decreased protein abundance in the brain in response to hypoxia. NBCn2 protein
levels generally fall in response to chronic continuous hypoxia (CCH) in the hippocampus, cerebral cortex, subcortex, and cerebellum of neonatal and adult mice (174).
Downregulation in hypoxia is also characteristic of NBCn1
and NDCBE, except that the downregulation of NDCBE
occurs in adults but generally not in neonates (175).
B) Lack of decreased protein abundance in response to
hypercapnia. Neither NBCn2 nor NDCBE protein abundance is increased in the brains of mice exposed to chronic
hypercapnia (463).
J) CONSEQUENCES OF NBCn2 DYSREGULATION.
As expected for a
gene most abundantly expressed in the central nervous system, most reported signs of NBCn2 ablation in mice and
pathologies linked to the SLC4A10 gene in humans relate to
the brain and bear on changes in neuronal excitability (e.g.,
reduced sensitivity to proconvulsants, epilepsy, autism).
Defective CSF secretion is also described in NBCn2-null
mice, but the molecular basis of this pathology appears to
be complex and may not be primarily due to loss of NBCn2
activity per se. A single report of a genetic linkage between
SLC4A10 and lung cancer bears on the consequence of
NBCn2 dysfunction outside of the brain.
I) General. A) Potential role in tumor growth. A report that
Na⫹-dependent Cl-HCO3 exchange activity attributed to
NBCn2 is important for pHi regulation, and therefore proliferation, in the breast cancer cell lines EMT6, MCF7, and
MDA-MB231 (1041), must be interpreted with caution.
910
II) Central nervous system. A) Reduced neuronal excitability in mice with a disrupted Slc4a10 gene. As noted above,
in the hippocampal CA3 region, the recovery of pHi from
an acid load is slower with an NBCn2-null than with a WT
mouse. Moreover, in both NBCn2-null and WT mice, the
frequency of 4-aminopyridine–induced seizure-like events
in the CA3 region is reduced by neuronal acidification
(429). Thus it is not surprising that the subsequent recovery
in the frequency of these seizure-like events is slower in the
knockout than in the WT mice (429). Consistent with this
indication of reduced neuronal excitability, NBCn2-knockout mice have an increased tolerance to seizure induction,
both in terms of latency until onset and in survival rate
(429).
B) Genetic linkage to epilepsy in humans. Gurnett et al.
(360) described a 13-year-old girl presenting with cognitive
dysfunction and complex partial epilepsy was determined
to have a balanced chromosomal translocation t(2;13)(q24;
q31) involving the SLC4A10 gene. The break point on
chromosome 2q24 disrupted SLC4A10 at a point between
exons 2 and 3, with the rest of the gene joined at a breakpoint in a gene desert on chromosome 13q31.79 If this
translocation event resulted in reduced NBCn2 activity due
to haploinsufficiency, then the neurological phenotype
would be inconsistent with the described phenotype of
Slc4a10 knockout mice, which have no behavioral abnormalities and a reduced, rather than increased, neuronal excitability (429). We consider five possible explanations for
this apparent disparity:
1) Systematic difference. A haploinsufficient human is not a
null mouse and chemically induced seizures are not complex partial epilepsy.
2) Creation of an uninhibited NBCn2. By real-time quantitative PCR (qPCR), Gurnett et al. (360) found that the level
of NBCn2 mRNA (specifically that encoded by exons 1–3)
was only about half of normal in lymphocytes from the
patient. Thus, although loss of NBCn2 protein could contribute to the disease phenotype, protein levels remain untested in this patient. In addition, it is similarly unknown
whether the relocated, telomeric end of SLC4A10 (i.e.,
exon 3 onwards), from its new locus, might be capable of
79
Build 37.1 of the human genome indicates that the FISH probes
used in this study to identify the break point in chromosome 13 in
fact bind to 13q22, rather than to the originally assigned 13q31.
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I) General. A) Inhibition of NBCn2 activity by PKA. As
expressed in 3T3 cells, NBCn2 activity is regulated by phosphorylation, such that 1) the action of PKA is inhibitory to
the functional expression of the transporter and 2) inhibition of PKA enhances functional expression of the transporter (562). It is unknown whether this phenomenon reflects a direct effect of PKA action on NBCn2.
The authors assumed that NBCn2 was the only HCO3⫺
transporter that could mediate recovery of pHi from an acid
load in a mammalian cell. In fact, any of the five NCBTs
could mediate such a pHi recovery. Subsequent studies have
identified NBCn1 as a major pH regulator in MCF7 cells
(546), although the presence of NBCn2 in these cells and in
other breast cancer cell lines cannot be discounted.
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
producing a truncated or alternative gene-product with enhanced function. It is noteworthy that Xenopus oocytes
expressing an Nt truncated NBCn2 (i.e., lacking sequence
encoded by exons 1–3) exhibit a pHi recovery rate twofold
greater than cells expressing the full-length transporter
(718). However, the only start codon identified in the
SLC4A10 gene to date is that in exon 1.
4) Position effects. The chromosomal translocation might
affect the transcription of genes other than SLC4A10, or of
microRNAs. Although the report by Gurnett et al. focuses
on the SLC4A10 locus on 2q24.2, it is worth noting that
13q22–13q31 is also a susceptibility locus associated with
seizures (376). The translocation of the broken arm of chromosome 13 to chromosome 2, and vice versa, might result in
altered expression of chromosome 2 or 13-translocated genes
such as SCN1A. SCN1A is located at 2q24.3, encodes a voltage-gated Na⫹ channel, and is implicated in epilepsy (669).
5) The phenotype is a function of reduced inhibitory signaling. The loss of NBCn2 could result in a reduced excitability
of inhibitory neurons, due to an inability to regulate pHi.
Alternatively, if the loss of NBCn2, which can act in an
NDCBE-like manner under some conditions, were either
directly or indirectly to result in a rise in neuronal [Cl⫺]i, the
result might be to convert glycine and GABA signals from
the usual inhibitory, to excitatory postsynaptic potentials.
Two other epileptic individuals have since been identified as
having an SLC4A10 haploinsufficiency. The first individual
has a de novo chromosomal deletion of 6.6 Mb that encompasses SLC4A10 and numerous downstream genes in 2q24.2–
2q24.3, including SCN1A, and is both epileptic and mentally
retarded (516). The deletion of epilepsy-associated SCN genes
in this individual confounds attempts to assess the contribution of NBCn2 loss to this pathology. The second individual
has a de novo chromosomal deletion of 6.4 Mb that encompasses SLC4A10 and numerous upstream genes in 2q24.1–
2q24.2. This individual is epileptic, autistic, and mentally retarded (516). Again, the deletion of other genes in this individ-
C) Genetic linkage to autism. A spontaneous deletion of exon
1 of the SLC4A10 gene has been identified in a pair of autistic
twins (857). What is presently unclear is 1) whether the two
phenomena are linked, 2) how perturbation of the SLC4A10
gene might result in autism, and 3) whether the deletion of
exon 1 would result in a haploinsufficiency of functional
NBCn2. As noted above, exon 1 includes the only reported
initiation codon for NBCn2. It is interesting to note that the
twins have not presented with epileptic symptoms.
A third individual (discussed two paragraphs above) has a
de novo chromosomal deletion of 6.4 Mb that encompasses
SLC4A10 and numerous upstream genes in 2q24.1–2q24.2
and is autistic, epileptic, and mentally retarded (516). The
contribution of NBCn2 loss to the autistic pathology is
difficult to assess inasmuch as autism, in some individuals,
is associated with genetic deletions in 2q24.1–2q24.2 that
do not encroach into the known extent of the SLC4A10
gene locus (679).
Another linkage to autism is to be found in the gene that is the
upstream neighbor of SLC4A10: a recent study found that the
TBR1 gene-product associates with the product of the AUTS2
autism-susceptibility candidate gene (69).
D) Defective CSF secretion in mice with a disrupted Slc4a10
gene. Mice with a targeted disruption of Slc4a10 exhibit a
78% decrease in brain ventricular volume (429). A subsequent study indicates that, in NBCn2-null mice, the deficit of
basolateral Na/base cotransport is at least partly compensated
by the relocation of normally apical NHE1 to the basolateral
membrane (216). Apical NHE1 is in turn replaced by an as yet
unidentified amiloride-insensitive NHE. Further preliminary
work by Damkier and Praetorius indicates that AQP1 and the
Na pump, transporters critical to CSF secretion, also have
reduced abundance in the choroid plexus of NBCn2-null mice,
reflecting a compensation that would favor cell survival (and
thus the integrity of the blood-brain barrier) at the expense of
CSF secretion (215). Thus, although the CSF secretion defect
in NBCn2-null mice is appropriate, given the location and
presumed role of NBCn2, the pathogenesis of this phenotype
in NBCn2-null mice is likely more complex than the simple
deletion of NBCn2.
E) Unproven genetic linkage to depression. Although 2 out
of 16 SNPs examined in a study of the human SLC4A10
gene locus were initially linked to major depressive disorder, the linkage was found to be not significant upon further
statistical analysis (846).
III) Sensory organs. A) Suggested genetic linkage to primary
open-angle glaucoma. Defective CSF secretion results in an
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3) Overcompensation. In the choroid plexus of mice, the
loss of NBCn2 is compensated by the redistribution of
NHE1 in its place (216). It is conceivable that a loss of
neuronal NBCn2 might be overcompensated in the proband by a more active population of Na⫹ and/or base transport mechanisms as is the case in NHE1-null mice. Although NHE1-null mice might be expected to be less sensitive to seizures because of their compromised neuronal pHi
regulation (1082), they in fact exhibit seizures (70) due to
an enhanced neuronal excitability, perhaps caused by the
observed compensatory increase in the functional expression of Na⫹ channels (355, 1051). The demonstrated
⬃60% decrease of the acid-loading AE3 protein in NHE1null mice (1060) could also contribute towards a higher pHi
and thus a lower seizure threshold (378).
ual makes it difficult to assess the contribution of NBCn2 loss
to this pathology. Nevertheless, NBCn2 remains a compelling
candidate in the pathogenesis of epilepsy.
MARK D. PARKER AND WALTER F. BORON
IV) Respiratory system. A) Genetic linkage to lung cancer.
A genetic linkage study found somatic mutations in
SLC4A10 in 2 of 11 lung carcinoma samples.80 One is a
P690L substitution at the distal end of EL3, close to the
extracellular end of TM6. What effect, if any, this alteration
might have on NBCn2 function remains untested. The second polymorphism is not predicted to change the NBCn2
protein sequence, being a synonymous change within the
codon for K901, a residue located at the intracellular end of
TM11. To date, the expression of SLC4A10 products in
healthy or cancerous lung tissue has not been demonstrated.
VI. RELATIVES OF NCBTs IN MAMMALS
Here we provide a brief overview of the anion exchangers
AE1–3; the three Na⫹-independent, electroneutral ClHCO3 exchangers, which are closely related both structurally and functionally to the NCBTs. This brief analysis
should assist in the interpretation of material presented
above. Interested readers might consult comprehensive AE
reviews, such as those by 1) Jennings (436), who provides
an excellent evaluation of the physiological studies that first
defined the molecular actions of AE1; 2) Alper (22), who
provides an extensive survey of current knowledge concerning the structure, function, splice variants, distribution,
physiological importance, and pathologies associated with
the AEs; and 3) Cordat and Casey (153) and Romero et al.
(805), who provide a thorough consideration of the physiological and pathological importance of HCO3⫺ transporters in general, including the AEs and NCBTs of the Slc4
family, as well as the anion exchangers of the Slc26 family.
80
The data were obtained from the Wellcome Trust Sanger Institute Cancer Genome Project website: http://www.sanger.ac.uk/
genetics/CGP/cosmic.
912
In the last part of this section, we summarize current knowledge concerning the two most recently described members
of the Slc4 family, Slc4a9 and Slc4a11. Slc4a9 is unique
inasmuch as it is like the electrogenic NCBTs in structure
but was named as if it mediated Cl-HCO3 exchange.
Slc4a11 is unique inasmuch as it is reported to have neither
AE-like nor NCBT-like activity, but instead retains the borate-transport activity common to fungal and plantal Slc4like products. However, the functions of both Slc4a9 and
Slc4a11 remain controversial.
A. Anion Exchangers (AE1–3; Slc4a1–3)
1. Summary
The Cl-HCO3 exchangers of the Slc4 family act as acidloaders (HCO3⫺ export mechanisms) and are the basolateral
counterparts of the apically distributed Slc26 family of anion exchangers (FIGURE 1). In erythrocytes, the HCO3⫺
fluxes mediated by AE1 contributes towards the Bohr effect. In addition, red cell AE1 acts as a scaffold protein
providing a linkage between the membrane and cytoskeleton, contributing towards maintenance of the structural
integrity of the circulating cell. In the kidney, AE1 action
contributes towards the maintenance of blood pH and supports urinary acidification. Thus AE1-related pathologies
include red cell fragility and whole body acidosis. AE2 exhibits the widest distribution of the three AEs and contributes towards pH balance in a variety of cell types. In the
kidney, AE2 contributes towards HCO3⫺ reabsorption in
the late PT81 and in the TAL. AE3 is expressed in the eye,
brain, and heart. AE3 dysfunction is associated with blindness, epilepsy, and cardiac hypertrophy.
2. Nomenclature
AE1, AE2, and AE3 are named for their anion exchange
function, which physiologically is the one-for-one exchange
of Cl⫺ for HCO3⫺. AE1 is the product of the Slc4a1 gene and
is the founder member of the family. AE1 is also referred to
as “band 3,” being the third largest protein band evident on
coommassie-stained gels of red cell membrane preparations. In older literature, AE1 is sometimes referred to as
capnophorin (literally “smoke carrier”). AE2 and AE3 are,
respectively, the products of the Slc4a2 and Slc4a3 genes.
