PHYSIOLOGICAL REVIEWS
Vol. 80, No. 1, January 2000
Printed in U.S.A.
Sodium-Potassium-Chloride Cotransport
JOHN M. RUSSELL
Department of Biology, Biological Research Laboratories, Syracuse, New York
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I. Introduction
II. Overview of the Cation-Coupled Chloride Cotransport Family
A. Na1-Cl2 cotransport
B. K1-Cl2 cotransport
III. Molecular Characterization of the Sodium-Potassium-Chloride Cotransporter
A. Isoform NKCC1
B. Isoform NKCC2
IV. History of the Discovery of the Sodium-Potassium-Chloride Cotransporter
A. Role of Na1 pump studies in the development of NKCC hypothesis: Na1 pump II
B. First steps: demonstration of Na1 and K1 coupling
C. Role of cell volume regulation studies
D. Linking Cl2 to the coupled Na1 and K1 movements
V. Fundamental Characteristics
A. Absolute cis-side requirement for all three co-ions
B. Bumetanide inhibition/binding
C. NKCC is electrically silent
VI. Stoichiometry/Thermodynamics
A. Stoichiometry of the transport process
B. Thermodynamics of the cotransport process
VII. Transport Model of the Sodium-Potassium-Chloride Cotransporter
A. Evidence for cooperative and ordered ion binding to the NKCC
B. Evidence that Cl2 binding sites are nonequivalent
C. Cation specificity
VIII. Bumetanide Binding Studies
A. Functional evidence for effects of external ions on bumetanide binding
B. [3H]bumetanide binding studies
IX. Regulation/Modulation of Cotransporter Activity
A. Role of ATP
B. Role of intracellular ions
C. Role of the cytoskeleton
X. Functions of the Sodium-Potassium-Chloride Cotransporter
A. Role in net Cl2 transport by epithelial tissues
B. To maintain [Cl2]i at higher than equilibrium values
C. Cell volume regulation and the NKCC
D. A role for the NKCC in the cell cycle?
XI. Questions Remaining to be Answered
Russell, John M. Sodium-Potassium-Chloride Cotransport. Physiol. Rev. 80: 211–276, 2000.—Obligatory, coupled
cotransport of Na1, K1, and Cl2 by cell membranes has been reported in nearly every animal cell type. This review
examines the current status of our knowledge about this ion transport mechanism. Two isoforms of the Na1-K1-Cl2
cotransporter (NKCC) protein (;120 –130 kDa, unglycosylated) are currently known. One isoform (NKCC2) has at
least three alternatively spliced variants and is found exclusively in the kidney. The other (NKCC1) is found in nearly
all cell types. The NKCC maintains intracellular Cl2 concentration ([Cl2]i) at levels above the predicted electrochemical equilibrium. The high [Cl2]i is used by epithelial tissues to promote net salt transport and by neural cells
to set synaptic potentials; its function in other cells is unknown. There is substantial evidence in some cells that the
NKCC functions to offset osmotically induced cell shrinkage by mediating the net influx of osmotically active ions.
Whether it serves to maintain cell volume under euvolemic conditons is less clear. The NKCC may play an important
role in the cell cycle. Evidence that each cotransport cycle of the NKCC is electrically silent is discussed along with
0031-9333/00 $15.00 Copyright © 2000 the American Physiological Society
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JOHN M. RUSSELL
Volume 80
evidence for the electrically neutral stoichiometries of 1 Na1:1 K1:2 Cl2 (for most cells) and 2 Na1:1 K1:3 Cl2 (in
squid axon). Evidence that the absolute dependence on ATP of the NKCC is the result of regulatory phosphorylation/
dephosphorylation mechanisms is decribed. Interestingly, the presumed protein kinase(s) responsible has not been
identified. An unusual form of NKCC regulation is by [Cl2]i. [Cl2]i in the physiological range and above strongly
inhibits the NKCC. This effect may be mediated by a decrease of protein phosphorylation. Although the NKCC has
been studied for ;20 years, we are only beginning to frame the broad outlines of the structure, function, and
regulation of this ubiquitous ion transport mechanism.
I. INTRODUCTION
1
The nomenclature used in this review for the various members of
the cation-coupled chloride cotransport family follows the lead of Forbush’s group. It has few, if any, conflicts with the nomenclature in use by
the Online Mendelian Inheritance in Man database (http://www3.
ncbc.nlm.nih.gov/Omim/).
II. OVERVIEW OF THE CATION-COUPLED
CHLORIDE COTRANSPORT FAMILY
The cotransport of Cl2 along with Na1 and/or K1 has
been reported for a variety of cells since the 1970s (see
sect. III) and was surmised even earlier than that (78). Not
long ago, it was difficult for even those working in the
field to know with any certainty whether a given K1dependent Cl2 transport process was a KCC or NKCC or
whether a given Na1-dependent Cl2 transport process
was NCC or NKCC. In fact, there were reports in the
literature which suggested that, in some situations, a single transport moiety could switch “modes” from one to
the other given certain stimuli, such as increased osmolarity of bathing solutions (77) or application of vasopressin (334). In addition to an uncertainty about the absolute
ion requirements for each of the putative ion cotransporters, there was confusion regarding their pharmacology.
Much of this uncertainty resulted from the simultaneous
presence of more than one coupled cotransporter in the
tissues/cells being studied. We can now be certain that all
three of these cotransporters are separate proteins encoded by the same gene family. The gene products of the
NCC (80) and the KCC (92, 139, 282) share ;45–50% and
25% identity, respectively, with the gene products of the
NKCC (277, 281).
Ion transport studies show all three of these cotransporters share an absolute requirement for Cl2 as well as
at least one cation (either Na1 and/or K1) and that all
three cotransport processes are electrically silent. Haas
(110) has proposed that this gene family be termed the
cation chloride cotransporter, or CCC, gene family. Phar-
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Probably the earliest suggestion of a coupled Na1Cl cotransport process was made by Shanes (318). Evidence for a tightly coupled Na1-K1-Cl2 cotransport
(NKCC) mechanism, blocked by so-called loop diuretics
and unaffected by ouabain, was first presented by Geck et
al. (90). At the time Geck et al. (90) presented their
findings, there had already been numerous reports of
cotransport of all the possible combinations of Na1, K1,
and Cl2 cotransport processes. For a while it was hoped
that all of the reported cation-coupled Cl2 cotransport
(CCC) processes merely represented different modes of a
single transport entity. However, as a result of tremendous progress in this field, in the last 5 years we now
know that K1-Cl2 (KCC) and Na1-Cl2 (NCC) cotransporters are separate gene products from one another as well
as from the NKCC (see sects. II and III). This review
focuses almost exclusively on the NKCC.
A by-product of the widespread interest in the NKCC
has been the appearance of a substantial number of review articles (e.g., Refs. 43, 50, 86, 109, 110, 114, 163, 171,
172, 239, 255, 266, 277, 281, 297, 311, 320). So why one
more? Previous reviews approach the subject from the
standpoint of a somewhat special interest within the field,
e.g., the properties of the transporter in some particular
cell type (red blood cells, Ehrlich ascites tumor cells, and
epithelial tissue), some functional aspect (cell volume
regulation), and, most recently, the molecular biology and
molecular structure of the NKCC.1 This review draws on
data from all these sources in an attempt to distill a
focused picture of our current understanding (and ignorance) of this important ion transport mechanism. Perhaps the best reason for a comprehensive review at this
time is that the field is poised on the brink of an exciting
new era that will undoubtedly yield interesting and unexpected insights into the workings of these CCC processes.
The past 5 years have seen the major energy in this
field being directed into molecular characterization of the
cotransporter. These efforts have been rewarded in that
two isoforms of the NKCC have been cloned as have two
2
isoforms of the KCC and one isoform of the NCC. We now
enter a period in which we can extend our understanding
of how these cotransporters work using the newly available molecular biological tools. This review will be a
success if it serves to help the reader distinguish between
what is well understood from what is poorly understood
about the NKCC, its transport mechanism, its regulation,
and its function. There are several excellent recent reviews that focus on the molecular biological information
that has just become available about this cotransporter
(110, 114, 163, 277, 281). Therefore, this review does not
attempt an exhaustive coverage of that information;
rather, a molecular biological framework is presented.
Na1-K1-Cl2 COTRANSPORT
January 2000
A. Na1-Cl2 Cotransport
Of the three members of the CCC family, this is the one
for which general recognition and acceptance was perhaps
TABLE
the slowest. This probably relates to the fact that in mammals its most definitive location is the early distal convoluted tubule in the kidney, a region where the NKCC is also
prominent. In addition, it was often mistaken for parallel
Na1/H1 and Cl2/HCO2
3 exchangers. The functional properties of the NCC were first definitively characterized by
Stokes et al. (332) using the urinary bladder of the winter
flounder, a preparation that apparently lacks the NKCC.