AE4 refers to the Slc4a9 gene product, which is discussed
separately below.
3. Molecular action
Under physiological conditions, all three AEs perform Na⫹independent, electroneutral Cl-HCO3 exchange (509, 587,
81
A basolateral Cl-HCO3 exchanger, thought to be AE2, also con⫺
reabsorption in the S3 segment of the proxtributes towards HCO3
imal tubule (506, 677). Although AE2 mRNA has been detected in
rat proximal tubule preparations (128), robust AE2 immunoreactivity has not been observed in any segment of the proximal tubules of
mice, rats, or humans (27, 158, 914).
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increased pressure differential between the CSF and intraocular compartment that may contribute to the development of glaucoma (briefly reviewed in Ref. 595). Because
NBCn2-null mice have reduced ventricle volume, it was
hypothesized that variations in the SLC4A10 gene locus
might be associated with the incidence of glaucoma in humans. However, a genetic linkage study did not establish a
link between affected individuals and seven common SNPs
in SLC4A10 (595), and no gross ocular phenotype has been
reported for Slc4a10-null mice (429). These data alone do
not preclude the possibility that defects in Slc4a10 contribute to glaucoma for four reasons: 1) NBCn2 is a major
contributor to CSF secretion and thus a link to glaucoma is
sensible, 2) seven SNPs are likely a minor sampling of the
true genetic variability among human SLC4A10 genes,
3) none of the 7 SNPs tested has been shown to affect the
functional expression of NBCn2, and 4) no studies that
describe the lack of ocular pathology in Slc4a10-null mice
have been reported.
⫺
TRANSPORTERS
Na⫹-COUPLED HCO3
Some have suggested, based on studies of red blood cells,
that AE1 is involved in the transport of alkali (i.e., Na and
Li) and heavy metals (i.e., Cd, Cu, Mb, Pb, and Zn) carried
in the form of anionic complexes (18, 303, 304, 323, 610,
886, 974). For example, Na⫹ is suggested to be transported
by AE1 in the form of NaCO3⫺, the same substrate predicted
by kinetic analysis to be carried by an NCBT from squid
axons (99). At present we can only speculate on how the
transport mechanisms of AEs and NCBTs are related, although at least NDCBE and NBCn2 are capable of performing anion exchange (i.e., consistent with NaCO3-Cl
exchange) under certain conditions and in principle the Na/
HCO3 cotransport mediated by NBCn1, NBCe1, and
NBCe2 could be achieved by variations on a NaCO3-HCO3
exchange mechanism.
After correction for transporter abundance, all three fulllength mammalian AE products mediate anion exchange at
similar rates (301, 551, 862). Among the three, AE2 is
uniquely pHi sensitive, becoming increasingly inactive as
pH decreases over the range 9 – 6 (401, 905). Multiple
groups have also reported variations in the relative efficacy
of anion transport inhibitors between pairs of AEs (e.g., see
Refs. 551, 862, 905), although conclusions appear to vary
between expression systems. Further distinctions between
the paralogs become apparent when we consider individual
gene variants and their distribution.
4. Genome
AE-encoding genes are considerably more compact
(⬃14 –20 kb) than NCBTs genes (⬃100 –360 kb), which is
mainly a function of shorter introns and 3=-UTR regions.
The human SLC4A1 gene that encodes AE1 covers ⬃20 kb
at chromosomal locus 17q21-q22. The SLC4A2 gene that
encodes AE2 covers ⬃17 kb at chromosomal locus 7q36.1.
The SLC4A3 gene that encodes AE3 covers ⬃14 kb at chromosomal locus 2q36. Only human chromosome 2 carries
more than one SLC4 gene (SLC4A3 and SLC4A10).
As previously discussed above, genes that encode AEs and
NCBTs share many common exon boundaries (FIGURE 7),
which indicates their relatedness. On the other hand, some
unique exon boundaries are shared only among AEs and are
not shared with NCBTs, indicating that the three AEs diverged from a common ancestor after the divergence of the
common NCBT ancestor.
5. Structural features and variants
AE proteins are predicted to have a similar structure to
NCBTs, due to their sequence similarity at the amino acid
level (see FIGURES 2 AND 3). AEs and NCBTs both have a
large cytosolic Nt, multiple transmembrane spanning segments, and a relatively short Ct. The crystal structure of the
AE1 Nt was first described in Reference 1091 and lowresolution structural reconstructions of the TMD have been
reported in References 1022, 1023, 1068, and 1069. One
group has suggested that the structure of the AE1 TMD
may be similar to that of prokaryotic ClC Cl/H antiporters
(1069). There are some key structural differences between AEs and NCBTs, among AEs and among variants of
each AE.
A) AMINO TERMINUS.
In contrast to the Nt of NBCe1, which is
absolutely required for Na/HCO3 cotransport activity
(276, 634), the Nt of AE1 is not at all required for basal
anion exchanger function (351, 353, 468, 568). However,
the Nt of AE1 contains important trafficking determinants
(975) and has many protein binding partners (recently reviewed in Ref. 141). The Nt of AE2 is not required for anion
exchange activity (587, 1095) but contains determinants
that influence the pH sensitivity of AE2 (527, 907, 1095).
The necessity of the Nt of AE3 for anion exchange function
is untested, but determinants in the Nt of AE3 contribute
toward its relatively poor plasma membrane accumulation
compared with AE1 and AE2 (301). The three-dimensional
structure of the AE1 Nt dimer at pH 4.8 has been solved at
2.6-Å resolution by X-ray crystallography (1091). A subsequent study of the AE1 Nt dimer in solution at neutral and
close-to-neutral pH indicates that the original, “low pH”
crystal structure is a good representation of the native AE1
Nt structure at physiological pH (1109).
B) TRANSMEMBRANE DOMAIN. The AEs have a short third extracellular loop compared with NCBTs. The shortest is that
of AE1, which lacks the glycosylation sites that are a common feature of AE2, AE3, and NCBTs. AE1 is also unique
in having a glycosylation site in its fourth extracellular loop.
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728), a capability not shared with NCBTs. AEs can also
perform futile cycles of HCO3⫺-independent Cl-Cl self-exchange, a mode often exploited in assays as a proxy for
physiological AE function (i.e., Cl-HCO3 exchange). Other
nonphysiological and minor transport modes described for
AEs, but never for NCBTs, include the exchange of monovalent anions such as Br⫺ and NO3⫺ and divalent anions,
which are cotransported with H⫹ thereby maintaining electroneutrality, such as SO42⫺ and oxalate2⫺ (e.g., Refs. 359,
401, 434, 439, and 861). In light of these observations, and
the observations of Boron and co-workers, which suggest
that at least NBCe1 and NDCBE are Na⫹-coupled CO32⫺,
as opposed to HCO3⫺, transporters, it is intriguing to speculate that the AEs could be considered to be H⫹-coupled
CO32⫺ transporters. The divergence of transporters that perform H⫹ versus Na⫹ coupled cotransport of a particular
substrate has been documented for other solute carrier orthologs. For example, members of the Slc23 protein family
in mammals are Na⫹ coupled, whereas bacterial Slc23-like
orthologs are H⫹ coupled (reviewed in Ref. 940). Due to the
permissiveness of the AEs, it is possible that they could be
capable of borate transport like their orthologs in plants
and fungi. There is some indirect evidence that borohydride
(BH4-) is a substrate of AE1 (435).
MARK D. PARKER AND WALTER F. BORON
C) CARBOXY TERMINUS.
The sequence of the Ct is well conserved among AEs and is ⬃40 amino acid in length, far
shorter than that of the NCBTs. The AE Ct lacks the characteristic Lys-rich stretches common to all NCBT Cts and
the class I PDZ binding motif characteristic of some
NCBTs. As is the case with the NBCe1 Ct, the Ct of AE1
contains vital trafficking determinants (184, 203, 300,
976).
D) AE VARIANTS. Each of the three AE genes produces one
full-length product and one to three additional truncated
variants, transcribed under the control of internal promoters. Thus all AE variants differ only in their extreme Nt
sequences (the variants discussed below are depicted in Appendix V). Posttranscriptional processing of AE transcripts
is not known to include the splicing that, for NCBTs, results
in the optional inclusion of protein cassettes within the Nt
and Ct, and variations in extreme Ct sequences.
SLC4A1 contains two alternative promoters. The first produces the full-length gene product erythrocyte AE1 (eAE1;
911 amino acid) and the second produces the truncated
kidney AE1 (kAE1; 846 amino acid) that lacks the first 65
amino acid of eAE1 (127, 520, 521) and thereby loses the
ability to bind ankyrin (251). A similar transcriptional
mechanism produces truncated versions of the AE2 and
NDCBE products (e.g., NDCBE-A versus NDCBE-C in FIGURES 36C AND 37).
SLC4A2 contains three alternative promoter regions: a, b,
and c (1030). The first produce the full-length gene product
AE2a (1,241 amino acid in humans, 1,237 amino acid in
mice). The second can produces one of two shorter products, AE2b1 or AE2b2, depending on which of two closely
positioned transcriptional start sites are utilized. In AE2b1,
the first 17 amino acid of AE2a are replaced by a novel
3-amino acid sequence. In AE2b2, the first 17 amino acids
of AE2a are replaced by a novel 8-amino acid sequence. In
mice, the third promoter region can produce one of two
914
even-shorter products, AE2c1 or AE2c2,82 again depending
on which of two closely positioned transcriptional start
sites are utilized. Mouse AE2c1 is the shortest of all AE2
products and is a truncated version of the other AE2 products, such that AE2c1 initiates at Met199 of AE2a. In
mouse AE2c2, the first 193 amino acid of AE2a are replaced
by a novel 27-amino acid sequence. The complex transcriptional and posttranscriptional mechanisms that produce
each variant are depicted in detail in References 550 and
637. In side-by-side comparisons, AE2b variants have a
greater functional expression than AE2a or AE2c1, whereas
AE2c2 activity was undetectable (527). It is possible that
these observations might at least in part be explained by
differences in surface expression; however, AE2c1 clearly
has an alkaline shifted pHo dependence (527) compared
with the other forms. Alternative promoter choice, resulting
in small, seemingly insignificant alterations in Nt sequence,
are also common to NBCn1 (e.g., NBCn1-A versus
NBCn1-B in FIGURES 31C AND 32).
SLC4A3 contains two alternative promoters. The first produces the full-length gene product brain AE3 (bAE3, aka
AE3fl; 1,232 amino acids). The second produces the shorter
cardiac AE3 (cAE3; 1,034 amino acids) from an alternative
transcription site that includes a novel ATG codon. Thus, in
cAE3, the first 271 amino acids of bAE3 are replaced by a
novel 73-amino acid sequence (588, 1080). One study
found that the intrinsic Cl-HCO3 activity of bAE3 is doubled compared with cAE3 and to a truncated AE3 that lacks
the unique sequence of both (905), as though the unique
longer sequence of bAE3 has a mildly autostimulatory effect. The transcriptional mechanism that produces bAE3
versus cAE3 is similar to that which produces NBCe1-B and
NBCe1-A. In fact, the point at which bAE3 and cAE3 sequences converge is only four amino acids downstream of
where NBCe1-A and NBCe1-B sequences converge.
6. Distribution
Whereas elsewhere we have considered the distribution of
NCBTs by organ system, in this section we consider each
AE in turn and consider whether its distribution overlaps
with that reported for any NCBT. In common with NCBTs,
the location of AEs in polarized cells is overwhelmingly
basolateral.
A) AE1.
eAE1 is prominently expressed in the plasma membrane of red blood cells, whereas kAE1 is located in the
basolateral membrane of ␣-intercalated cells in the renal
collecting duct (24, 262). Red blood cells have not been
demonstrated to express any NCBT. Some researchers describe NBCn1 (694, 1014) and AE4 (498) immunoreactiv82
In rats, AE2c1 and AE2c2 transcripts both encode the AE2c1
polypeptide (1030). Genomic differences may mean that the rabbit
and human AE2 genes lack the capacity to produce AE2c transcripts
(527).
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As is the case for the NCBTs, glycosylation of AE1 is not
essential for transport (155, 352). The AEs lack the four
conserved cysteines in EL3 common to NCBTs, although
AE2 has a single Cys in this loop. A comparative study of
the accessibility of substituted cysteines in the latter half of
the TMD indicates structural differences between AE1 and
NBCe1 in this region (1112). Lysine-rich motifs, also found
in the NCBTS, at the extracellular ends of putative TMs 5
and 13 contribute to the stilbene sensitivity of the AEs (63,
435, 699). Finally, a glutamate in putative TM8 forms an
important part of the transport gate of AE1 (437, 438, 440,
443). The three-dimensional structure of the AE1 TMD
dimer has been solved at 7.5-Å resolution using cryo-electron microscopy (1068, 1069). However, this resolution is
not sufficient to visualize all TMs nor to assign amino acid
sequence to regions of electron density.
⫺
TRANSPORTERS
Na⫹-COUPLED HCO3
ity in the basolateral membranes of ␣-intercalated cells in
the medullary segments of the collecting duct. However, we
are unaware of the direct colocalization, even of any two of
these three Slc4 proteins, by the same authors in the basolateral membrane of the ␣-intercalated cell.
In many cases the distribution of AE2 overlaps with that of
an NCBT. For example, sites of AE2 expression that match
those of NBCe1, presented in separate reports, include the
basolateral membranes of salivary parotid acinar cells (372,
818), pancreatic acinar cells (816), duodenal enterocytes
(25), proximal colon enterocytes (25), late PT, and epididymal epithelia (446). The distribution of AE2 overlaps with
that of NBCn1 in the basolateral membrane of mTAL epithelia, and with NBCn2 in the basolateral membrane of
choroid plexus epithelia (see cartoon in FIGURE 28 AND Ref.
587). In other cell types, AE2 is coexpressed with NBCe2
but in polar opposite membranes. For example, in choroid
plexus epithelia (basolateral AE2, apical NBCe2, see cartoon in FIGURE 28) and in hepatocytes (apical AE2, basolateral NBCe2; see cartoon in FIGURE 29 AND Ref. 46).
Reports conflict as to whether NBCe1 is present in ameloblasts, which also express AE2 (see cartoon in FIGURE 20 as
well as Refs. 456 and 706). Despite these apparent overlaps
in reported distribution, we are not aware of any publications in which the same set of authors has visualized NCBTs
and AE2 protein in the same cell.
There is no evidence that AEs and NCBTs are capable of
forming heterodimers. However, functional coupling of
AEs, specifically AE2, and NCBTs has been proposed (571,
1019). For example, NBCn1 in the basolateral membrane
of the mTAL presumably mediates the uptake of Na⫹ and
HCO3⫺ (571). In parallel, some of the HCO3⫺ exits the cell in
exchange for Cl⫺ via AE2. To the extent that the HCO3⫺
fluxes of the two transporters balance, the net effect is NaCl
uptake.
C) AE3. AE3 is expressed in neurons and glia throughout the
central nervous system (378, 463, 502, 509) and in heart
preparations (588, 761), specifically in the sarcolemma and
T tubules of myocytes (31). Thus the expression of AE3
potentially overlaps with all NCBTs in the CNS and with
NBCe1 and NBCn1 in cardiac myocytes. Again, despite the
apparently overlapping distribution, NCBTs and AE3 have
7. Physiological roles
Here we discuss the generally complementary, but sometimes similar, physiological roles of AEs and NCBTs, and
how their different molecular actions impact these roles.
A) GENERAL: PHI REGULATION.
AEs typically operate as acidloaders, exchanging intracellular HCO3⫺ for extracellular
Cl⫺, tending to restore pHi after an alkaline load (995).
NCBTs on the other hand typically operate as acid-extruders, tending, as is the case with Na-H exchangers, to restore
pHi after an acid load. The concerted action of these three
transport mechanisms over a range of pHi values is nicely
demonstrated in a study of ventricular myocytes by Leem
and coworkers (563).
B) AE1: MAINTENANCE OF RED CELL MORPHOLOGY.
The cytosolic
Nt of AE1 forms extensive interactions with cytoskeletal
proteins, tethering the red cell cytoskeleton to the red cell
membrane. Not only do these interactions help the red cell
maintain its biconcave shape, thereby maximizing its surface area-to-volume ratio for gas exchange, but they also
allow each cell to be temporarily deformed, rather than
sheared, as it passes through the microcirculation. The importance of AE1 for red cell morphology is reviewed by
Burton and Bruce (141).
C) AE1: PROMOTION OF GAS EXCHANGE ACROSS THE RED CELL
MEMBRANE. Carbon dioxide entering red blood cells in the
systemic capillaries is hydrated to H⫹ plus HCO3⫺ via the
action of CA II. The HCO3⫺ exits into the plasma via AE1,
maintaining a driving force for CO2 entry into the red blood
cell and maximizing the CO2-carrying ability of the blood.
The H⫹ generated by the CA reaction is buffered by hemoglobin (Hb), which may be tethered to the cytoplasmic Nt
domain of AE1 (475). The binding of H⫹ to Hb reduces the
affinity of Hb for O2, thereby promoting O2 release (the
Bohr effect). The Bohr effect also plays a role in the pulmonary capillaries, where HCO3⫺ entry into the red cell plasma
via AE1, maintains a driving force for CO2 exit from the red
cell, and promotes H⫹ consumption thereby increasing the
affinity of Hb for O2. The relationship between red cell pH
and gas exchange is reviewed in Reference 444.
⫺
D) AE1 AND AE2: HCO3 REABSORPTION/H
⫹
SECRETION. In the basolateral membrane of mTAL epithelia (FIGURE 34), AE2 is
predicted to move HCO3⫺ from cell to the blood, thereby
contributing towards reabsorption of residual HCO3⫺ from
the mTAL lumen into the blood (269). HCO3⫺ exit across
the basolateral membrane also promotes the generation of
intracellular H⫹, which stimulates H⫹ secretion into the
lumen. This secreted H⫹ either titrates HCO3⫺ in the lumen
(HCO3⫺ reabsorption) or titrates NH3 and other nonHCO3⫺ buffers (H⫹ excretion). Further along the nephron,
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B) AE2. Of the AEs, AE2 is by far the most widely distributed.
Locations include the basolateral membranes of epithelia
that line the gastrointestinal tract (e.g., see cartoons in FIGURES 21 AND 22 as well as Refs. 25 and 813) and also the
basolateral membranes of some renal tubule segments,
most prominently in the mTAL (see cartoon in FIGURE 34 as
well as Refs. 27, 158, 298, and 914). The AE2a and AE2b
variants have a similar broad distribution, whereas robust
AE2c expression appears to be restricted to the stomach
(25, 550, 1030).
not, to our knowledge, been formally colocalized by immunocytochemistry in the same cell.
MARK D. PARKER AND WALTER F. BORON
in the basolateral membrane of collecting duct ␣-intercalated cells, AE1, rather than AE2, performs a similar function (282, 904). The renal actions of AE2 and AE1 both
counter metabolic acidosis in the blood. In ameloblasts and
osteoclasts, AE2-mediated HCO3⫺ efflux across the basolateral membrane supports H⫹ secretion across the apical
membrane, contributing to tooth (456) and bone (1048)
remodeling (see FIGURES 20 AND 33).
⫺
E) AE2: HCO3 SECRETION/H
⫹
A consequence of the HCO3⫺ transport function of AEs and NCBTs is that AEs import Cl⫺,
whereas NCBTs generally import Na⫹. As noted earlier,
both of these consequences can contribute towards the vectoral transport of NaCl, together with osmotically obligated H2O, across epithelia. In airway, duodenal, and colonic epithelia, the functionally coupled action of AE2 and
an NCBT (e.g., AE2 and NBCe1 in FIGURE 22) performs,
along with NKCC1, Na⫹ and Cl⫺ influx across the basolateral membrane that supports CFTR-mediated Cl⫺ secretion across the apical membrane (312, 397, 1019). Knocking out murine AE2 or NBCe1 is associated with a presumably compensatory increase in NKCC activity in intestinal
epithelia (312, 313).
F) AE2: SALT SECRETION.
G) AE2: VOLUME REGULATION.
The intracellular alkalinization
that follows the shrinkage-induced activation of NHE1
subsequently activates AE2. The resulting net influx of Na⫹
and Cl⫺, followed by water, tends to restore cell volume
(449, 873). One group has suggested that shrinkage-induced activation of NDCBE might also tend to restore cell
volume. In the red blood cells of trout, swelling opens a
cryptic solute channel within AE1 protein (285). The efflux
of ions and uncharged solutes through trout AE1 would
tend to restore cell volume. Regulated volume decrease is
common to the red blood cells of many species, but the
physiological involvement of AE1 in such a pathway is not
well demonstrated in mammals (357).
H) AE3: CONTROL OF NEURONAL EXCITABILITY.
The acid-loading
action of AE3 tends to dampen neuronal excitability, con-
916
8. Causes of AE upregulation
Here as well as in the following section, we mainly consider
the effect upon AEs of those perturbations that have been
described elsewhere to affect NCBT functional expression.
Typically, acid-extruding NCBTs are upregulated by metabolic and respiratory acidosis, the consequence of which is
defense of pHi. Even NBCe1, acting as an acid-loader in the
proximal tubule, is upregulated by acidosis, increasing
HCO3⫺ reabsorption, the consequence of which is defense of
plasma pH.
⫺
A) AE1. AE1 plays a role in support of renal HCO3
reabsorption/H secretion in the CCD. Indeed, AE1 transcript and
protein abundance increase in response to metabolic acidosis (282, 398, 763, 828, 1002), which is the appropriate
response. One study reports that the red blood cells of individuals permanently living at high altitude (e.g., Bolivians) contain 50% more AE1 protein than red blood cells of
individuals permanently living at sea level (e.g., Danes, see
Ref. 457).
⫹
B) AE2. In the mTAL, AE2 protein abundance is increased
in response to metabolic acidosis, a compensatory mechanism that could increase HCO3⫺ reabsorption/H⫹ secretion by this nephron segment (778). AE2 protein abundance in the mTAL is also elevated by NaCl loading,
consistent with its role in support of salt secretion (778),
as discussed above.
9. Causes of AE downregulation
A) AE1. In keeping with its upregulation during acidosis, AE1
protein abundance in the collecting duct is reduced during
metabolic alkalosis (828).
B) AE3.
AE3 protein abundance is reduced in the brains of
rats following a 2-wk exposure to 12% CO2 (463), a response that is consistent with the reduced usefulness of an
acid-loading transporter under hypercapnic conditions.
10. Consequences of AE dysfunction
Interested readers might refer to the reviews of others for
extensive discussions of the pathological consequences of
AE1–3 dysfunction (e.g., see reviews in Refs. 22 and 1036).
Here we discuss pathologies that are relevant to defects in
both AEs and NCBTs and how differences between the
transporters may impact the sequelae.
A) AE1: DISTAL RENAL TUBULAR ACIDOSIS AND HEMOLYTIC ANEMIA.
Individuals with mutations in the SLC4A1 gene (130) and
mice with a disrupted Slc4a1 gene (904) have a lower-than-
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REABSORPTION. Although AEs typically support HCO3⫺ reabsorption, in three instances, AE2
is positioned to support HCO3⫺ secretion: 1) AE2 exhibits
an apical distribution in cholangiocytes and 2) hepatocytes
(46, 987) and 3) AE2 exhibits a lateral distribution in
ameloblasts, becoming exposed to the apical compartment
during ameloblast maturation by a rearrangement of tight
junctions (456). In contrast to AEs, NCBTs typically support HCO3⫺ secretion. Two notable exceptions in the case
of NCBTs are NBCe1-A in the basolateral membrane of the
proximal tubule and NBCe2 in the apical membrane of the
choroid plexus, both of which are predicted to operate with
the unusual apparent Na⫹:HCO3⫺ stoichiometry of 1:3,
thereby mediating HCO3⫺ reabsorption.
sistent with the association of AE3 dysfunction with epilepsy.
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
B) AE2: BONE AND ENAMEL DEFECTS.
Mice and cattle with AE2
insufficiency exhibit signs of osteoclast dysfunction, such
as growth retardation and osteopetrosis (432, 455, 643,
1048). On its free or contralacunar membrane surface
(facing bone interstitium), the osteoclast expresses AE2
(FIGURE 33). The Cl-HCO3 exchange activity acidifies the
cell, thereby promoting H⫹ secretion across the ruffled
border by the vacuolar-type H⫹ pumps. As the H⫹ enters
the resorption lacuna (the space between the ruffled border and calcified bone), the acidity promotes bone resorption (solubilization of bone mineral and hydrolysis of
matrix proteins). Osteoclasts in which NBCn1 abundance has been reduced by antisense technology also exhibit reduced bone-resorption function. NBCn1 appears
to be expressed at high levels in the ruffled-border membrane that faces the resorption lacuna, where the action
of H⫹ on CaCO3 forms HCO3⫺. Presumably the NBCn1
would move this newly formed HCO3⫺ from resorption
lacuna to the cytosol of the osteoclast for removal by AE2
into the interstitium (797).
AE2-null mice are toothless, exhibit growth retardation,
and die prematurely (314). Mice that are unable to express
the a and b variants of AE2 also have defective tooth enamel
(126, 616) because AE2 supports H⫹ secretion in ameloblasts. NBCe1 dysfunction is similarly associated with
enamel defects, although the underlying mechanism in that
case has yet to be resolved.
C) AE2: INFERTILITY. AE2-null mice do not live to breeding age,
but male mice that lack only the a and b variants of AE2 are
infertile due to defects in spermiogenesis (638).
D) AE2: GASTRIC ACID SECRETION DEFECTS. Gastric secretions in
AE2-null mice are not acidic due to a combination of loss of
AE2-mediated HCO3⫺ efflux across the basolateral membrane of parietal cells (which normally supports H⫹ secretion), loss of parietal cells, and ultrastructural defects in
remaining parietal cells (314).
E) AE3: EPILEPSY.
A mutation, A679D, in AE3 reduces the
per-molecule activity of the transporter (1004) and is associated with idiopathic generalized epilepsy in humans
(830). Furthermore, mice lacking AE3 have a reduced seizure threshold in response to proconvulsive agents (378).
The neurons of mice lacking the acid-loading AE3 exhibit
an elevated pHi that likely contributes to neuronal hyperexcitability (378). Note that these features of AE3-null mice
are opposite to the phenotype of mice that lack the acidextruding transporters NDCBE and NBCn2, which have an
increased seizure threshold.
F) AE3: BLINDNESS. AE3-null mice exhibit signs of reduced
inner retina function and increased apoptosis of photoreceptor cells (30). A similar phenotype is observed in certain
strains of NBCe2-null and NBCn1-null mice, the common
denominator perhaps being an inability to regulate pHi in
these cells, assuming that this is not a side effect of the
expression of misfolded protein fragments expressed from
the disrupted genes.