They demonstrated 1) a clear interdependency between
Na1 and Cl2 effects on net absorption of Cl2 and Na1,
respectively; 2) that the transport process did not require
K1; and 3) that the NCC was inhibited by thiazide diuretics
but not by the so-called loop diuretics (which, as we shall
see, are the most specific inhibitors of the NKCC) such as
furosemide, nor by stilbenedisulfonic acid derivatives
(which inhibit the KCC and Cl2/HCO2
3 exchange, among
other anion transport processes). However, there is functional evidence for bumetanide-sensitive K1-independent
NaCl cotransport in trachea (207, 241) and Necturus gallbladder (196). The later confusion about whether the NCC
could be converted into a NKCC probably arose because in
some epithelial tissues, as mentioned above, the two transporters spatially coexist (e.g., Ref. 143).
There is some functional evidence for this cotransporter in nonepithelial tissue. In the rabbit heart, treatment with chlorothiazide caused a reduction of cell volume (57). This cell shrinkage effect of thiazide treatment
was tentatively attributed to the inhibition of a NCCmediated uptake process. However, in vascular smooth
muscle, thiazides activate a Ca21-activated K1 channel
1. Comparison of some known general properties of NKCC, KCC, and NCC
Property
NKCC
KCC
Cis-side ion requirement
Direction of net cotransport
Effect of hypertonic media
Effect of hypotonic media
Na1, K1, Cl2 (sect. IVA)
Influx
Stimulates (sect. IXC)
No effect or inhibits (sect.
Loop diuretic sensitive
Bumetanide . furosemide (sect. VIIA)
Bumetanide K0.5 > 1 3 1027 M (see
Table 2)
No effect (77, 264)
No effect (125, 260, 319)
Thiazide sensitive
Disulfonic acid stilbene
sensitive
ATP effect
Cloned
Isoforms: tissue distribution
(Northern blots)
Tissue distribution:
functionally identified
Amino acid identity, %
(relative to NKCC1)
Effects of intracellular Cl2
Electrically silent
IXC)
Stimulates (sect. VIIIA)
Yes (sect. X)
NKCC2(BSC1): kidney (TAL)
NKCC1(BSC2): kidney, stomach, heart,
lung, brain, skeletal muscle
RBC; smooth, skeletal, and cardiac
muscle; epithelia; fibroblasts; Ehrlich
ascites tumor cells; neurons
NKCC1: 100% (sect. X)
NKCC2: ;60% (280)
Inhibits (sect. VIIB1)
Yes (sect. IVC)
NCC
K1, Cl2 (34)
Efflux
Inhibits
Stimulates KCC1, but not KCC2
(185, 279)
Furosemide . bumetanide
Bumetanide K0.5 > 1 3 1024 M
(199)
?
Inhibits (53)
Na1, Cl2 (332)
Influx
?
?
Inhibits
Yes (92, 283)
KCC1: brain, colon, heart, kidney,
liver, lung, spleen, and stomach
KCC2: brain
RBC; nerve; epithelia; Ehrlich
ascites tumor cells; neurons
?
Yes (79, 80)
NCC1: urinary bladder; distal
convoluted tubule (80,
359)
mammalian kidney; flounder
urinary bladder
25% (sect. X)
45–50% (sect. X)
Stimulates (51)
Yes (161)
?
Yes (332)
No effect (332)
Inhibits? (196, 241, 334)
Inhibits (332)
No effect (332), inhibits (6)
NKCC, Na1-K1-Cl2 cotransporter; KCC, K1-Cl2 cotransporter; NCC, Na1-Cl2 cotransporter; K0.5, mean affinity constant; RBC, red blood cell;
TAL, thick ascending limb.
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macologically, they are somewhat distinguishable. Only
the NCC is blocked by thiazide diuretics, whereas only the
KCC is blocked by disulfonic acid stilbenes, such as DIDS.
The loop diuretics (bumetanide, furosemide) inhibit both
the NKCC and the KCC but are much more potent in their
action against the NKCC. In addition, there may be an
NCC that is inhibited by loop diuretics (e.g., Refs. 196,
241, 334), although there is some disagreement about
whether the NCC blocked by bumetanide is the same as
that blocked by thiazides (134).
On the basis of a combination of functional and genetic
results, both the NKCC and the KCC are found in a wide
variety of tissue types. In general, the NCC seems to be
largely confined to epithelial tissue such as kidney
(especially in the distal nephron), where it participates in the
net movement of Na1 and Cl2 across an epithelial barrier.
Functionally, the KCC has been best characterized in red
blood cells but has also been described in other tissues. The
KCC has been associated with regulatory volume decrease
in response to cell swelling. In nervous tissue, where it has
been recently identified (282), it may participate in maintaining an intracellular Cl2 concentration ([Cl2]i) at lower than
electrochemical equilibrium levels. What follows is a brief
description of the properties of the KCC and the NCC. Table
1 summarizes the key similarities and differences among
these three CCC.
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B. K1-Cl2 Cotransport
During the same series of studies that began the
linkage between cell volume regulation and NKCC (see
sect. IVC), Kregenow (185), also made several critical
observations for our understanding of the KCC. The KCC
mediates the coupled cotransport of K1 and Cl2 across
plasma membranes. Although reversible, it is thermodynamically poised to effect net efflux. Because of this net
loss of K1 and Cl2, it can promote regulatory volume
decrease. Thus it is activated by a cell volume increase
(135). It may also be involved in “pumping” [Cl2]i to lower
than equilibrium levels in neurons (279, 282). At present,
there are two known isoforms of the KCC, the “housekeeping” form (KCC1) that is found in a variety of tissues
(e.g., Ref. 92) and a neuron-specific isoform (KCC2, Ref.
282). Structurally, both of these isoforms differ more from
the NKCC than does the NCC (92, 282). They have an
estimated molecular mass of 120 –125 kDa (92, 282).
Functional studies have confirmed that the KCC is
found in several cell/tissue types, including ascites tumor
cells (182) and mammalian kidney epithelial cells (65,
294). However, by far the most detailed functional studies
have been performed in the red blood cell (RBC). These
properties have been well covered in recent reviews
(RBC, Ref. 199; all cells, Ref. 134). I highlight only the key
properties that differentiate the KCC from the NKCC. As
already mentioned, the KCC is activated by cell swelling,
and as we shall see (sects. IIIC and IXC), the NKCC is
activated by cell shrinkage. Activation of the KCC by
swelling leads to the net loss of K1 and Cl2 along with an
osmotic equivalent of water. This results in a reduction of
cell volume (“regulatory volume decrease”). Another intriguing functional difference between the KCC and the
NKCC is that dephosphorylation activates KCC (e.g., Ref.
154), whereas it inactivates the NKCC (see sect. IXA). The
KCC differs from the NKCC in its response to an increase
of [Cl2]i as well. Whereas the NKCC is inhibited by such
an increase (e.g., Refs. 28, 93; see sect. IXB1), the KCC is
stimulated (51, 197, 198). Unlike either the NKCC or the
NCC, the KCC can be inhibited by the disulfonic acid
stilbenes such as DIDS (52). Because it is unlikely that
DIDS crosses the plasmalemma, this property suggests
that the DIDS-sensitive site of the KCC is externally accessible.
III. MOLECULAR CHARACTERIZATION OF THE
SODIUM-POTASSIUM-CHLORIDE
COTRANSPORTER
Since 1994, all three members of the CCC family have
been cloned from a variety of species (see Refs. 50, 163,
277, 281). A quantitative comparison of the derived amino
acid sequences of members of this family with other
known proteins suggests that it is a unique family, perhaps distantly related to the amino acid-polyamine-choline family of transporters (277). Full-length clones of two
isoforms (see below) of the putative NKCC have been
reported from rat kidney (79), shark rectal gland (358),
inner medullary collecting duct of the mouse kidney (53),
the loop of Henle from rabbit kidney (280), human colon
(283), mouse kidney (143), human kidney (325), and bovine aortic endothelium (360). The deduced amino acid
sequences of these clones have revealed a protein whose
molecular mass varies from ;120 to ;130 kDa. A very
similar protein has been reported as encoded by the gene
YBR235w of chromosome II of the yeast Saccharomyces
cerevisiae (18). Hydropathy profiles of the deduced proteins (using the Kyte-Doolittle algorithm) have shown
there are three general regions. A central hydrophobic
region of ;50 kDa flanked by an amino- (;20 –30 kDa)
and a carboxy-terminal (;50 kDa) region. These latter
two regions are more hydrophilic than the central region
of the molecule. Based on such analyses, Xu et al. (358)
suggested that there are 12 a-helical membrane-spanning
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(37). It is quite possible the cell shrinkage observed in the
heart muscle after thiazide treatment was due to K1 (Cl2)
loss and not to inhibition of an NCC. Also, high-stringency
Northern blots failed to reveal any NCC mRNA in rat heart
(80). There were early reports of apparent NCC activity in
Ehrlich ascites tumor cells (136), but subsequent functional evidence showed that this cell has only the NKCC
(89, 155, 202) and the KCC (182). Finally, Northern blot
analysis of mammalian tissues (including human tissues)
localize it mainly to kidney, but also possibly to small
intestine, placenta, prostate, colon, and spleen (in humans, Ref. 45). In rat tissues, high-stringency assays revealed the NCC to be only in the kidney (79). In fact, in
situ hybridization probes (79) and mRNA localization
(359) reveal that the NCC is exclusively localized to the
distal convoluted tubules of the kidney. This result fits
very well with what is known about thiazide-sensitive
NaCl absorption by the kidney.
The winter flounder urinary bladder behaves functionally much like the mammalian kidney distal tubule
and, as we have seen, was the first site of functional
characterization of the NCC. The flounder urinary bladder
NCC was the first of the CCC to be cloned (80). The
human NCC has also been cloned (40) and expressed
(225).
Gitelman’s syndrome is a human genetic disease associated with a mutation of this thiazide-sensitive cotransporter in the renal distal convoluted tubule (326). The
syndrome is characterized by hypokalemic metabolic alkalosis, hypocalcuria, hypomagnesemia, and natriuresis.
The mutation, located on chromosome 16, is believed to
lead to a loss of function of the NCC.
Volume 80
January 2000
Na1-K1-Cl2 COTRANSPORT
whereas the NKCC2 is localized to chromosme 2 (289).
The human NKCC1 is localized to chromosome 5 (283).
A. Isoform NKCC1
The NKCC1 isoform is by far the most widely distributed of the two currently identified isoforms. In addition
to being found on the basolateral membrane of secretory
epithelia, Northern probe studies have indicated this is
the isoform found in the plasmalemma of a wide variety of
cell types, including most nonepithelial cells (283, 358).
There is some evidence for tissue-specific variants of the
NKCC1. For example, skeletal muscle presents a somewhat smaller mRNA transcript than is found in other
tissues (6.7 vs. 7–7.5 kb, Ref. 283). Because it is found on
the basolateral membrane of epithelial cells, this isoform
is often referred to as the “secretory” isoform. However,
when it is found in the membrane of nonpolar cells, it may
be referred to as the housekeeping isoform.
Figure 1 is a general model based on the hydropathy
profile for human colonic NKCC1. As already mentioned,
the molecule has three regions. The amino-terminal region has the lowest amino acid identity of the three
regions among NKCC1 from different species. There is at
least one phosphorylation site in this region. This site has
the greatest degree of amino acid identity found in the
amino terminus across species (213, 358). On the other
hand, the carboxy terminal region is relatively highly
conserved among species extending from Cyanobacterium to human (281). The carboxy-terminal end has several consensus phosphorylation sites that have a relatively high degree of identity across species (see sect.
IXB2). In addition, there are several hydrophobic regions
in the carboxy terminus that might also be embedded in
1
1
2
FIG. 1. Model of Na -K -Cl cotransport (NKCC) isoform 1 protein based on its hydropathy profile. NKCC1
protein is ;1,200 amino acids with 12 transmembrane
(TM)-spanning domains, numbered TM1-TM12. Transmembrane domains 1, 3, 6, 8, and 10 are rather highly
conserved between NKCC1 and NKCC2. In addition, intracellular loop linking transmembrane domains 2 and 3 is
highly conserved. Virtually entire difference in molecular
mass between NKCC1 (;195 kDa) and NKCC2 (;121
kDa) can be accounted for by an additional 80 amino acids
on NH2 terminus of NKCC1. Site-directed mutagenesis
studies have shown that changes in cation affinity result
from changes in TM2, whereas Cl2 affinity was affected by
changes in TM4 to TM7. Bumetanide binding appears to be
associated with transmembrane TM2 to TM7 and TM11 to
TM12. [Adapted from Payne and Forbush (281).]
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regions. These putative transmembrane regions are highly
conserved between isoforms, with 75–90% identity between the NKCC1 and NKCC2 isoforms. It has been suggested that alternative topological models (e.g., 14 membrane-spanning regions) might better conform to other
predictors of membrane protein topology (277). Both isoforms have consensus N-linked glycosylation sites on a
putative extracellular loop that fits with biochemical evidence that the cotransport protein is significantly glycosylated (293).
As mentioned above, there are two isoforms of the
NKCC. The isoform initially found on the basolateral
membrane of the shark rectal gland is known as NKCC1
(Ref. 358; also known as SLC12A2). The same isoform
was also identified in mouse kidney by Delpire et al. (53)
but named BSC2 (for bumetanide-sensitive cotransporter
2). The NKCC nomenclature is used in this review. The
other isoform is referred to as NKCC2 (or BSC1; also
known as SLC12A1). The NKCC1 isoform is the larger of
the two with ;1,200 amino acid residues and a transcript
size of ;7.4 kb. It has an overall 58% amino acid identity
with NKCC2. The NKCC2 isoform is somewhat smaller
than NKCC1, containing ;1,100 amino acid residues with
a transcript size of ;5 kb. The difference in molecular
size is almost entirely accounted for by an additional 80
amino acids at the amino terminus of the NKCC1. In
contrast, the carboxy end of the molecule is relatively
well conserved, exhibiting .65% identity among the two
isoforms and between the same isoforms from different
sources (277). Given the difference in transcript size and
the relatively low overall amino acid identity between the
two isoforms, it is not surprising that they are products of
two different genes. Delpire et al. (53) showed in the
mouse that the NKCC1 is localized to chromosome 18,
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JOHN M. RUSSELL
B. Isoform NKCC2
The NKCC2 (BSC1) isoform has thus far been identified only in the medullary regions of the kidney (rat, Ref.
79; rabbit, Ref. 281). This anatomic location of the NKCC2
corresponds with the location of the loop of Henle and the
juxtaglomerular apparatus. Thus NKCC2 is believed to
play a critical role in two of the crucial homeostatic
functions of the kidneys, namely, regulation of extracellular fluid volume and osmolarity.
In studies on the rabbit kidney NKCC2, Payne and
Forbush (280) identified three distinct variants that differed only by an alternately spliced 96-bp exon. With the
use of such a hydropathy plot model very similar to that
for NKCC1 (refer to Fig. 1), it was shown that these exons
coded for the amino acids in TM2 as well as 13 amino
acids that are contiguous with TM2, but which extend into
the intracellular compartment (280). A particularly interesting addendum to this observation was that high-stringency Northern probes indicated these three variants had
very distinct regional localization within the rabbit
nephron; one was found only in the renal cortex, one only
in the renal medulla, and the third in both the cortex and
medulla. Igarashi et al. (143) have made a similar observation for the mouse kidney. This finding is consistent
with a proposal made by Knepper and Burg (177) that
there are some differences between NKCC function in
different regions of the thick ascending limb of the loop of
Henle. It is possible that the proposed regional differences in Na1 reabsorption along the thick ascending limb
might be mediated by the different isoforms of the
NKCC2. This interpretation fits well with the finding that
TM2 is a critical component of Na1 and K1 affinities for
the NKCC (148, 149).
The NKCC2 has also been identified in the renal
juxtaglomerular apparatus (143, 163). Igasrashi et al.
(143) showed that one of the alternately spliced isoforms
(NKCC2B) is found in macula densa cells. Macula densa
cells are involved with two important renal homeostatic
processes: tubuloglomerular feedback and secretion of
renin. LaPointe’s group (194, 195) has presented evidence
that strongly suggests a key role for the NKCC in the
tubuloglomerular feedback process. LaPointe et al. (195)
suggest that the NKCC (NKCC2B?) in the macula densa
cells is very near its thermodynamic equilibrium point
such that small changes in the NaCl concentration in the
nephron tubule lumen could affect the net direction of
NKCC transport and thereby change the equilibrium potential of Cl2 (ECl). This in turn is hypothesized to affect
the resting membrane potential (Vm) of the basolateral
membrane of the macula densa cells which somehow
transmit this membrane voltage information to the granule cells of the afferent artery. Evidence for this function
of the NKCC2 comes from reports that luminal furosemide inhibits tubuloglomerular feedback (152) and renin secretion (209).