B. AE4 (Slc4a9)
1. Summary
Despite its reported function as a Cl-HCO3 exchanger in
heterologous systems, Slc4a9 is more closely related, at the
level of exon-boundary structure and deduced amino acid
sequence, to the Na⫹-coupled members of the Slc4 gene
family. The molecular action and subcellular distribution of
Slc4a9 products remains controversial and may even be
species-specific. AE4 expression appears to be mainly restricted to the kidney, most likely in the basolateral membranes of non-␣-type intercalated cells of the collecting
duct. As discussed earlier, the Slc4a9 gene seems to have
arisen from a recent duplication (the most primordial
Slc4a9 known appears in two frog genomes) of an electrogenic NCBT-encoding gene. Among the 10 vertebrate Slc4s,
Slc4a9 orthologs have the most divergent sequences. It is
possible that this recently duplicated gene is still in the process of diverging.
2. Nomenclature
Originally termed hSBC5 (human sodium bicarbonate
cotransporter 5) in an early GenBank submission, the
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normal blood pH and [HCO3⫺] and excrete an unusually
alkaline urine. Underlying these signs is loss of per-molecule
function and/or reduced accumulation of AE1 in the basolateral membrane of collecting duct ␣-intercalated cells
(130). The result is an impaired ability of the collecting duct
to reabsorb HCO3⫺/secrete H⫹, processes that normally
support H⫹ secretion into the duct lumen. Similarly, genetic
defects in NBCe1 result in proximal renal tubular acidosis
(pRTA), although underlying the acidosis in this case is a
failure of HCO3⫺ reabsorption/H⫹ secretion at the level of
the PT. Individuals with AE1-associated distal renal tubular
acidosis (dRTA) typically have a less severe deficit in plasma
[HCO3⫺] because the PT, which is responsible for ⬃80% of
the HCO3⫺ reabsorption, is intact. However, patients with
an AE1 defect do relatively poorly in acidifying their urine.
Individuals with NBCe1-associated pRTA typically have a
severe deficit in plasma [HCO3⫺]. Thus the filtered load of
HCO3⫺ is low enough that the intact distal nephron can
reabsorb the HCO3⫺ and also lower urine pH. (e.g., see Ref.
253). AE1-associated dRTA also have a different set of
extrarenal sequelae from NBCe1-associated pRTA because
of the different sites of AE1 and NBCe1 expression. For
example, dRTA is sometimes accompanied by loss of AE1
from red blood cells, resulting in hemolytic anemia (see
review in Ref. 1036) and, secondary to the anemia, cardiac
hypertrophy (31, 708).
MARK D. PARKER AND WALTER F. BORON
Slc4a9 product was renamed AE4 (anion exchanger 4) following a report that the rabbit Slc4a9 product mediates
Cl-HCO3 exchange (982).
3. Molecular action
A cDNA encoding an Slc4a9 product was first reported by
Tsuganezawa et al. (982). There are three reports of ClHCO3 exchange activity mediated by mammalian AE4.
The authors also reported that Xenopus oocytes expressing
rabbit AE4 mediate a Na⫹-independent and DIDS-insensitive 36Cl uptake in the nominal absence of CO2/HCO3⫺.
Because they did not examine oocytes in the presence of
CO2/HCO3⫺, this result cannot be taken as evidence of ClHCO3 exchange.
2) Ko et al. (498) demonstrate that HEK-293 cells transiently transfected with rat AE4 cDNA alkalinize in response to the removal of bath Cl⫺ in the presence of CO2/
HCO3⫺. This pHi increase appears to be predominantly
CO2/HCO3⫺ dependent. The alkalinization is unaffected by
lowering of bath Na⫹, but is strongly inhibited by the application of H2DIDS. Evidence of electroneutrality of the
transport process is provided by the lack of effect of valinomycin and elevated [K⫹]o on the alkalinization.
3) Xu et al. (1054) expressed mouse AE4 in Xenopus
oocytes, using the fluorescence of BCECF to monitor pHi.
They reported that, following a 20 –30 min equilibration in
CO2/HCO3⫺-containing solution, oocytes expressing AE4
alkalinized upon removal of bath Cl⫺. These authors did
not discuss whether pHi recovered from the CO2-induced
acid load when the oocytes were exposed to Cl⫺ or whether
the alkalinization induced by Cl⫺ removal was DIDS sensitive. An unusual aspect of the data was that the pHi of the
control oocyte (i.e., the one not expressing AE4) in CO2/
HCO3⫺ was ⬃7.2 (i.e., roughly the pHi expected in the
absence of CO2/HCO3⫺). We would have expected the ap-
918
In one preliminary study on Xenopus oocytes, Parker et al.
(716) observed that Xenopus oocytes expressing a human
AE4 splice variant exhibited a small but significant pHi
recovery from a CO2-induced acid load. Moreover, the removal of extracellular Na⫹ produced a very small but significant pHi decrease, consistent with electroneutral Na/
HCO3 cotransport activity (716).
We conclude that the functional data on AE4 are inconsistent, possibly due to the use of cDNAs from different species, the use of different heterologous expression systems,
different and nonoverlapping protocols, and different experimental approaches. We cannot rule out the possibility
that the pHi increases observed after removal of Cl⫺ required a protein endogenous to the host cell.
4. Genome
The human SLC4A9 gene occupies at least 22 exons spread
over 16 kb at the chromosomal locus 5q31 (591, 720),
making it similar in size to the genes encoding AE1, AE2,
AE3, and BTR1 but considerably more compact than genes
encoding verified NCBTs, which are typically ⱖ100 kb.
However, Slc4a9 shares more common exon boundaries
with Slc4a4 and Slc4a5, genes that encode electrogenic
NCBTs (FIGURE 7). Putative promoter elements are located
upstream of the transcriptional starting position of human
SLC4A9, including a CCAAT box, a GC box, and a TATA
box (720). A region upstream of the first exon of mouse
AE4 has basal promoter activity and contains a consensus
motif for binding the transcription activator Foxi1. Indeed,
AE4 transcription is enhanced 100-fold by Foxi1 (528), and
mice lacking Foxi1 also lack AE4 (87) as well as subunits of
the H⫹-pump (1003). Sequences with promoter activity are
also found within the mouse Slc4a9 gene upstream of exon
3 and upstream of exon 6 (377).
5. Structural features and variants83
A 2001 survey of human ESTs describes 14 distinct AE4
transcripts, most of which are not predicted to encode a
functional transporter/stable membrane protein, owing to
the presence of stop codons or the absence of transmembrane spans (591). Compared with the most complete reported mammalian transcript, a 15th human transcript
(720) lacks part of exon 9 (i.e., leading to the absence of the
11-amino acid “LFGGLIQDVRR” in the cytosolic Nt domain close to TM1) as well as part of exon 12 (i.e., leading
to the absence of the 3 amino acid “VSM” in the 3rd extracellular loop). Northern blots of human, mouse, and rabbit
83
GenBank protein accession numbers for the variants discussed
in this section are provided in Appendix IV.
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1) Tsuganezawa et al. (982) report that 60% of COS-7 cells
transiently transfected with rabbit AE4 cDNA rapidly and
reversibly alkalinize in response to the removal of bath Cl⫺
in the presence of CO2/HCO3⫺. The 60% figure is consistent
with the 68% transfection efficiency calculated for these
cells. Supposed evidence for electroneutrality is provided in
experiments in which the authors used a whole cell patch to
monitor Vm while applying CO2/HCO3⫺ and then, in the
continued presence of CO2/HCO3⫺, removing Na⫹. They
observed no substantial Vm changes. It is not clear how this
protocol could address the issue of whether the putative
Cl-HCO3 exchanger is electroneutral; a better approach
would have been to monitor an electrical parameter while
removing Cl⫺ in the presence versus the absence of CO2/
HCO3⫺. Only the switch to 130 mM K⫹ produced a Vm
change, although the depolarization was slow and poorly
reversible.
plication of 5% CO2 to cause pHi to fall to ⬃6.9 and not
recover much from there (e.g., see Ref. 725).
⫺
TRANSPORTERS
Na⫹-COUPLED HCO3
kidney AE4 RNA demonstrate a multiplicity of bands in
these organisms (377, 591, 982). Only two rabbit AE4 variants, AE4a and AE4b, have been cloned, and both have a
full complement of transmembrane spans, AE4b differing
from AE4a only in the absence of a 16-amino acid sequence
within the cytosolic Nt domain of AE4b (982). In mice,
Slc4a9 transcription can initiate at multiple points, resulting in the production of Nt truncated AE4 splice variants
that are shorter than the longest reported AE4 protein by
157 and 251 amino acids (377). The effect of such truncation on the function and/or trafficking of AE4 remains untested.
E) CIRCULATORY SYSTEM. A study of laser-captured rat brain
microvessels demonstrated the presence of AE4 protein
by ICAT (isotope-coded affinity tagging) nanoLC-MS/MS
(366).
F) MUSCULOSKELETAL SYSTEM. We are not aware of any reports
of AE4 expression in the musculoskeletal system.
G) UPPER DIGESTIVE SYSTEM. I) Salivary gland. AE4 immunoreactivity is reported in the basolateral membranes of duct
cells from the mouse submandibular gland (498).
II) Stomach. AE4 transcripts are detected in northern
blots of stomach RNA preparations from mouse, rabbit,
and rat (498, 1054). Specifically, rabbit AE4 transcripts
were detected in preparations from gastric mucous and
parietal cells (1054). A 2003 immunohistochemical study
reports the presence of AE4 protein in the apical membranes of mouse and rabbit gastric surface mucous cells
(1054).
6. Distribution
H) LOWER DIGESTIVE SYSTEM.
AE4 is predominantly expressed in the kidney. The apparent AE4 distribution in specific organ systems is discussed
below. Some reports of protein distribution must be regarded with caution due to inadequate characterization of
the antibodies used.
A) CENTRAL NERVOUS SYSTEM.
AE4 immunoreactivity has been
detected in the apical membranes of ciliated ependymal cells
in the third ventricle of the choroid plexus of mice and rats
(755).
B) SENSORY ORGANS.
I) Intestines. AE4 immunoreactivity is reported in the apical villus membranes of human,
rabbit, and mouse duodenum (1054). However, the evidence presented for the presence of AE4 protein in duodenal
cells hinges on the specificity of the antibody, which, in
mouse preparations, does not immunoreact with a protein
of the molecular weight expected for AE4 (see FIGURE 4B of
Ref. 1054). Furthermore, others report that AE4 transcripts
are absent from mouse duodenum preparations (482, 887).
Thus the presence of AE4 in duodenum remains controversial.
AE4 transcripts have been reported in rat cecum (498).
As far as we are aware, there are currently no reports of AE4 expression in the eye, ear, or olfactory system.
II) Liver. An NCBI-curated database suggests that the mouse
liver is a minor site of AE4 transcription (Appendix VI).
C) PERIPHERAL NERVOUS SYSTEM.
I) ENDOCRINE SYSTEM.
D) RESPIRATORY SYSTEM.
J) LYMPHATIC AND IMMUNE SYSTEMS. We are unaware of any
reports of AE4 expression in the lymphatic or immune systems.
As far as we are aware, there
are currently no reports of AE4 expression in the peripheral
nervous system.
AE4 transcripts are detected in cultured human nasal epithelial cells, increasing in abundance
as the cells grow to confluence and project cilia (878). The
authors of that study report that “immunofluorescent staining was seen along the whole cell membrane, which suggests
that AE4 is localized in both the luminal and basolateral
membranes.” However, the immunocytochemistry presented in the study, performed on permeabilized cells, does
not support the stated conclusion for several reasons. 1) The
cells are of undemonstrated confluence and polarity. 2) The
confocal microscopic image, which is only in the x-y plane,
shows predominantly perinuclear staining, with no evidence of a signal at the plasma membrane. 3) The specificity
of the commercial anti-AE4 antibody is not demonstrated.
We are unaware of any reports of AE4
expression in the endocrine system.
K) URINARY SYSTEM.
I) Kidney. Northern blotting studies
demonstrate that AE4 transcripts are predominantly renal
in humans (591, 720), rats (498), and rabbits (982). In
mice, the kidney-specific transcription factor Foxi1 is responsible for the predominantly renal expression of AE4
(377). In microdissected preparations of rat kidneys, AE4
transcripts are most abundant in the cortical collecting duct
(498). AE4 mRNA and immunoreactivity are also detected
in the rat renal collecting duct cell line RCCD1 (798). Reports, individually considered in Appendix VIII, conflict as
to the precise location of AE4 protein within the collecting
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The Nt domain of AE4 includes a leucine-zipper– consensus
sequence that may contribute towards protein oligomerization or interactions with binding partners (498, 720). The
third extracellular loop includes the four cysteines that are
common to NCBTs, and includes multiple putative glycosylation sites, although an in vitro study failed to demonstrate N-glycosylation of human AE4 (720).
MARK D. PARKER AND WALTER F. BORON
duct. Although the consensus is that the AE4 protein is
expressed predominantly in intercalated cells, it is not
agreed whether AE4 is located in ␣- vs ␤-intercalated cells
(see Ref. 498 versus Refs. 87, 982, and 1054). There is also
controversy as to whether AE4 is localized to the apical
versus basolateral membrane (see Refs. 762, 982, and 1054
versus Refs. 87 and 498), although we note that the data
pointing to an apical location are all from rabbits. These
disparate observations have been suggested to represent interspecific differences, although they might equally be explained by two other factors.
2) Putative sites of AE4 expression are designated as either
␣- or ␤-intercalated cell subtypes, but none of the studies
considers the substantial subpopulation of intercalated cells
in the collecting ducts of mice, rabbits, and rats that are
non-␣/non-␤ types (270, 487).84
The evidence discussed in Appendix VIII is consistent with
the hypothesis that AE4 is expressed in the basolateral
membranes of both ␤-intercalated cells and non-␣/non-␤
intercalated cells (collectively known as non-␣ types) in rats
and mice. The expression of AE4 in ␣-intercalated cells is
not well demonstrated. It is unclear why anti-AE4 antibodies immunoreact with epitopes in the apical membranes of
rabbit intercalated cells.