Mutations of NKCC2 have been linked to some forms
of Bartter’s syndrome, a severe human genetic disease
involving hypokalemic alkalosis with hypercalcuria
largely attributable to dysfunction of the NKCC2 in the
thick ascending limb of the loop of Henle (325). The
authors reported a variety of mutations, all in the putative
transmembrane region of the molecule. The interested
reader is directed to a thoughtful discussion of the roles
of the NKCC and NCC in Bartter’s and Gitelman’s syndromes by Hebert and Gullans (129).
IV. HISTORY OF THE DISCOVERY OF THE
SODIUM-POTASSIUM-CHLORIDE
COTRANSPORTER
The NKCC is distinguished by the fact that it transports Na1, K1, and Cl2 stoichiometrically by means of a
tightly coupled mechanism that can be blocked by loop
diuretics. Although this distinguishing “fingerprint” can be
stated briefly, it took nearly 15 years for workers to put
the pieces together (90). Three key elements were required: 1) a conceptual breakthrough; 2) a laborious,
empirical characterization of Na1 and K1 fluxes; and 3) a
reason to consider that Cl2 transport might be directly
linked to cation transport. The conceptual breakthrough
occurred in the early 1960s with Crane’s seminal formu-
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the membrane. It is hypothesized that the central ;500amino acid residue region is arranged into 12 transmembrane domains with connecting loops. The hydrophobic
membrane-spanning region is not only highly conserved
between isoforms, but also between the same isoform
from different species (e.g., Ref. 281). There are two
consensus N-linked glycosylation sites on the extracellular loop between transmembrane (TM) segments 7 and 8.
It is currently unknown whether a functional NKCC is a
monomer or an oligomer.
Structure-function studies have just begun. In a recent series of studies, Forbush’s group made chimeras
from shark and human NKCC1. They also made some
point mutations. These studies show that ion binding and
transport as well as bumetanide binding depend on the
;500 amino acids believed to comprise the conserved
hydrophobic transmembrane regions (147–150). These
studies have provided strong evidence that the second
transmembrane region (TM2; a region highly conserved
across the entire CCC family) is an important site in
determining cation and bumetanide binding, but not Cl2
binding. Chloride binding determinants appear to reside
in TM4 and TM7. Bumetanide binding determinants appear to be somewhat more diffuse than those for the ions,
being in TM2, -7, -11, and -12.
Volume 80
January 2000
Na1-K1-Cl2 COTRANSPORT
A. Role of Na1 Pump Studies in the Development
of NKCC Hypothesis: Na1 Pump II
Hoffman and Kregenow (137) demonstrated in
ouabain-treated human RBC that there was a fraction of
Na1 efflux that was stimulated by intracellular Na1,
blocked by ethacrynic acid, and which might not use ATP
as an energy source. To explain this finding, they hypothesized that in addition to the ouabain-sensitive sodium
pump, there was a second, energy-dependent mechanism
capable of moving Na1 against its electrochemical gradient. They called this mechanism pump II to distinguish it
from the ouabain-sensitive Na1/K1 exchange pump
(pump I). Over the next 8 years, the pump II hypothesis
was ultimately disproven. However, it served well to stimulate much-needed research into the properties of coupled Na1-K1 cotransport processes. In the interim, some
workers concluded that the ouabain-insensitive Na1 efflux was mainly a Na1/Na1 exchange process (60, 210),
whereas others concluded that these fluxes were simply
manifestations of different properties of pump I, depending on the particular experimental conditions (309).
critical series of studies that linked K1 uptake with Na1
uptake via a ouabain-insensitive mechanism. He showed
that (in the presence of ouabain) there was a Na1 uptake
dependent on external K1 that could be blocked by 1 mM
furosemide. Under the appropriate set of conditions, he
could demonstrate a small net efflux of Na1 (in the presence of ouabain) that was also blocked by furosemide.
This is the first published report of linking K1-dependent
Na1 fluxes to inhibition by a loop diuretic. However,
because he and others had demonstrated that furosemide
could inhibit pump I (Na1-K1-ATPase) fluxes, Sachs (309)
concluded that the effects seen in the presence of ouabain
were probably mediated by pump I, which had different
properties under the experimental conditions being used.
Wiley and Cooper (355) were the first to demonstrate
the obligatory coupled cotransport of Na1 and K1. They
extended Sach’s finding by demonstrating furosemideinhibitable, mutually dependent Na1 and K1 fluxes occurring in the presence of ouabain. Figure 2 illustrates their
finding that the furosemide-inhibited Na1 influx in human
RBC required the presence of extracellular K1. Figure 3
shows that a portion of the ouabain-insensitive K1 uptake
required the presence of extracellular Na1 and could be
inhibited by furosemide. This was the first demonstration
that the furosemide-sensitive components of the unidirectional influxes of both Na1 and K1 required the presence
of the other cation. They also noted, as has had Glynn et
al. (95), that there was a saturable component of the
ouabain-insensitive Na1 uptake. This saturable component of Na1 uptake was inhibited by furosemide and had
a Michaelis constant (Km) for external Na1 of 24 mM.
Furthermore, and this was very important at the time,
they demonstrated net, furosemide-sensitive fluxes. This
observation, coupled with the observation that furosemide inhibited ouabain-insensitive effluxes of K1 and
Na1 in the absence of extracellular Na1 and K1, showed
B. First Steps: Demonstration of Na1
and K1 Coupling
By the late 1960s, Na1 pump workers began to notice
that K1 uptake, in the presence of ouabain, was saturable
(e.g., Refs. 84, 310). This was a perplexing finding since it
was generally believed at that time that K1 movements
consisted of only two pathways: 1) via the Na1 pump and
2) via electrodiffusive leaks (the pump-leak hypothesis).
Thus, in the presence of ouabain, K1 influx was not
expected to be saturable as a function of extracellular K1
concentration ([K1]o), unless there was a hitherto unknown mechanism for K1 uptake.
Evidence for such an unknown mechanism was provided by Sachs (309). Using human RBC, he performed a
1
FIG. 2. Inhibition by 1 mM furosemide of Na influx into human red
blood cells bathed either in K1-containing or K1-free media. Each
isotonic medium contained 10 mM NaCl plus requisite concentration of
indicated cation as chloride salt. Ouabain (0.1 mM) was present in all
media. Values are means 6 SD. [From Wiley and Cooper (355).]
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lation of the sodium gradient hypothesis and the concept
of secondary active transport (see Refs. 46, 47) whereby
the transmembrane electrochemical gradient (maintained
by a primary active transport process) of one solute (usually Na1) provides the energy for the thermodynamically
uphill movement of a second solute. It is difficult to
overemphasize the importance of this concept to the
study of cell solute homeostasis. The labor-intensive characterization of Na1 and K1 fluxes was performed by a
variety of workers whose original intent was to characterize further the operation of the Na1 pump. The reasons
to consider Cl2 linkage to Na1 and K1 movements were
almost serendipitous. As we shall see, the Cl2 linkage
idea grew out of studies designed to characterize amino
acid transport mechanisms.
217
218
JOHN M. RUSSELL
Volume 80
3. Inhibition by 1 mM furosemide of ouabain-insensitive K1
influx into human red blood cells bathed in either Na1-rich or Na1-free
media. Each isotonic medium contained 6 mM KCl plus requisite concentration of indicated cation as chloride salt. Ouabain (0.1 mM) was
present in all media. Values are means 6 SD. [From Wiley and Cooper
(355).]
FIG.
that the transport process they were studying was not
simply isotopic exchange. The conclusion of these workers was that the RBC possessed a cotransport mechanism
that moves both Na1 and K1 and that there was a codependency in their transport. This represented a very critical stage in the development of the idea of the NKCC. It
is important to remember that at that time the idea of
secondary active transport in general, and cotransporters
in particular, was relatively new and by no means universally accepted.
D. Linking Cl2 to the Coupled Na1
and K1 Movements
1. Evidence in RBC
Since the early 1970s, red cell workers were aware
that the coupled Na1-K1 movements they observed were
accompanied by Cl2 movements in the same direction
(see above). However, it was not until the late 1970s that
investigators directly addressed a critical question: Are
the Cl2 movements obligatorily linked to the cation movements or are they functionally linked via changes in the
electrochemical driving forces? Although the question
seems straightforward now, at the time there were con-
C. Role of Cell Volume Regulation Studies
The long and continuing association between what
has come to be recognized as the NKCC and cell volume
regulation that occurs in response to cell shrinkage began
with several critical observations by Kregenow (185, 186).