L) REPRODUCTIVE SYSTEM.
An NCBI-curated EST database
suggests that human and mouse testes are a minor site of
AE4 transcription (Appendix VI).
7. Physiological roles
Inasmuch as the expression of AE4 is mainly restricted to
the kidney and inasmuch as AE4-null mice do not have an
obvious renal phenotype, the physiological role(s) of AE4 is
presently unknown. Four possible roles for AE4 have been
proposed.
I) Suggested role in gastric HCO3⫺
secretion. A 2003 study reported AE4 immunoreactivity in
the apical membranes of mouse stomach epithelia, where
A) UPPER DIGESTIVE SYSTEM.
84
⫺
(i.e., lumen to blood)
␣-Type intercalated cells reabsorb HCO3
and are characterized immunologically by an apical presence of a
vacuolar-type H⫹ pump and a basolateral presence of AE1 (mediat⫺
⫺
efflux). ␤-Type intercalated cells secrete HCO3
(i.e., blood
ing HCO3
to lumen) and are characterized immunologically by an apical pres⫺
ence of pendrin (Slc26a4, mediating HCO3
efflux), a basolateral
presence of a vacuolar H⫹ pump, and a lack of basolateral AE1.
Non-␣/non-␤-intercalated cells (sometimes refered to as ␥-subtypes) have an apical presence of pendrin and H⫹ pump and lack
basolateral AE1.
920
B) LOWER DIGESTIVE SYSTEM.
I) Suggested role in duodenal
HCO3⫺ secretion. A 2003 study detected AE4 immunoreactivity in the apical membranes of mouse and rabbit duodenal epithelia where HCO3⫺ secretion mediated by an apical
Cl-HCO3 exchanger might play a role in mucosal protection (87). However, 1) others do not detect AE4 transcripts
in duodenal epithelial cells of mice (887), 2) Cl-HCO3 exchange is unperturbed in the apical membranes of duodenal
villus cells from AE4-null mice (887), and 3) later work
suggests that an Slc26a6 product is the apical Cl-HCO3
exchanger in these cells (887). Thus, if AE4 is indeed in the
apical membranes of these cells, its role remains undemonstrated.
I) Suggested role in support of renal H⫹
secretion. The apparent localization of AE4 to the basolateral membranes of collecting-duct ␣-intercalated cells led to
the suggestion that AE4 (acting as a Cl-HCO3 exchanger)
could act in parallel with basolateral AE1 to support H⫹
secretion (498). However, experimental evidence for such a
physiological role is lacking, inasmuch as the presence of
AE4 in ␣-intercalated cells is not well demonstrated, and
AE4 protein levels are not compensatorily increased in AE1
null-mice (904).85
C) URINARY SYSTEM.
II) Suggested role in support of renal HCO3⫺ reabsorption
in acidosis. A preliminary study suggests that, in collecting
duct ␤-intercalated cells isolated from rabbits (see footnote
84), acidosis reduces the abundance not only of pendrin at
the apical membrane but also the abundance of AE4 mRNA
as well as the abundance of AE4 immunoreactivity in a
subapical compartment (762). Inasmuch as ␤ cells mediate
transepithelial HCO3⫺ secretion and pendrin mediates the
apical step (HCO3⫺ efflux into lumen), it is tempting to
speculate that AE4 might normally contribute to HCO3⫺
secretion in rabbits. If this speculation is correct, and if the
AE4 protein is present in the apical membrane of these cells,
then AE4 would have to be a Cl-HCO3 exchanger. On the
other hand, if AE4 exhibits a basolateral distribution in
rabbit ␤-intercalated cells, as is the case in other model
animals, then AE4 would have to mediate HCO3⫺ transport
coupled to Na⫹ influx (i.e., Na/HCO3 cotransport), rather
than to Cl⫺ efflux (i.e., Cl-HCO3 exchange), to contribute
85
The defect in AE1-null mice may be partly compensated by
upregulation of the AE1-colocalized transporter Slc26a7, which
some investigators describe as a Cl-HCO3 exchanger (921) (see
footnote 2).
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1) None of the anti-AE4 antibodies that are considered in
Appendix VIII recognizes a single band of the appropriate
molecular weight in western blots of kidney preparations.
Thus the specificity of these antibodies is not demonstrated.
HCO3⫺ secretion mediated by an apical DIDS-sensitive ClHCO3 exchanger would contribute to mucosal protection
(87). However, later work by the same group suggests that
an Slc26a9 product, not an Slc4a9 product, is responsible
for the apical Cl-HCO3 exchange activity in stomach epithelia (1055). Thus, if AE4 is indeed at the apical membrane
of these cells, its role is unclear.
⫺
TRANSPORTERS
Na⫹-COUPLED HCO3
to HCO3⫺ secretion. We note that NCBT activity has not
been reported in ␤-intercalated cells.
8. Causes of AE4 upregulation
A) CIRCULATORY SYSTEM.
I) Increased protein abundance in
ischemia/reperfusion. Haqqani et al. (366) used a proteomic approach (ICAT-nanoLC-MS/MS) to identify proteins, among them AE4, whose expression level was altered
in the microvasculature of rats following global cerebral
ischemia and reperfusion (366). Microvascular AE4 protein
abundance was transiently increased 1 h after ischemia/
reperfusion but returned close to normal levels after 6 h.
The physiological relevance of this phenomenon is unknown.
A) URINARY SYSTEM. I) Downregulation in Foxi1-deficient
mice. Foxi1-deficient mice lack properly differentiated ␣and ␤-intercalated cells, are afflicted with distal renal tubular acidosis, and not surprisingly lack renal AE4 (87). Due
to the morphological abnormalities in the tubules, and because Foxi1-null mice also lack AE1 and the H⫹-pump
subunit ATP6B1, defects in either of which alone is sufficient to cause dRTA (130, 473), the acidosis in these mice
cannot be uniquely linked to the loss of AE4.
II) Decreased mRNA abundance and disturbance of AE4
protein distribution in acidosis. A preliminary study suggests that in collecting duct ␤-intercalated cells acidosis reduces the abundance not only of pendrin at the apical membrane but also the abundance of AE4 mRNA as well as the
basolateral presence of AE4 protein (762).
10. Consequences of AE4 dysfunction
There are no reports of pathologies linked to the SLC4A9
locus in humans, nor are we aware of any reports of phenotypical consequences related to the loss of AE4 in mice.
C. BTR1 (Slc4a11)
2. Nomenclature
BTR1, bicarbonate transporter related protein 1 (720), is
the most divergent member of the vertebrate Slc4 family
and also the last member to be cloned. An alternative name,
NaBC1, Na-coupled borate cotransporter 1 (712), was proposed following a report that BTR1 is a borate transporter.
For the purposes of the present review, we will continue to
refer to Slc4a11 products as BTR1 because NaBC1 is unfortunately similar in name to NABC1, a breast cancerassociated gene (198) that is located on the same human
chromosome as SLC4A11, and the acronym NaBC1 does
not usefully distinguish Na-borate cotransporters from Nabicarbonate cotransporters.
3. Molecular action
The function of mammalian BTR1 as a borate transporter
remains controversial. One group suggests that BTR1 has a
dual action (712). In the absence of borate, BTR1 is proposed to function as an electrogenic Na/OH cotransporter,
or Na-H exchanger, which is thermodynamically equivalent, that carries two or more OH⫺ per Na⫹. However, in
the presence of borate, BTR1 is proposed to function as an
electrogenic Na/B(OH)4 cotransporter that carries two or
more Na⫹ per borate, a Na/anion stoichiometry that is
opposite to that for Na/OH cotransport.
Three main observations support the borate-independent
action of BTR1.
1. Summary
In mammalian genomes, Slc4a11 is the singleton representative of a third subgroup of Slc4 genes. The exon boundaries of Slc4a11 are distinct from those of genes that encode
NCBTs and AEs (FIGURE 7). The demonstration that Slc4like family members from Arabidopsis thaliana (BOR1)
and Saccharomyces cerevisiae (Bor1p) promote boron efflux from cells (943) led to the suggestion that BTR1, too, is
a boron transporter (299). However, the apparent clustering of BOR1 and Bor1p with mammalian BTR1 protein
sequences on a cladogram (299) owes more to their collective lack of identity to mammalian AEs and NBCs than any
1) BTR1-expressing HEK-293 cells acidify to a greater extent than control cells upon removal of extracellular Na⫹.
This NHE-like activity in BTR1-expressing cells is not
blocked by 10 ␮M EIPA nor by 500 ␮M DIDS. These cells
also acidify in response to removal of extracellular K⫹,
which could either be interpreted as K-H exchange or, because that maneuver would tend to hyperpolarize these
cells, outward electrogenic Na/OH cotransport.
2) BTR1 expression reduces the ability of HEK-293 cells to
defend pHi from increases or decreases in pHo, as if the cells
have an increased flux of OH–/H⫹.
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9. Causes of AE4 downregulation
specific identity among BOR1, Bor1p, and BTR1 (TABLE 3).
Nonetheless, a subsequent report provided indirect evidence that BTR1 is an electrogenic Na/borate cotransporter
(712). Although BTR1 exhibits a wide distribution, genetic
defects in BTR1 so far are associated only with a number of
corneal dystrophies. Despite the uniqueness of BTR1, there
is much to be learned from BTR1 about AEs and NCBTs.
For example, pathological mutations occur at many of the
same conserved sequence positions in BTR1, AE1, and
NBCe1. Thus the numerous mutations that have been described in BTR1, but not (yet) in NBCe1, could point at
critical residues for NBCe1 structure-function analysis.
MARK D. PARKER AND WALTER F. BORON
3) Elevating [K⫹]o causes a reduction of [Na⫹]i in BTR1expressing cells. Assuming that the rise in [K⫹]o shifts Vm in
the positive direction, we do not understand how this observation can be interpreted in light of the electrogenic
Na/OH model (point 1), which should, by itself, have
caused [Na⫹]i to rise.
Three main observations support the borate-dependent action of BTR1.
2) The presence of borate stimulates a small inward current
in BTR1-expressing HEK-293 cells and oocytes.
3) In the presence of borate, the removal of extracellular
Na⫹ elicits a larger outward current in BTR1-expressing
HEK-293 cells and oocytes than in control cells. It is this
result that requires a Na⫹:borate stoichiometry greater
than 1:1.
It must be noted that the above study presents no direct
evidence of borate flux. Moreover, the concentrations of
borate (i.e., 5 mM) are two orders of magnitude greater
than physiological concentrations of this trace element
(plasma [B] values are reviewed in Ref. 403). Finally, we are
surprised that that BTR1 would bind 1 Na⫹ plus 2 anions in
the absence of borate but 2 Na⫹ plus 1 anion in the presence
of borate.
Another group (1005), in a paper about BTR1 trafficking,
reports (citing unpublished data) that they are unable to
replicate the data of the previous group.
At present, no data are available concerning the ability of
AEs or NCBTs to transport borate in place of bicarbonate
or carbonate. Likewise, the ability of BTR1 to transport
HCO3⫺ has not been directly measured.
4. Genome
The human SLC4A11 gene, which contains 20 exons that
occupy 12 kb on chromosome 20 (720), is the shortest of
the 10 mammalian Slc4 genes. The gene locus was originally assigned to position 20p12 (720), but subsequent
refinements of the human genome map now place
SLC4A11 at 20p13. The absence of a TATA-box and
presence of a downstream promoter-element–like sequence suggests SLC4A11 gene expression is under the
control of a TATA-less promoter (720).
922
Three full-length transcripts are reported to be transcribed
from the SLC4A11 gene. The three differ in the inclusion of
alternative extreme Nt sequences. The archetypal BTR1
sequence, which we provisionally term BTR1-a, is a 3.1-kb
transcript derived from exons 3–20 of SLC4A11. In
BTR1-b, 30 amino acids encoded by part of exon 3 in
BTR1-a are replaced by 57 amino acids encoded by exon 2.
In BTR1-c, the 30 amino acids of BTR1-a are replaced by
14 amino acids encoded by exon 1.
In theory, any of the aforementioned transcripts could encode two proteins variants of ⬃100 kDa, inasmuch as there
are two initiating methionine codons, corresponding to
Met1 and Met36 of BTR1-a. The second of the two start
codons is preceded by the consensus Kozak sequence
“CCACC.” Interestingly, the shorter variant in each case
would begin Met-Ser-Gln-Xaa-Gly; the same sequence that
initiates from Met1 in BTR1-a. It is unknown what fraction
of protein product expressed from BTR1-a mRNA initiates
with Met1 versus Met36.
Hydropathy analysis predicts that BTR1 has a similar topology to other Slc4 family members (720, 1005). The Nt
domain is shorter than most other Slc4 members and contains a number of consensus PKA and PKC phosphorylation sites, together with an unusually high proportion, for
an Slc4, of cysteine residues.
As noted above, BTR1 does not appear to be blocked by
DIDS (712). On the other hand, the BTR1 protein binds to
H2DIDS and SITS affinity columns (1005), and BTR1 does
include a putative DIDS-interaction motif “KGTVK” at the
extracellular end of TM5.
Cell-free translation in canine pancreatic microsomes (720)
and western blots of BTR1 expressed in HEK-293 cells
(1005, 1010) reveal that BTR1 is N-glycosylated on at least
one of the two consensus glycosylation sites in its third
extracellular loop. This loop lacks the four conserved cysteines characteristic of NCBTs.
6. Distribution
BTR1 expression is widespread and in many cases is expressed in cell types that also express NCBTs. The distribution of BTR1 in specific organ systems is discussed below.
A) CENTRAL NERVOUS SYSTEM. I) Brain. BTR1 transcripts have
been detected in mouse whole brain preparations (378) but
not in rat cerebellum preparations (755). It is possible that
the brain BTR1 transcripts are derived from choroid plexus,
rather than neurons or glia (see next section).
II) Choroid plexus. BTR1 immunoreactivity is evident in
the apical membranes of human choroid plexus epithelial
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1) In BTR1-expressing HEK-293 cells, the presence of “borate, i.e., an equilibrated mixture of H2BO3 and the anion
B(OH)4–, exaggerates the pHi decrease caused by removing
extracellular Na⫹ or by lowering pHo. If we assume that
H3BO3 is freely diffusible, this acidification would be expected if B(OH)4– export caused the intracellular reaction
H3BO3 ⫹ H2O ^ B(OH)4– ⫹ H⫹ (pK ⬵9.2) to shift to the
right.
5. Structural features and variants
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
cells, although BTR1 transcripts are reportedly absent from
mouse choroid plexus (755).
G) LOWER DIGESTIVE SYSTEM.
B) SENSORY ORGANS. I) Eye. BTR1 transcripts (1011) and
protein (214, 608) are detected in corneal endothelium, as is
a BTR1-lacZ fusion protein86 in a transgenic mouse (340).
The polarity of BTR1 expression in these cells has not been
described, and thus BTR1 is not shown in FIGURE 19. The
absence of BTR1 transcripts has been reported in the human retina (see supplemental information in Ref. 1011).
Except for a weak and occasional presence in the anterior
corneal squamous epithelium (214, 608),87 the presence of
BTR1 has not been reported in any other ocular structures.
II) Liver. According to an NCBI-curated EST database, the
mouse liver is a site of BTR1 transcription (Appendix VI).
III) Intestines. BTR1 transcripts have been detected in preparations of ileum and jejunum from pigs (583) and from
colonic extracts from mice (512).
H) ENDOCRINE SYSTEM.
We are aware of no reports of BTR1
expression in the endocrine system.
I) LYMPHATIC AND IMMUNE SYSTEMS. I) Bone marrow. According to an NCBI-curated EST database, the bone marrow of
mice is a site of BTR1 transcription (Appendix VI).
II) Spleen. BTR1 protein has been detected in extracts from
rat spleen (712).
J) URINARY SYSTEM.
BTR1 transcripts (720, 755, 1011) and
protein (712) are abundant in whole kidney extracts and in
the HEK-293 and MDCK renal cell lines (712).
C) RESPIRATORY SYSTEM.
BTR1 transcripts have been detected
in RNA preparations from human trachea (720). According
to an NCBI-curated database of ESTs, the olfactory mucosa
of mice and lung of humans and mice are additional sites of
BTR1 transcription (Appendix VI).
In the renal cortex, BTR1 immunoreactivity is demonstrated in glomerular podocytes and in the basolateral
membranes of proximal tubule epithelia (214). Immunoreactivity also is reported in the apical membranes of cortical
collecting duct epithelia (214).
D) CIRCULATORY SYSTEM.
In the renal medulla, BTR1 transcripts are detected in the
inner medulla, including preparations that are enriched in
IMCDs (986) and in microdissected segments of the thin
descending (754) and thick ascending (694) limbs of the
loop of Henle. In outer medullary collecting ducts, BTR1
immunoreactivity is reported in the apical membranes of
intercalated cells. However, in inner medullarly collecting
ducts, BTR1 is in the basolateral membranes of intercalated
cells (214).88
According to an NCBI-curated EST
database, BTR1 transcripts are present in RNA preparations derived from human blood (Appendix VI). BTR1 immunoreactivity is present in blood vessels in rat submandibular salivary glands (712).
E) MUSCULOSKELETAL SYSTEM. We are unaware of reports of
BTR1 expression in the musculoskeletal system.
F) UPPER DIGESTIVE SYSTEM. BTR1 transcripts have been detected in salivary gland extracts (720). BTR1 protein has
been detected in both parotid and submandibular salivary
gland preparations from rats and mice as well as in a rat
submandibular cell line (712). In rat submandibular glands,
BTR1 immunoreactivity is reported to be in the basolateral
membranes of acinar cells (i.e., in the same membrane as
NBCe1 in FIGURE 21A) but not duct cells (712).
86
A soluble, ⬃320-amino acid fragment of the BTR1 NH2 terminus fused to ␤-galactosidase.
87
Groger and co-workers find no evidence of BTR1 expression in
the anterior corneal epithelium in mice using an anti-BTR1 antibody
(340), nor is BTR1 promoter activity disclosed in these cells by
␤-galactosidase assays of corneal sections from BTR1-lacZ transgenic mice (340).
A different renal distribution of BTR1 gene expression is
suggested by studies of transgenic mice that express a
BTR1-lacZ fusion protein. In these mice, the ␤-galactosidase reporter activity is absent from renal cortex, but present in the renal papilla and also in structures that are reported, by process of elimination, to represent the thin descending limbs of Henle’s loop (340). Thus the antibody
and the lacZ data are consistently positive for BTR1 only in
the case of the thin descending limb.
88
BTR1 transcripts have been detected in inner medullary preparations, apparently decreasing in abundance in segments closest to
the papilla, according to a semiquantitative study (754), but oddly
were undetectable in microdissected inner medullary duct preparations (754).
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II) Ear. BTR1 immunoreactivity is detected throughout the
inner ear, specifically in the fibrocytes of the spiral ligament
that underlie the stria vascularis of the cochlea (340, 608),
together with a lesser presence in the spiral limbus of the
cochlea and in the stroma that underlies the sensory epithelia of the macula of saccule in the vestibular system (608).
According to an NCBI-curated database, more ESTs have
been isolated from mouse inner ear preparations than from
any other organ (Appendix VI).
I) Pancreas. BTR1 protein is detected in extracts from rat pancreas (712).
MARK D. PARKER AND WALTER F. BORON
K) REPRODUCTIVE SYSTEM. BTR1 protein has been detected in a
human cervical cancer cell line (712), and ESTs have been
detected in preparation of human ovary (Appendix VI).
7. Physiological roles
The United States Department of Agriculture does not currently classify boron as an essential nutrient,89 but several
nutritional studies suggest that boron deficiency can have
detrimental consequences for mammalian physiology. The
underlying cause to most of the pathologies associated with
boron deficiency (reviewed in Refs. 402 and 403) is the
increased activity of enzymes that are normally inhibited in
the presence of boric acid. Such enzymes include serine
proteases (e.g., those released by activated leukocytes) and
vitamin D-24-hydroxylase (the enzyme that catalyzes the
first step in the inactivation of vitamin D3). Thus “boron”
has anti-inflammatory action and also potentiates the effects of vitamin D3, promoting Ca2⫹ reabsorption and increasing insulin sensitivity (402, 403). Furthermore, boric
acid is a ligand for molecules such as ribose, S-adenosylmethionine, ATP, ADP, cAMP, NAD⫹, and NADH, although
the consequences of boric acid binding for the bioactivity of
these molecules has not been investigated (402, 403).
Even given the usefulness of boron, it is reasonable to ask if
a borate transporter would be useful in mammals. Dietary
insufficiency of boron is rare. In fact, boron is so pervasive,
and its normal dietary level so low, that it is technically
challenging to reduce the boron content in animal feed to
lower-than-normal levels. Although, boric acid is freely diffusible across artificial lipid bilayers (256), this observation
does not address boric acid permeability of living cell membranes. In plants, aquaporins are responsible for boron uptake, whereas Slc4-like molecules are responsible for boron
extrusion (FIGURE 9). Even if certain mammalian AQPs
could provide a pathway for passive boric acid fluxes across
membranes, BTR1 might still be useful for concentrating,
or, alternatively, preventing toxic buildup of, boron inside
cells.
89
Boron is one of 11 “ultratrace elements” that have a dietary
requirement of ⬍1 ␮g/g body wt, and for which pathological consequences of dietary insufficiency have not been adequately demonstrated (683). Boron is, however, an essential nutrient for plants.
924
8. Causes of BTR1 upregulation
A) LOWER DIGESTIVE SYSTEM. I) Increased transcript abundance
following dietary boron supplementation. In a study of
pigs, a doubling of dietary boron content, maintained over
18 days, resulted in a threefold increase in BTR1 transcript
abundance in jejunal preparations but no increase in ileal
preparations (583). A quadrupling of normal dietary boron
intake had no greater effect on BTR1 transcript abundance
in either the ileum or jejunum (583). The consequence of the
upregulation is not known, but is consistent with a role of
BTR1 in boron homeostasis. If BTR1 was normally involved in boron secretion, upregulation of BTR1 would
enhance boron loss under conditions of excess boron intake.
9. Causes of BTR1 downregulation
A) SENSORY ORGANS. I) Reduced transcript abundance in the
cochlea in response to acoustic trauma. A preliminary
qPCR study reveals that BTR1 transcript abundance is reduced in the cochlear lateral wall of mice in response to
acoustic trauma, an observation that the authors of that
study link to a consequence of hypoxia (1073).
B) LOWER DIGESTIVE SYSTEM. II) Reduced transcript abundance
following probiotic treatment. Probiotic treatment of mice
is a model for investigating the molecular mechanism underlying the health benefits associated with probiotic treatment of inflammatory bowel disorder and ulcerative colitis.
Kotka and co-workers (512) report a fourfold decrease in
BTR1 transcript abundance in mouse colon 24 h after treatment with a probiotic mix (512).
C) URINARY SYSTEM.
I) Decreased transcript abundance following dietary boron supplementation. In the same study of
pigs as was mentioned above, a doubling of dietary boron
content, maintained over 18 days, resulted in a twofold
decrease (rather than the increase observed in the jejunum)
in renal BTR1 transcript abundance (583). A quadrupling
of normal dietary boron intake had no greater effect on
renal BTR1 transcript abundance (583). The consequence
of the downregulation is not known, but is consistent with
a role of renal BTR1 in boron homeostasis. If BTR1 was
normally involved in boron reabsorption, downregulation
of BTR1 would enhance urinary boron loss under conditions of excess boron intake.
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BTR1 has no demonstrated physiological role, although as
discussed in the following sections, defective BTR1 expression is associated with a number of pathologies. Assigning a
hypothetical role for BTR1 is difficult because 1) no consensus has yet been reached concerning the molecular action or polarized distribution of BTR1 and 2) it is unlikely
that the human pathologies associated with BTR1 defects
are solely the result of a functional deficit in BTR1. However, we can at least speculate on the role of borate transport, the suggested role of BTR1.
Studies that address the ability of mammalian organs to
accumulate, or defend themselves from overaccumulation,
of boron are difficult to reconcile among themselves because of differences among species, and individuals of different maturity. However, these studies provide indications
that boron is not passively distributed throughout the body
but instead is selectively accumulated or eliminated by certain cell types (781). There is insufficient data to determine
whether BTR1 is involved in these processes.
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
10. Consequences of BTR1 dysfunction
A) GENERAL. I) Cell proliferation. One study found that
siRNA knockdown of BTR1 in HeLa cells resulted in a
reduction in cell proliferation that could be rescued by increasing the concentration of extracellular boron (712). In
individuals with SLC4A11 defects, a decreased proliferative ability may be expected to contribute towards the severity of dystrophies involving cell types in which BTR1 is
normally expressed.
B) SENSORY ORGANS.
BTR1 is abundantly expressed in the corneal endothelium,
a monolayer of squamous/low-cuboidal epithelial cells that
forms the inner surface of the cornea. The endothelium
plays a role in maintaining stromal deturgescence (i.e., corneal transparency) by reabsorbing fluid that moves by osmosis from the aqueous humor into the stroma. This reabsorption prevents disruption of the transparent crystalline
array of collagen fibers and proteoglycans that constitute
the stromal matrix (267). The manifestations of the dystrophy are a loss of endothelial cell density, stromal thickening,
corneal clouding, and visual impairment (reviewed in Ref.
496).
Interestingly, gene-trap disruption of Slc4a11 in mice
(caused by the random insertion of a neomycin-resistance
cDNA) causes no major corneal phenotype, other than a
slight thickening of the basal cell layer of the corneal anterior epithelium (608). A separate study of transgenic mice in
which Slc4a11 was disrupted with ␤-galactosidase also revealed a slight thickening of the epithelium as well as a
doubled thickness of the corneal endothelium, Descemet’s
membrane, and stroma (340). Furthermore, the endothelial
layer was vacuolized and the stroma included Na- and Clenriched crystalline deposits (340). The clarity (or lack
90
A database of SLC4A11 mutations is curated at the Leiden Open
Variation Database (https://grenada.lumc.nl/LOVD2/mendelian_
genes/home.php?select_db⫽SLC4A11).
An alternate possibility is that the absence of transport
function (whatever the nature of that function) is not the
main cause of the corneal endothelial dystrophy in humans
with mutations in SLC4A11. With a few possible exceptions identified in individuals with late-onset dystrophy
(794), all of the human mutant BTR1 proteins tested to date
accumulate in the ER when overexpressed in mammalian
cells (1005, 1011, 1012). It is possible, as has been demonstrated for other mutant proteins that misfold in the corneal
endothelium (274), that the expression of large amounts of
misfolded mutant protein causes ER stress (the “misfolded
protein response”) and ultimately death of corneal endothelial cells. However, this hypothesis remains to be tested.
II) Hearing: loss. Slc4a11-null mice exhibit a reduced response to auditory stimuli, an observation that is consistent
with the genetic link between mutations in SLC4A11 and
the perceptive deafness associated with Harboyan syndrome (340, 608).