He showed that duck RBC, shrunken by exposure to a
hypertonic medium, could (in the continued presence of
the hypertonic medium) recover toward their original
volume. This recovery depended on [K1]o being elevated
somewhat above normal levels and the presence of extracellular Na1. In the absence of ouabain, cell volume recovery was accompanied by a net uptake mainly of K1
and Cl2 plus the osmotically obligated water. However,
Kregenow (186) showed that the volume regulation did
not require a functioning Na1 pump (Fig. 4). With the Na1
pump inhibited, the main increase was seen in the Na1
and Cl2 contents, with a somewhat smaller increase of
K1. This important observation implied that the primary
solute transport mechanism responsible for the volume
FIG. 4. Lack of effect of ouabain on response of duck red blood cells
to exposure to a hypertonic medium. Duck erythrocytes were incubated
in isotonic or moderately hypertonic fluids containing either 2.5 or 19
mM K1. Ouabain (1024 M) was added just before beginning experiment.
[K1]o, extracellular K1 concentration. [From Kregenow (186).]
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increase moved Na1 (1Cl2) in addition to K1 (-Cl2).
Kregenow (186) supported this interpretation by showing
that cell shrinkage was accompanied by an increase in the
ouabain-insensitive unidirectional isotopic influx and efflux of both Na1 and K1. However, in keeping with the
cationocentric point of view of those times, the increase
of Cl2 content was believed to be “passive,” in response
to changes in membrane potential caused by the cation
movements. Thus Kregenow’s findings showed that cell
shrinkage activated a ouabain-insensitive Na1 and K1
transport pathway or pathways that moved these two
cations into the cell (K1 against its electrochemical gradient) to effect a net uptake of osmotically active particles. For the first time, Cl2 was also implicated in the
process (albeit in an apparently passive role).
January 2000
Na1-K1-Cl2 COTRANSPORT
could support furosemide-sensitive net Na1 transport. In
their earlier report (316), they had reported that reducing
both [Cl2]i and [Cl2]o to 20 mM (using acetate as the
replacement) still permitted a norepinephrine-stimulated
uptake of Na1 and K1 without any change of [Cl2]i. In the
earlier work, this observation had been interpreted to
mean there was no absolute Cl2 requirement by the cotransporter. When this experiment was repeated in the
presence of DIDS, it was found that only Cl2 or Br2
would support the net uptake of the cations (121). Furthermore, in DIDS-treated cells, they were able to show a
furosemide-sensitive net uptake of Cl2 that equaled the
furosemide-sensitive net uptake of Na1 plus K1. In a
particularly convincing series of studies summarized in
Figure 5, they also showed that the direction of the transmembrane Cl2 chemical gradient dictated the direction of
the furosemide-sensitive net flux of Na1 when the membrane potential was “clamped” with valinomycin (142).
Thus this study not only demonstrated that Cl2 was transported by the cotransporter, but also that the chemical
gradient for Cl2 could “energize” net cotransport-mediated fluxes of the cations.
The original misinterpretation of their results was
reinforced by an analysis of the thermodynamic equilibrium of the putative Na1-K1 cotransporter that agreed
with their actual results. That one could arrive at the
correct final thermodynamic expression starting with incorrect initial assumptions was the result of two pieces of
bad luck: 1) Cl2 is distributed at thermodynamic equilibrium and 2) Cl2 is a transported species. In the first report
(316), they interpreted their results as being due to an
electrogenic Na1-K1 cotransport with Cl2 following via
an electrodiffusive pathway. Starting with this assumption, they derived an equilibrium equation for such a
model as follows.
At thermodynamic equilibrium, the net electrochemical potential (Dm̃net) is
D m̃ net 5 D m̃ Na 1 D m̃ K
(1)
where Dm̃Na and Dm̃K are the electrochemical potentials
for Na1 and K1, respectively. Knowing that Cl2 is distributed at electrochemical equilibrium (i.e., Vm 5 ECl) allows
one to write the following expression for the net electrochemical potential
D m̃ net 5 RT ln
@Na 1 # o @K 1 # o @Cl 2 # 2o
@Na 1 # i @K 1 # i @Cl 2 # 2i
(2)
Unfortunately, the exact same equation can be derived
starting with the assumption that the cotransporter is
electroneutral, that is, there are one Na1, one K1, and two
Cl2 transported per cycle, i.e.
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ceptual and technical roadblocks both to formulating as
well as to addressing this question. The conceptual roadblock was the then-current view (based on results from
RBC and frog skeletal muscle) that Cl2 crossed membranes only by electrodiffusive pathways and was distributed at electrochemical equilibrium across all cell membranes. According to this view, Cl2 simply traversed
membranes through very high permeability pathways in
response to primary changes in the electrochemical driving forces as set by the movements of cations. Moving
beyond this concept required overcoming two technical
roadblocks. One was the difficulty of measuring Cl2
movements. The principal radioisotope of chlorine (36Cl)
has a half-life of over 300,000 years, meaning that it can
only be produced with a relatively low specific activity,
and it is expensive. The measurement of net Cl2 movements, while possible, was laborious. The second roadblock had to do with a property of the membrane of RBC:
it has both a high Cl2 conductance and a high Cl2 exchange flux via the band 3 anion exchanger (AE1).
Both the problems outlined above came together to
influence the interpretation of the results of an elegant
study by Schmidt and McManus (316). Using duck RBC,
they examined the role of Cl2 in the shrinkage-induced
volume increase that had first been demonstrated by Kregenow (185, 186). The duck RBC is an excellent subject to
study cell-volume regulation and the associated Na1 and
K1 fluxes. However, like other RBC, it is ill-suited for the
study of Cl2 fluxes associated with volume regulation,
because the Cl2 flux via the Cl2/HCO2
3 exchanger (band
3 or AE1) is orders of magnitude greater than the Cl2 flux
via the NKCC. As a result of this problem, Schmidt and
McManus (316) were unable to measure any Cl2 fluxes
directly. Instead, they looked at effects of varying Cl2
concentrations and concluded that the Cl2 movements
during cell reswelling were in response to the change in
the electrochemical driving force on this anion, caused by
the net cotransport of Na1 and K1. There is an additional
circumstance that contributed importantly to the conclusion that Cl2 movements were passive and not obligatorily linked to those of Na1 and K1. That is the fact that
Cl2 is distributed at thermodynamic equilibrium across
the RBC membrane. This means the ECl is equal to the Vm.
Thus, as long as the process(es) responsible for this passive distribution were operating, measurements of net Cl2
fluxes could never reveal Cl2 flux via the NKCC.
In a later publication from the same laboratory (121),
the question of whether Cl2 was directly linked to cation
transport was readdressed using duck RBC whose membrane potential was “voltage-clamped” (using valinomycin) and whose band 3 fluxes were blocked by DIDS.
Under these conditions, they completely replaced Cl2
with permeant anions across the duck red cell membrane.
Now, with Vm unable to change and band 3 unable to
exchange anions, they showed that only Cl2 and Br2
219
220
JOHN M. RUSSELL
Volume 80
D m̃ net 5 D m̃ Na 1 D m̃ K 1 2D m̃ Cl
(3)
Thus we see that although the initial assumptions were
quite different (Eqs. 1 and 3), the final equation (Eq. 2) is
the same. This identity contributed to the initial misinterpretation by Schmidt and McManus (316). It took the
development of the voltage-clamped RBC treated with
DIDS (to block the band 3 anion exchanger) to distinguish
between the two very different models.
Three other groups studying Na1 and K1 fluxes in
human RBC reported a close relationship between
ouabain-insensitive cation influxes and the presence of
extracellular Cl2. In a preliminary note, Kregenow and
Caryk (189) reported that Cl2 was cotransported with
Na1 and K1 during volume regulation in duck RBC. Dunham et al. (59) showed that ;75% of the ouabain-insensitive influx of K1 was dependent on [Cl2]o in a concentration-dependent manner. They further showed that 1 mM
furosemide abolished this [Cl2]o-dependent K1 influx.
Similarly, they showed that a portion of the ouabaininsensitive Na1 influx required the presence of external
Cl2 and that this fraction of the Na1 influx was eliminated
by treatment with furosemide. The fact that the magnitude of the [Cl2]o-dependent Na1 influx was significantly
smaller than that of the same K1 influx as well as the fact
that Wiley and Cooper (355) had shown that furosemide
blocked a substantial portion of K1 influx into cells
bathed in Na1-free media caused them to conclude that
they were studying a KCC. At about the same time, Chipperfield (43) also reported a [Cl2]o-dependent K1 influx,
blocked by furosemide, but noted that “direct evidence
for Cl2 transport by the same system is lacking.” Clearly,
the inability to measure non-band 3-mediated Cl2 fluxes
in the RBC was a critical impediment to correctly assessing the role of Cl2 in the RBC cotransporter.