C) URINARY SYSTEM. I) Polyuria. A study of mice in which
Slc4a11 was disrupted with a lacZ gene revealed that these
mutant mice excrete more urine per day than wild-type mice
and that the urine of mutant mice has a lower osmolarity
and [Ca2⫹] compared with that excreted by wild-type mice
(340). Gröger and co-workers explain the polyuria by suggesting that BTR1, in the thin descending limb of the loop of
Henle, normally mediates Na⫹ influx and thereby contributes towards the efficacy of the countercurrent multiplier.
However, no direct evidence for such a role is provided in
that study, and the role of BTR1 elsewhere in the kidney is
not considered. Furthermore, the authors report a significant decrease in NKCC2 mRNA in one of two data sets
from these mice (see Supplementary Table 2 that accompanies Ref. 340), an alteration that could potentially contribute to a polyuric phenotype (939).
VII. CONCLUDING REMARKS
A. Summary
In this section we consider the common themes that emerge
as we revisit the structure, actions, and roles of the five
NCBTs. Our consideration reveals a number of unresolved
issues as well as several emerging topics that are understudied. In this section we summarize these points using the
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I) Vision: corneal dystrophy. To date,
nearly 60 mutations90 have been identified, scattered across
the length of the BTR1 molecule, among individuals with
corneal dystrophies (19, 20, 161, 244, 375, 450, 522, 640,
782, 794, 867, 919, 1011, 1012). Pathological SLC4A11
mutations are most frequently inherited in a homozygous
recessive manner, although numerous cases of compound
heterozygous inheritance of SLC4A11 mutations have been
described (20, 244, 375, 782, 919). There are three
SLC4A11-associated corneal endothelial dystrophies:
1) congenital hereditary endothelial dystrophy (CHED2),
first associated with SLC4A11 in Reference 1011; 2) Harboyan syndrome, also known as corneal dystrophy and perceptive deafness (CDPD), first associated with SLC4A11 in
Reference 244; and 3) late-onset Fuchs’ endothelial corneal
dystrophy (FECD4), first associated with SLC4A11 in References 330 and 1012.
thereof) of the mouse cornea was not reported in this study.
Thus neither of these mice are demonstrated to adequately
model all of the features of human corneal dystrophy. Others have suggested that these gene-disrupted mice do not
have a clear CHED phenotype due to factors such as the
proliferative ability of mouse endothelial cells, functional
redundancy with other transporters, incomplete inactivation of the gene, or other undefined mouse/human differences (340, 608, 1012).
MARK D. PARKER AND WALTER F. BORON
same subject areas that we used in organizing sections V
and VI.
B. Nomenclature
1. Nonmammalian
2. Mammalian
In the mammalian realm, the initially confusing nomenclature is now generally settled both for the transporters themselves (NBCe1, NBCe2, NBCn1, and NDCBE) as well as for
the variants of each (e.g., NBCe1-A, NBCe1-B). However,
the nomenclature for Slc4a9, Slc4a10, and Slc4a11 products is controversial because the molecular actions of these
transporters, which determines their acronyms, is not universally agreed upon. For example, AE4 does not mediate
Cl-HCO3 exchange in the hands of all investigators, nor
does BTR1/NaBC1 mediate boron transport in the hands of
all investigators. Furthermore, the action of the Slc4a10
product is reportedly different for the human (“NBCn2”)
versus the mouse and rat (“NCBE”) protein. Until these
matters are resolved, caution must be exercised in the use of
these acronyms. We recommend that papers on mammalian
clones always refer at least once, preferably in a prominent
way near the beginning of the manuscript, to the Slc4 designation. Investigators in doubt about the phenotype might
use the Slc4 designation exclusively.
C. Molecular Action
The first two topics in this section deal with the diversity of
NCBT gene products, either multiple NCBTs performing
different actions, or multiple NCBTs performing the same
action.
926
Among the five mammalian NCBTs, are engendered at least
four distinct molecular actions: 1) electrogenic Na/HCO3
cotransport, 2) electroneutral Na/HCO3 cotransport, 3)
electroneutral Na/HCO3 cotransport with a HCO3⫺-independent conductance, and 4) Na⫹-driven Cl-HCO3 exchange. Each action has its own physiological niche.
In the kidney, the basolateral step of HCO3⫺ reabsorption
could not be effected by an electroneutral NCBT, which,
driven by prevailing ion gradients, would contribute to
HCO3⫺ secretion. Only a Cl-HCO3 exchanger (e.g., AE2 in
the TAL, and AE1 in the ␣-intercalated cells) driven by an
inwardly directed Cl⫺ gradient, or an electrogenic NBC
(i.e., proximal tubule) driven by Vm in addition to prevailing ion gradients, could contribute to HCO3⫺ reabsorption.
Another example of the advantage of being electrogenic
versus electroneutral is illustrated in the case of NBCe1 in
neurons. Neuronal acidification, which could dampen neuronal excitability during repetitive firing, is avoided due to
an NBCe1-mediated DIA because depolarization promotes
electrogenic HCO3⫺ import.
On the other hand, being electroneutral is sometimes advantageous. For example, electroneutral NCBTs can counter the effects of a whole body acidosis on neuronal pHi,
and thereby maintain neuronal excitability, without influencing or being influenced by Vm.
Being coupled to Cl⫺ transport also has important consequences. A study of NCBT action in nematode neurons
indicates that Cl⫺ efflux mediated by the action of an
NDCBE plays an important role in the maturation of the
CNS: by lowering [Cl⫺]i, it make ECl more negative than
Vm, rendering GABAergic and glycinergic signaling inhibitory. On the other hand, NBCn1, which is not coupled to
Cl⫺, regulates neuronal pHi without influencing [Cl⫺]i.
The only molecular action of NCBTs that has no currently
demonstrated role is the HCO3⫺-independent conductance
mediated by NBCn1. In theory, this conductance could
make Vm more positive in neurons, influencing the action of
ion channels and electrogenic transporters. For example, a
more positive Vm in neurons would tend to inactivate voltage-gated Na⫹ channels, rendering these cells less excitable.
Benefits of having NCBTs with functional redundancy.
There appears to be no discernible difference in the molecular actions of NBCe1 and NBCe2 or between the net transport activities of human NBCn1 and NBCn2. It is unclear,
for example, why NBCe1 could not take the place of
NBCe2 in hepatocytes, or why NBCe2 could not take the
place of NBCe1 in proximal tubules. However, genetic redundancy has obvious potential benefits in providing
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Even within the vertebrate realm, where the homology
among Slc4-like genes is sufficiently high to permit adherence to a consistent system of trivial nomenclature, some
investigators have created their own nomenclature. An example is the frog ortholog of BTR1, which has been dubbed
XNBC2 (1102). Outside the vertebrate realm, where direct
orthologs of the 10 vertebrate Slc4 genes simply do not
exist, no consistent system of trivial nomenclature is possible, a situation exacerbated by the lack of functional data
that would normally inform the nomenclature. These nomenclature issues will likely cause confusion as the body of
literature expands. We recommend that each study of an
Slc4-like gene includes reference to GenBank or Ensembl
DNA and protein accession numbers, as well as a list of any
previous terminologies applied to the same gene/gene products in related organisms by others. Of course, wherever
possible, the guidelines laid down by the nomenclature
committees that oversee the genomes of those organisms
should be followed.
1. Benefits of having multiple NCBTs with distinct
molecular actions
⫺
TRANSPORTERS
Na⫹-COUPLED HCO3
backup in the case of haploinsufficiency. An example may
be CNS neurons that contain both NBCn1 and NBCn2
(195). Moreover, the presence of both NBCe1 and NBCe2
(or NBCn1 and NBCn2) provides the opportunity for differential regulation during development or in response to
stresses, or differential expression in different parts of the
cell.
2. Transporters with controversial action
Another aspect to bear in mind is that some transporters
exhibit different molecular actions in difference species. For
example, some Slc4 proteins from fishes exhibit conductive
features that are not shared with their mammalian counterparts (e.g., trout versus human AE1, Ref. 285).
How does the molecular action of an electrogenic NCBT
compare with that of an electroneutral NCBT? So far, all
that is known of the determinants of electrogenicity versus
electroneutrality is that critical amino acid residues reside in
EL4.
4. Unusual features of NBCn1
Three questions arise when we consider the action of
NBCn1.
What is the relationship between the Na/HCO3 cotransport
activity and the HCO3⫺-independent conductance of
NBCn1?
Why is NBCn1 relatively insensitive to blockade by DIDS,
despite retaining a seemingly intact DIDS-binding motif on
TM5?
Furthermore, why does DIDS block the NCBT activity attributed to NBCn1 in mesenteric arteries and trigeminal
ganglion neurons–is it possible that differences in posttranslational processing or local environment can impact DIDS
sensitivity?
3. Novel substrates
5. Undetermined inhibitor binding sites
Slc4-like proteins from invertebrate species have been suggested to transport non-HCO3⫺ species such as silicate and
valproic acid, although direct evidence is lacking. Cl-HCO3⫺
versus CO32⫺ versus NaCO3–: even for extensively characterized NCBTs, there remain many key questions concerning
molecular action.
Do NCBTs carry HCO3⫺ or CO32⫺? Preliminary studies indicate that, at least in the case of NBCe1 and NDCBE as
expressed in oocytes, CO32⫺ is the dominant, if not the only,
carbon-containing substrate.
With the assumption that a transporter carries CO32⫺, does
the CO32⫺ move in the form of the NaCO3⫺ ion pair, as may
be the case for the Na⫹-driven Cl-HCO3 exchanger in the
intact squid giant axon?
What is the mechanism by which electrogenic NCBTs appear to be able to switch between a 1:2 and a 1:3 stoichiometry?
What is the relationship between the molecular mechanisms of
the exchangers/antiporters (e.g., AEs, NDCBE) and the presumed cotransporters/symporters (e.g., NBCe1, NBCn1)?
Could all Slc4s be antiporters? For example, the electroneutral
Although the binding site for reversible DIDS inhibition is
well described for NBCe1, the irreversible binding determinants are unknown as are the binding determinants for
other drugs such as tenidap.
6. The K/HCO3 cotransporter
The protein(s) responsible for this activity are undescribed.
Does an NCBT working in an unusual mode contribute?
Intriguing in this regard is the observation that the Na⫹
versus Li⫹ specificity of certain NCBTs appears to be celltype specific. An alternative explanation is that K/HCO3
cotransport could be mediated by another transporter family (e.g., SLC12, which includes the KCC K/Cl cotransporters).
7. Three-dimensional structure
Critical to understanding the molecular action of any
NCBT will be a high-resolution three-dimensional structure
together with molecular dynamic simulations. However, no
high-resolution structure is available for any Slc4 or Slc4like protein.
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The molecular actions of Slc4a9 (AE4) and Slc4a11 (BTR1/
NaBC1) remain controversial, as does the action of
SLC4A10/Slc4a10 (NBCn2/NCBE). NCBTs may behave
differently in diverse heterologous expression systems and
when heterologously versus natively expressed. All systems
include endogenous factors (e.g., ion channels) that can
interfere with electrophysiological measurements. The benefits of performing transporter characterization in a heterologous system are many, but it is important, where possible, to reconcile the transport properties and inhibitor profile of the heterologously expressed transporter with the
properties of the native protein.
cotransport of one Na⫹ and one HCO3⫺ would be thermodynamically equivalent to, and difficult to distinguish from, the
cotransport of two Na⫹ and one CO32⫺, or the exchange of
one Na⫹ and one CO32⫺ (or 1 NaCO3– ion pair) for one HCO3⫺.
MARK D. PARKER AND WALTER F. BORON
NBCe1, NBCn1, and NBCn2 all have the capacity to encode a Ct that terminates with a PDZ domain. Note that the
Ct of the three AEs is not known to be variable.
D. Genome
1. Diversity
Humans, mice, and rats have 10 Slc4 genes. Other mammals likely have the same number, but we have yet to truly
appreciate the diversity of Slc4-like genes in nonmammalian species.
2. Gene clusters
3. Promoter characterization
An emerging and underexplored area in the study of mammalian Slc4 genes is the mapping and characterization of
Slc4 promoter regions, the understanding of which will impact our knowledge of the consequence of the use of alternative promoters, NCBT dysregulation in disease, and the
factors that are responsible for altering NCBT abundance in
response to diverse physiological and non-physiological
stimuli.
E. Structural Features and Variants
3. Influence of the Nt upon the TMD
An emerging theme is that many NCBT splice cassettes
include docking sites for protein partners (e.g., IRBIT) that
can influence NCBT activity. Furthermore, the Nt of
NBCe1 appears to be a binding partner for the TMD of
NBCe1, and this interaction is necessary for Na/HCO3
cotransport activity. There are a number of important
mechanisms that have yet to be elucidated in this regard.
For example, how do the Nt, the ASD, the AID, and IRBIT
exert their effects on Na/HCO3 cotransport by the TMD?
One possibility is that the cytosolic domain acts as a scaffold for the TMD and that structural rearrangements in the
Nt influence the ability of the Nt to scaffold the TMD in a
transport-competent conformation, as has been proposed
for the cytosolic domain of a ClC from red alga (283).
4. Isolated Nt variants
1. Role of UTRs
At the level of Slc4 transcripts, we do not yet understand the
consequences of alternative 5=- and 3=-UTR inclusion.
These sequences likely include many determinants that impact the stability and efficiency of translation of the transcript, such as miRNA target sites. Because NCBT overabundance is linked to the poor outcomes in cancer, heart
disease, and stroke, understanding how to manipulate
NCBT transcript abundance would be valuable.
2. The diversity of NCBT protein variants
At the level of protein sequence, most NCBTs exhibit similar patterns of variation. For example, all NCBTs, with the
current exception of NBCe2, have variants that include an
autoinhibitory Nt appendage.