2. Evidence in other cells
Thus the first definitive experiments showing the
direct linkage between the furosemide-sensitive Na1 and
K1 fluxes with Cl2 fluxes were performed in a different
preparation (Ehrlich ascites tumor cells) and for a completely different reason than the series of studies we have
just outlined. In the mid 1970s, Heinz et al. (131) were
attempting to determine the energy sources for amino
acid uptake. In the course of those studies, his group
noticed an apparently paradoxical movement of Cl2
(131). They had demonstrated a strong electrogenic Na1
pump component to the Vm of the Ehrlich cells. Thus, as
[K1]o was increased from 0 to 15 mM, the Vm became
increasingly negative, an effect largely blocked by
ouabain. According to the prevailing dogma of those
times, Cl2 is distributed passively and can rapidly move to
maintain Cl2 electrochemical equilibrium (Vm 5 ECl). It
was expected that Cl2 content would decrease in response to the intracellular negativity. In fact, they reported that the Cl2 content of the Ehrlich cells actually
increased as a function of [K1]o, and this response was
exaggerated in the presence of ouabain. Furthermore,
they showed that this apparently paradoxical Cl2 uptake
could be blocked by furosemide. These findings were
followed (87, 88) by a demonstration that furosemide
blocked the unidirectional fluxes of Cl2 and K1.
Finally, in a landmark paper, Geck et al. (90) demonstrated virtually all the basic properties of the NKCC (see
sect. V). As seen in Figure 6, they first showed that when
K1-depleted cells were reexposed to external K1, they
took up K1, Na1, and Cl2 in a ratio of 1:1:2. Using a
membrane potential-sensitive probe, tetraphenylphosphonium, they showed that these ouabain-insensitive fluxes
had no effect on the membrane potential of the cells.
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FIG. 5. Demonstration in duck red blood cells
that Cl2 chemical gradient can drive furosemidesensitive Na1 fluxes at a constant membrane potential (calculated to be 212.4 mV over entire range of
[Cl2]o, where subscript o refers to extracellular).
Cells pretreated with 1025 M DIDS (to block band 3
anion exchanger) were incubated with 2 3 1026 M
valinomycin (to “voltage-clamp” membrane potential), ouabain (to block Na1 pump), and norepinephrine (to stimulate NKCC). In addition, media
contained 30 mM Na1 and 100 mM K1 6 1 mM
furosemide. Initial intracellular concentrations
were as follows (in mmol/l cell water): Na1 5 7.8;
K1 5 174.9; Cl2 5 95.6. Methylsulfate was used to
substitute for external Cl2. Subscript c refers to
cytosolic. [From Haas et al. (121).]
January 2000
Na1-K1-Cl2 COTRANSPORT
221
Conversely, they also showed that changing the membrane potential had no effect on the apparent cotransport
fluxes. Thus the cotransporter was electroneutral. Using a
coupling analysis derived from irreversible thermodynamics, they showed that the net movements of all three ions
were tightly coupled to one another and to an isosmotic
water flow. From these results, they explicitly proposed
that there existed a strongly coupled NKCC in the membrane of the Erhlich ascites tumor cell. They further
speculated that reports from experiments on RBC and
several epithelia could be explained by the existence of a
coupled triple cotransporter, the NKCC, rather than either
Na1-K1, K1-Cl2, or Na1-Cl2 cotransporters as originally
postulated.
Although for many years the widely accepted view of
Cl2 distribution was that it was at equilibrium and
crossed membranes easily and quickly, results from a
number of tissues had never been able to fit into that
model (e.g., heart muscle, Ref. 191; nerve cells, Ref. 165;
muscle, Ref. 238). For technical reasons, data from the
squid giant axon were the most convincing in this regard.
As early as 1939 chemical analysis of extruded axoplasm
had shown [Cl2]i of the giant axon to be very high (;120 –
150 mM) (23). (It must be remembered that the ionic
composition of the squid’s extracellular fluid is similar to
seawater, i.e., [Cl2]o is ;500 mM.) Keynes (166) made a
careful study of the [Cl2]i in the axon to rule out several
potential technical problems and very clearly showed that
at 120 –130 mM, [Cl2]i was at least three times greater
than expected if it were distributed at electrochemical
equilibrium (i.e., Vm 5 270 mV; ECl 5 235 mV). Furthermore, he made the intriguing observation that 2,3-dinitrophenol, an inhibitor of oxidative phosphorylation, significantly reduced 36Cl influx into axons. In addition, the
conductive (electrodiffusive) pathways for Cl2 transmembrane movement across the squid axolemma are very
small. Adelman and Taylor (1) showed that the Cl2 con-
ductance was so small that it could account for no more
than 10% of the “leakage” conductance across the voltageclamped squid axolemma. Thus it seemed clear that some
sort of active transport process must be responsible for
this high, nonequilibrium distribution of Cl2 in squid axoplasm. The search for the mechanistic basis of this nonequilibrium Cl2 distribution resulted in a series of studies
that used the internally dialyzed squid axon preparation
(see sect. VA). These studies showed that the mechanism
responsible for the high [Cl2]i 1) required intracellular
ATP (5, 302), 2) was inhibited at high [Cl2]i (28, 302, 303),
3) involved the cotransport of Na1 and was blocked by
furosemide (303), and 4) involved the cotransport of K1
and was blocked by bumetanide (306).
In the early 1980s, several labs provided evidence
that the NKCC process was found in the thick ascending
limb of the loop of Henle (101, 103), in the distal tubule of
the kidney (245, 246), as well as in the intestine (242). It
was demonstrated that these epithelial transport processes required all three co-ions (101, 242, 246) and were
blocked by the sulfamoyl benzoic acid diuretics (see sect.
VB, Ref. 245).
Thus, by the early 1980s, investigators had reported
functional evidence of the NKCC in a wide variety of cell
types. This was a period during which much of the functional characterization of this coupled cotransporter was
obtained.
V. FUNDAMENTAL CHARACTERISTICS
There are three functionally defining and unique
characteristics of the NKCC. 1) Ion translocation by the
NKCC requires that all three ions (Na1, K1, and Cl2) be
simultaneously present on the same side of the membrane. 2) Bumetanide and its congeners (5-sulfamoyl benzoic acid loop diuretics) bind to the cotransporter protein
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FIG. 6. Furosemide-sensitive net ion
movements. Ehrlich ascites tumor cells, previously K1 depleted and Na1 enriched, were
incubated for periods up to 5 min in KrebsRinger phosphate buffer with 38 mM K1 and
93 mM Na1. Furosemide-inhibitable net ion
movement is defined as difference in intracellular ion content (mmol/g dry wt) between cells incubated in presence of 1 mM
ouabain and those incubated in 1 mM
ouabain and 2 mM furosemide. This furosemide-inhibitable net movement is plotted
against incubation times on left panel. Right
panel shows ratio of Cl2 to K1 net movements and ratio of Na1 to K1 movements.
[From Geck et al. (90).]
222
JOHN M. RUSSELL
and inhibit ion translocation of all three ions. 3) Under
normal ionic conditions, all three ions are translocated,
and the translocation process has an electrically silent
stoichiometry: 1 Na1:1 K1:2 Cl2 for most cells or 2 Na1:1
K1:3 Cl2 for at least one cell (the squid giant axon). As a
minimum, these three criteria should be met before a
given process can be functionally identified as being mediated by the NKCC. In the following section, we consider
the evidence for these widely accepted properties and
what these properties tell us about the NKCC.
A. Absolute Cis-Side Requirement
for All Three Co-ions
ley and Mullins (31). Briefly, it involves threading a miniature dialysis capillary longitudinally down the axis of
the squid axon. The dialysis capillary used has a molecular mass cutoff of ;1,000 kDa, quite satisfactory for the
control of small inorganic ions, organic solutes, and ATP.
This gives the investigator two powerful advantages for
studies on ion transporters. First, it is possible to control
the internal milieu, and thereby maintain a steady-state
condition in terms of solute concentrations that cannot be
maintained in nondialyzed cells. Second, it permits one to
directly measure either the unidirectional influx, or the
efflux, simply by placing the relevant isotope in the extracellular fluid or the intracellular (dialysis) fluid, respectively, and collecting the fluid from the opposite side of
the axolemma.
This technique has been used to examine a number of
functional properties of the NKCC, including the requirement for cis-side ions. For the latter studies, the transside fluid was designed so that at least one of the necessary co-ions was absent, thereby reducing the possibility
of isotopic exchange fluxes (see sect. VIIB). In a series of
papers, Russell and co-workers have demonstrated the
absolute requirement for the cis-side presence of all three
ions both for unidirectional influx (303, 306) as well as for
efflux (8). Each of the three co-ions was systematically
removed while measuring the bumetanide-sensitive influx
of the other two, using a double-label flux approach. The
results of three representative experiments are shown in
Figure 7. Figure 7A shows the effects on 36Cl and 24Na
influx when an axon was sequentially superfused with
external fluids that were, in turn, K1 free, K1 containing,
and finally K1, 10 mM bumetanide containing. In this
axon, it can be seen that providing extracellular K1 increased the influx of both Cl2 (by ;21 pmolzcm22zs21)
and Na1 (by ;14 pmolzcm22zs21) and that both these
increases were reversed by the application of extracellular bumetanide. Figure 7, B and C, shows the same general protocol applied to examine the external Na1 (Fig.
7B) and Cl2 requirement. In each case, the increase in the
influx of the two co-ions caused by supplying the third
one is reversed by bumetanide treatment. These results
show that the cis-side absence of any one of the three ions
is the same as treating with bumetanide. Thus it is clear
that the NKCC requires all three ions be present for
transport to occur. In addition, this particular demonstration also shows that after “delivering” the three ions to the
inside of the membrane, the cotransporter can return to
an outward-facing conformation even though only K1 is
present on the inside. Because there is no evidence of
bumetanide-sensitive K1/K1 exchange in the squid axon
(i.e., K1 influx in Fig. 7, B and C, is the same in either the
absence of the third co-ion or in the presence of bumetanide), this suggests that the reorientation of the binding
sites to the outward-facing conformation must not require
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Wherever it has been possible to rigorously examine
the individual fluxes of Na1, K1, and Cl2, it has been
shown that the NKCC-mediated fluxes of all three ions
require the presence of the other two ions on the side of
the membrane from which the flux originates (termed the
cis-side; e.g., Refs. 8, 90, 121, 229, 303, 306).
Although it would seem to be a simple, straightforward matter to test for this property, using the sensitivity
of each flux to bumetanide, it is now clear that complications can arise. Care must be exercised to remove, as
much as possible, contributions from other transport
pathways for the ion whose flux is being measured. For
example, McManus’ group initially believed Cl2 was not
directly involved because the band 3 anion exchanger
obscured the net Cl2 fluxes via the NKCC (see sect. IVD1).
Prevention of this kind of error requires a preknowledge
and understanding of the ion transporters that exist in the
particular cell type one is investigating.
Unidirectional flux studies (using radioactive tracers) can be subject to a subtle kind of complicating error:
isotopic exchange. In some RBC, the NKCC can mediate
isotopic exchange fluxes under the appropriate set of
ionic conditions (e.g., Refs. 38, 58, 69, 118, 218; but see
Refs. 160, 161, 181; see sect. VIIA for further details). This
property can lead to errors when determining the apparent stoichiometry of the cotransport process. It can also
lead to misidentification of NKCC as KCC or as NCC. The
important thing to remember is that because of this possibility one cannot rule out the presence of NKCC just
because the unidirectional flux is not reduced by removal
of one of the three cotransported ions. One must strive to
arrange conditions that will not permit exchange fluxes of
the ion whose flux one is measuring.
Both of these potential problems have been overcome using the internally dialyzed squid giant axon. Because of its large size (;500 mm in diameter and 6 –7 cm
in length), it has been possible to develop a technique to
control the intracellular as well as the extracellular solute
composition while measuring unidirectional fluxes. The
technique of intracellular dialysis was developed by Brin-
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January 2000
Na1-K1-Cl2 COTRANSPORT
223
intracellular binding, translocation, and extracellular release of ions.
B. Bumetanide Inhibition/Binding
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1
1
2
FIG. 7. Dependence of cotransport influxes of Na , K , and Cl
on simultaneous presence of all 3 ions in external fluid. Experiments
were carried out on internally dialyzed giant squid axons. Internal fluid
in all cases contained the following (in mM): 400 K1, 0 Na1, 0 Cl2,
8 Mg21, and 4 ATP (pH 7.3, osmolality 5 970 mosmol/kgH2O). Ouabain
(1025 M) and tetrodotoxin (1027 M) were present at all times. A: effect
of external K1 on 24Na and 36Cl influxes. After a control period in 0 mM
[K1]o [N-methyl-D-glucamine (NMDG) replacement], 10 mM K1 was
applied and both influxes increased; 36Cl influx increased by ;21
pmolzcm22zs21 and 24Na influx by ;14 pmolzcm22zs21. Finally, 10 mM
bumetanide was applied causing both fluxes to decrease to levels very
near that seen under 0 [K1]o conditions. [Na1]o 5 425 mM; [Cl2]o 5 561
mM. B: effect of external Na1 on 42K and 36 Cl influxes. After a control
period in 0 mM [Na1]o (NMDG substitution), 425 mM Na1 was applied
leading to increases of both 42K (;8 pmolzcm22zs21) and 36 Cl (;20
pmolzcm22zs21) influxes. Subsequent application of 10 mM bumetanide
caused influxes to decrease to levels near those observed in absence of
extracellular Na1. [K1]o 5 10 mM; [Cl2]o 5 561 mM. C: effect of external
Cl2 on 42K (;7.5 pmolzcm22zs21) and 22Na (;15 pmolzcm22zs21)
influxes. After a control period in 0 mM [Cl2]o (gluconate substituted
for Cl2), 561 mM Cl2 was applied leading to increases of both 42K
and 22Na influxes. Subsequent application of 10 mM bumetanide caused
influxes to decrease to levels near those observed in absence of
extracellular Cl2. [Na1]o 5 425 mM; [K1]o 5 10 mM. (From A. A.
Altamirano, G. E. Breitwieser, and J. M. Russell, unpublished observations.)
Bumetanide is the prototype for the loop diuretics.
They are called loop diuretics because the diuresis (increase in urine production) they produce is the result of
their action in the thick ascending limb of the loop of
Henle. This region of the renal nephron reabsorbs Na1
and Cl2 from the tubular fluid and is responsible for the
establishment of the hypertonic interstitium of the renal
medulla. Thus, when this isoform of the NKCC (NKCC2)
is inhibited, a large increase in urine flow is observed, and
the urine is nearly isosmotic with plasma, regardless of
the hydration state of the individual, i.e., the individual
loses the ability to excrete either a concentrated or a
dilute urine. Other representatives of this group (5-sulfamoylbenzoic acid derivatives) are furosemide, an agent
used in early studies on the NKCC, but only rarely used
experimentally nowadays, and piretanide, a congener that
has been used mainly in Europe. Both are chemically
closely related to bumetanide but less potent and less
specific for the NKCC (e.g., Ref. 313) than bumetanide.
It is difficult to overstate the importance of the loop
diuretics to the development of our present level of understanding of the NKCC. They are routinely used to
identify fluxes mediated by the NKCC. This latter use has
contributed in a major way to our learning that the NKCC
is found in a very wide variety of cells (e.g., Ref. 269).
They have played an important role in the identification of
the protein and the subsequent cloning of the cotransporter, and they have provided information that has been
used to interpret ion binding to the NKCC.
Given this importance, it is necessary to emphasize
that these agents are not the NKCC’s equivalent of the
Na1 pump’s ouabain. In fact, they are far from it. Although these agents inhibit the NKCC with a reasonably
high affinity (see below), they can also inhibit other anion
transport processes when used in higher concentrations,
2
e.g., Cl2/HCO2
channels (63),
3 exchange (108, 153), Cl
and KCC (199). Thus, used alone and/or in high concentrations, not even bumetanide can provide positive proof
that a given function is mediated by the NKCC. For example, Kracke et al. (180) showed in human RBC that
bumetanide inhibits components of Na1 and K1 efflux
that are neither dependent on cell Cl2 nor on the mutual
presence of the other cation. So one must know the
properties of one’s experimental subject well before using
these “specific” agents, especially for binding studies designed to identify the NKCC. A good general rule of thumb
would be when used at concentrations at or below 10 mM,
bumetanide is specific for the NKCC.
Bumetanide reversibly inhibits the NKCC in a variety
224
JOHN M. RUSSELL
2. Representative data for bumetanide affinity for
the NKCC under physiological ionic conditions
TABLE
Function
Measured
Preparation
Turkey erythrocytes
Duck erythrocytes
Duck erythrocytes
Duck erythrocytes
Human erythrocytes
Microsomes from
squid optic ganglia
C6 glioma cells
Rat astrocytes
Purified plasma
membranes from
MDCK cells
Vascular endothelial
cells
HT29 cells (human
adenocarcinoma
cells)
Shark rectal gland
Basolateral
membrane vesicles
from rabbit colon
Basolateral
membrane vesicles
from rabbit colon
Rabbit parotid
basolateral
membrane vesicles
LLC-PK1/C14 cells
Dog kidney cortex
membranes
Vesicles from dog
kidney outer
medulla (NKCC2?)