Working with multiple cDNA libraries, investigators have
amplified transcripts predicted to encode an isolated NCBT
Nt domain (i.e., without a TMD). Such transcripts are produced by the Slc4a7, Slc4a8, and Slc4a10 genes. The abundance, stability, and relevance of protein expressed from
these transcripts has yet to be described. Because the Nt
does not appear in Slc4 evolution until the emergence of
animals, the isolated Nt transcripts may represent the vestigial expression of the original “isolated Nt” open-reading
frame that was appended to the “isolated TMD” transporter gene. The origin and original function of the ancestral isolated Nt is unknown. Note that the archetypal-Nt
gene product need not have had the same open reading
frame as the modern Nt.
F. Distribution
All NCBTs, with the possible exception of NBCe2, have
variants that are stimulated by interaction with the soluble
protein IRBIT.
NBCe1, NBCn1, and NBCn2 all exhibit variation in their
Nt loop region. Note that this region is not known to be
variable in the three AEs.
928
1. Overview
The distribution of NCBTs in the mammalian body is likely
broader than is presently appreciated. In some instances,
the location, but not the identity, of an NCBT is known as
is the case with the DIDS-sensitive electroneutral NCBT
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SLC4A7 and SLC4A10 are both neighbored by T-box transcription factor genes (FIGURES 31 AND 39), indicating a
long-standing relationship between the two gene families.
Furthermore, growth factor genes are often located at similar chromosomal loci to Slc4 genes (e.g., TGFA and
SLC4A5, see Ref. 591).
The commonality between these gene variations is evident
when we compare the primary structure of the variants side
by side as illustrated in Appendix V. Despite the wealth of
variants encoded by the NCBTs genes, we can be certain,
based on variation between EST sequences, that more are
yet to be described. In most cases, the physiological consequence of such variance is unknown, as are the mechanisms
that dictate the presence or absence of the splice cassettes in
specific tissues.
⫺
TRANSPORTERS
Na⫹-COUPLED HCO3
activity in platelets (315). A thorough analysis of NCBT
distribution would require the use of variant-specific and
variant-independent primers and antibodies that are still in
development, together with their application in normal and
stressed tissues that have yet to be probed. In this respect,
the study of transgenic animals that express reporter genes
under the control of NCBT promoters could be useful.
2. Apparently overlapping distribution
3. The polarity of NCBT expression
With few exceptions, the epithelial polarity of NCBTs, and
indeed all Slc4s, is basolateral, complementing the usually
apical distribution of Slc26 proteins (FIGURE 1). Some unusual epithelia express basolateral markers in their apical
membranes, two examples that pertain to NCBT expression being the choroid plexus (with apical NBCe2) and the
retinal pigment epithelium (with apical NBCe1). However,
other reports of apical NCBT expression ought to be regarded with caution, pending independent confirmation.
One example is the unusual apical NBCn1 immunoreactivity in renal tubules disclosed by the “anti-NBC3” antibody,
the use of which is documented in Appendix VII. This distribution has not been confirmed by the use of any other
anti-NBCn1 antibodies, which react with basolateral targets. The reason for this disparity remains unclear.
G. Physiological Roles
1. Overview
NCBTs fulfill three main roles: pHi regulation, HCO3⫺ secretion, and Na/HCO3 reabsorption. In most cell types
NCBTs mediate Na⫹ and HCO3⫺ influx. Indeed, when expressed in the basolateral membranes of polarized cells
(e.g., NBCn1 in salivary acinar cells in FIGURE 21), NCBTs
support ion and fluid secretion. In several studies, this contribution becomes apparent only following inhibition of
intracellular CAs, consistent with the idea that it is the CAs
(e.g., in conjunction with Na-H exchangers) that are dominant in generating HCO3⫺ for secretion under unstimulated
conditions. In the CPE, apical NBCe2 could support HCO3⫺
secretion by operating with an apparent 1:3 stoichiometry
to support HCO3⫺ secretion across the apical membrane
into the CSF (FIGURE 28).
NBCe1, by operating with an apparent 1:3 stoichiometry in
the basolateral membranes of PT epithelia(FIGURE 23), is the
only NCBT demonstrated to support HCO3⫺ reabsorption.
2. Inferences from phenotypes of transgenic mice
Many of the physiological roles ascribed to NCBTs are
inferred from the signs of mice and humans with disrupted
NCBT genes. A potential complication is that, in some
cases, the pathological signs may have more to do with the
expression of misfolded protein (e.g., a partial transmembrane domain) rather than the absence of the physiological
NCBT function per se. The observed signs may also depend
on the nature of the NCBT disruption and the genetic background of the disrupted gene. In this regard, it will be informative to observe the phenotypes of multiple strains of
mice in which the Slc4 has been disrupted in diverse ways
(e.g., knockout versus gene-trap versus knocked-in mutation). The unintended consequences of dysregulation of
other genes could also contribute to the observed phenotype, in some cases requiring that the investigators systematically examine the expression and activity of other transporters expected to contribute to the phenotype.
H. Causes of Upregulation
As depicted in Appendix V, NBCe1-B/C, NBCn1, NDCBEA/B, and NBCn2 all include both autoinhibitory domains
and modules in their Nt appendage that are predicted to
render them sensitive to stimulation by IRBIT. However,
although the physiological cues that activate IRBIT with
respect to NCBTs are unknown, the abundance of NBCe1,
NBCn1, and NDCBE transcripts are all increased during
acidosis. In addition, NBCe1 and NBCn1 protein abundance is increased during hypercapnia. These observations
are consistent with the role of these NCBTs in maintaining
pH within a narrow physiological range.
Lacking from our current knowledge are the full details of
the molecular mechanism(s) by which the NCBTs are upregulated by acidosis/hypercapnia. In principle, upregulation could occur at any or all of four levels: transcript abundance, total protein abundance, plasma-membrane protein
abundance, and per-molecule protein activity. At least in
the case of NBCe1-B, a pH-responsive element has been
identified in the promoter region, and in the case of
NBCe1-A, some of the signaling components that enhance
HCO3⫺ reabsorption in response to respiratory acidosis
have been elucidated (e.g., Refs. 824, 890, 1104, and 1107).
Characterization of promoter regions and the signaling cascades that enhance functional expression of NCBTs would
be helpful towards the goal of understanding how the activity of multiple transporters are coordinated in a cell type.
For example, an emerging theme is that IRBIT stimulates
ion and fluid secretion by coordinated upregulation of multiple transporters.
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Existing studies suggest that all five NCBTs are present in
the central nervous system, the choroid plexus, and the
kidney. The reason for such apparent redundancy is unknown. It is also unknown if two NCBTs coexpressed in the
same cell type, such as NBCe1 and NBCn1 in duodenal
villar cells (FIGURE 22), are capable of heterodimerizing to
create a transporter with novel properties.
In neurons and glia, HCO3⫺ transport mediated by electrogenic NCBTs (the direction of which appears to depend
upon prevailing Vm and ion gradients) tends to maintain
neuronal excitability.
MARK D. PARKER AND WALTER F. BORON
Rather than stimulating transport per se, the action of CAs
in the vicinity of NCBTs could minimize pH changes close
to the plasma membrane, thereby minimizing adverse effects on the activities of other membrane proteins. The
metabolon controversy is reviewed in Reference 102.
I. Causes of Downregulation
The abundance of NBCe1 and NBCn1 protein falls under
alkalotic conditions, consistent with a reduced requirement
for reabsorption and cellular influx of HCO3⫺. The abundance of NBCe1, NBCn1, NDCBE, and NBCn2 protein
falls under hypoxic conditions, consistent with energy conservation and possibly also reflecting a response to respiratory alkalosis in these animals. The perturbation of NBCe2
abundance in response to hypoxia/alkalosis has not been
reported. With the exception of the pH-responsive element
located within the NBCe1-B/C promoter, the mechanisms
that result in reduced NCBT abundance remain to be elucidated. An emerging and underexplored theme is that NCBT
abundance can be reduced by miRNAs.
J. Consequences of Dysfunction
1. Transgenic mice
Recurring phenotypes observed in NCBT-null mice are acidosis (NBCe1 and NBCe2), hypertension (NBCn1 and
NBCe2), reduced neuronal excitability (observed for
NBCe2, NDCBE, NBCn2, and inferred for NBCe1), impaired CSF secretion (NBCe2 and NBCn2), and ocular defects (NBCe1, NBCe2, and NBCn1). In the majority of
cases, these phenotypes accord well with the known distribution and inferred physiological roles of each NCBT.
Somewhat surprising, given the abundance of NBCe1 in the
pancreas, NBCe2 in the liver, and NBCn1 in the mTAL,
NBCe1-null mice lack an obvious pancreatic phenotype,
930
NBCn1-null mice lack an obvious renal phenotype, and
NBCe2-null mice lack an obvious hepatic phenotype. Presumably these mice have upregulated unknown compensatory pathways or have not been subjected to the appropriate
challenges. In the case of NBCe1-null mice, another possibility is that the animals, which die shortly after weaning,
have not lived long enough to exhibit a phenotype. In this
respect, studies of conditional and inducible knockouts,
which have not been reported for any NCBT, will be illuminating, as should be the application of antisense RNA
technology in animals.
One potentially confounding aspect of existing NCBT
knockout mice is the interpretation of their phenotypes. For
example, as mentioned above, especially in the case of genetrapped mice, some phenotypes may be consequences of the
expression of partial, misfolded NCBT protein rather than
consequences of the absence of the NCBT activity per se.
Such mice may be better models for the pathological consequences of NCBT mutation in humans because some
signs in affected individuals may be specifically due to misfolded protein response. Studies of targeted transgenic mice
that carry orthologs of human mutations, the NBCe1-mutant W516X mouse that mimics a human pRTA is the only
example to date, are the most appropriate in regard to modeling pathologies associated with human disease. In terms
of investigating the results of the loss of NCBT transport
activity, a transgenic mouse that expresses a nonfunctional,
yet full-length NCBT would perhaps provide the clearest
picture. Attempts to disrupt an NCBT gene close to its
initiator Met include the potential hazard of permitting normal transcription of alternative gene products.
2. Linkage studies
Numerous GWAS studies implicate variation at NCBT gene
loci with susceptibility to various traits and disorders, such as
autism, substance abuse, hypertension, and cancer. These associations are consistent with known roles for NCBTs in control of neuronal excitability, Na⫹ reabsorption, and in countering the apoptotic effects of acidosis. These studies will need
to be followed up with deep sequencing, the results of which
would provide a statistically significant link (or lack thereof) of
the trait to a specific SNP within an NCBT gene. The next step
would be to study, in a heterologous system or transgenic
animal, the effect of the SNP on the functional expression or
regulation of that NCBT.
In conclusion, it seems likely, given the widespread abundance of NCBTs, and the effects of pH on almost all physiological processes, that NCBT action modifies a wide array
of complex genetic traits. Our current knowledge concerning the diverse molecular actions, distribution, regulation,
physiological roles, and pathophysiological roles of NCBTs
provides only a glimpse of their potential importance.
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On the topic of activating binding partners, CAs are proposed to bind to the Ct and EL4 of NBCe1 and to the Ct of
NBCn1, forming a “metabolon.” The concept is that CAs
supply HCO3⫺ to the outer face of the transporter and remove HCO3⫺ from the inner face of the transporter, or vice
versa, depending on the direction of movement, thereby
speeding transport. This theory remains controversial for
four major reasons: 1) NBCe1 stimulation by CAs is observed only in some studies; 2) the binding of CAs to the Ct
of NCBTs is observed only in some studies; 3) if, as preliminary data suggest, NCBTs transport CO32⫺, rather than
HCO3⫺, it is not clear that CAs would enhance NCBT action
substantially; and 4) modeling data suggest that CAs would
have only a minor effect on HCO3⫺ or CO32⫺ transport rates
(Ref. 342 and Rossana Occhipinti, personal communications).
⫺
Na⫹-COUPLED HCO3
TRANSPORTERS
For Appendices I–VIII, the online version of this article
contains supplemental material.
Appendix I: Annotated Protein Sequence
Alignments of Human SLC4s (See Figures 3
and 15)
Appendix II: GenBank or Ensembl Protein
Accession Numbers for Nonmammalian
Slc4-like Transporters (See Figures 4 and 8)
Parker), DK30344 (to W. F. Boron), DK81567 (to W. F.
Boron), NS18400 (to W. F. Boron), HD032573 (to Gabriel
G. Haddad/project 2 and W. F. Boron), and HL090969 (to
Alanna C. Morrison).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared
by the authors.
REFERENCES
Appendix IV: GenBank Protein Accession
Numbers for Mammalian Slc4 Variants (See
Sects, V and VI)
Appendix V: Annotated Protein Sequence
Alignments of Human NCBT Variants (See
Sect. V)
Appendix VI: The Distribution of Expressed
Sequence Tags for Humans and Mouse
NCBTs, AE4, and BTR1 (See Sects. V
and VI)
Appendix VII: Locations of “Anti-NBC3”
Immunoreactivity (See Sect. V)
Appendix VIII: Locations of Renal Anti-AE4
Immunoreactivity (See Sect. VI)
ACKNOWLEDGMENTS
We thank Dennis Brown for his patient editorial oversight.
We thank the two reviewers for their thorough reading of
the manuscript and their helpful comments. We also thank
members of the Boron lab past and present as well as all
those investigators whose studies and personal communications have contributed to fabric of this review.
Address for reprint requests and other correspondence:
M. D. Parker, Dept. of Physiology and Biophysics, Case
Western Reserve University School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106-4970 (e-mail: mark.d.
[email protected]).
GRANTS
This work was supported by National Institutes of Health
Grants EY021646 (to Michael L. Jennings and M. D.
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