Flounder intestine
Cortical thick
ascending limb
tubules
Cultured mouse
medullary thick
ascending limb
cells (NKCC2)
HSWP human
fibroblasts
Xenopus oocytes
[3H]bumetanide
binding
Unidirectional 36Cl
efflux
[3H]bumetanide
binding
86
Rb uptake
86
Rb influx
[3H]bumetanide
binding
2 3 1026
0.6 2 2 3 10
Reference
No.
264
27
117
1 3 1027
111
27
111
1 3 10
27
2.6 3 10
27
319
6 3 10
6 3 1026
83
135
8.7 3 1026
135
27
1 3 10
3.1 3 10
27
27
8
9
5 3 10
5.5 3 1027
1.3 3 1026
41
340
106
[3H]bumetanide
binding
[3H]bumetanide
binding
1.3 3 1027
249
27
76
Cl2 secretion
Cl uptake
5 3 1026
1.4 3 1027
269
353
[3H]bumetanide
binding
1.3 3 1027
353
[3H]bumetanide
binding
2.8 3 1026
345
86
Rb uptake
[3H]BSTBA
4.5 3 1027
6.1 3 1028
17
112
[3H]bumetanide
binding
4.5 3 1028
72
[3H]bumetanide
binding
Short-circuit
current
3 3 1027
256
27
313
86
Rb influx
1.8 3 1027
160
86
Rb influx
1 3 1027
260
Rb influx
28
337
36
86
1.3 3 10
2 3 10
7 3 10
MDCK, Madin-Darby canine kidney; BSTBA, 4-benzoyl-5-sulfamoyl-3-(3-phenyloxy)benzoic acid.
of preparations by a concentration-dependent mechanism, with a half-inhibitory constant of ;1 3 1027 M (see
Table 2). From the early days of work in this field, there
have been attempts to relate the potency of these agents
to the concentration of the three cotransported ions.
Some workers have observed that the apparent inhibitory
potency is inversely related to the [Cl2] of the bathing
media (117, 264) and positively correlated with [K1] and
[Na1] (116, 264; see sect. VIIIA for further discussion of
this important observation).
Lytle and Forbush (213–215) have suggested that
there might be significant differences in bumetanide sensitivity between NKCC found in secretory versus those
found in absorptive epithelia. Thus they point out that
absorptive epithelia such as renal thick ascending limb of
the loop of Henle and flounder intestine have apparent
dissociation constants (KD) that are significantly ,1 mM,
whereas the apparent affinities for some secretory epithelia such as rectal gland and parotid gland are .1 mM. We
now know that these absorptive epithelia express NKCC2
on their apical membranes, whereas the secretory epithelia express NKCC1 on their basolateral membranes. Thus
this suggestion implies a difference in bumetanide binding affinity between the two NKCC isoforms. This interpretation is borne out by recent studies in which NKCC1
and NKCC2 were expressed in HEK-293 cells. Isenring et
al. (150) showed an about threefold difference in the
inhibition constant (Ki) for bumetanide-sensitive 86Rb
flux via the NKCC2 isoform (0.08 mM) compared with the
NKCC1 isoform (0.28 mM). Table 2 is a collation of a
published Ki values measured in situ in a variety of cells.
Most of these cells would be expected to express NKCC1.
For such NKCC1 cells, the apparent Ki values span a
range of almost two orders of magnitude, from 8.7 mM
(Ehrlich ascites cells) to 0.06 – 0.07 mM (duck erythrocytes and Xenopus oocytes). By comparison, dog kidney
outer medulla vesicles, which are very likely to to be
enriched with NKCC2, had a Ki value of 0.045 (Table 2).
Thus, although the pattern of a somewhat higher bumetanide affinity for the NKCC2 isoform appears to hold for
in situ measurements, there is more variability within the
NKCC1 cohort than between the NKCC1 and NKCC2 isoforms. Clearly, one could not identify the isoform based
on its bumetanide sensitivity except in the more extreme
cases.
These compounds have a very high lipid solubility
(ether/water partition coefficient 5 0.25 at pH 7 and 0.04
at pH 8; Ref. 73). Thus they would be expected to partition
easily into and cross biological membranes. This raises
the question of their site of inhibitory action. Nitschke et
al. (244) addressed this question by constructing two
high-molecular-weight congeners of piretanide. One was
dextran-sulfonylurea piretanide (Pir-Dex) and the other
was polyethylene glycol piretanide (Pir-PEG). Both had
molecular weights of ;5,300. Thus they were presumably
too large to cross the membrane and thereby gain access
to the cytoplasmic-facing face of the membrane. These
agents as well as the parent compound isothiocyanopiretanide (Iso-Pir) were tested for their effectiveness in inhibiting NKCC activity in the isolated, perfused thick
ascending limbs of rabbit kidneys. In this preparation, the
NKCC is located on the apical membrane that faces the
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Human erythrocytes
Ehrlich ascites tumor
cells
Ehrlich ascites tumor
cells
Squid giant axon
cAMP-stimulated
86
Rb influx
Cl2-dependent net
Rb1 uptake
([Cl2]o 5 100
mM)
[3H]bumetanide
binding
22
Na flux
inhibition
22
Na flux
inhibition
Net Na1 efflux
Net Cl2 influx
Apparent
Affinity, M
Volume 80
January 2000
Na1-K1-Cl2 COTRANSPORT
C. NKCC is Electrically Silent
Given that the NKCC mediates the transmembrane
movement of three ions, Na1, K1, and Cl2, there is the
possibility that the overall transport process generates a
transmembrane current and/or is affected by the membrane potential. This would be most obvious if the ion
transport stoichiometry was such that a net charge was
translocated with each turnover of the cotransporter. The
term electrically silent as used here means that the overall transport process of Na1, K1, and Cl2 via the cotransporter is not driven by the cellular transmembrane voltage, nor does the transport process directly generate a
membrane current and thereby affect the transmembrane
voltage. (This does not exclude the possibility that the
“carrier” is charged and hence affected by membrane
voltage in some, as yet, unrecognized way.) In fact, it was
the movement of Cl2 against its electrochemical gradient
(“paradoxical movement of Cl2”) that first led Geck et al.
(90) to undertake the studies that resulted in the initial
published description of NKCC (see sect. IVD2). The electrical silence of the NKCC is a fundamental characteristic
of the NKCC that is now well accepted by workers in the
field, yet the direct evidence for the assertion is surprisingly sparse. Electrical silence is often inferred from the
apparent stoichiometry (see sect. VI) of the cotransport
process, but arriving at this conclusion can potentially
involve circular reasoning.
Conclusive proof of electrical silence requires direct
measurements of membrane potential combined (ideally)
with simultaneous measures of cotransporter-mediated ion
transport. For obvious technical reasons, such studies are
rare. For example, Geck et al. (90) varied extracellular Na1,
K1, and Cl2 concentrations while measuring furosemidesensitive water movements. The membrane potential was
estimated using the transmembrane distribution ratio of the
lipid-soluble cation [3H]tetraphenylphosphonium. They observed that wide variations of the extracellular ion concentrations caused large changes of the furosemide-sensitive
water fluxes but had no effect on the distribution ratio of the
tetraphenylphosphonium, i.e., on the membrane potential.
Although this result was interpreted to mean that cotransport was voltage insensitive, it also implies that the Vm was
quite insensitive to very large variations in the electrochemical gradients of the three major extracellular ions, a surprising result. Thus either the cells were very “leaky” or the
tetraphenylphosphonium distribution was not accurately reflecting the membrane potential. Either way, this finding
casts a serious doubt on the overall interpretation of the
result.
Haas et al. (121) addressed the question of whether
the effects of varying [Cl2]o on the norepinephrine-stimulated, furosemide-inhibitable Na1 and K1 net fluxes in
the duck RBC were direct (via a coupled cotransporter)
or indirect, via a change of Vm. By comparing the effects
of variations of [Cl2]o on changes of cell Na1 content (i.e.,
measuring net Na1 fluxes) under conditions in which Vm
was expected to vary widely, these authors concluded
that changes of Vm played no role in the Cl2-dependent
net fluxes of Na1 (Fig. 8). Thus, in the presence of valinomycin and DIDS (to block Cl2 conductance through
the band 3 anion transporter), Vm is expected to be
“clamped” to the K1 equilibrium potential because, (as
the authors showed) under these conditions, the membrane permeability to K1 is about seven to eight times
larger than the membrane permeability to Cl2. In a similar
experiment, they also showed that the K1 dependence of
the norepinephrine-stimulated net Na1 fluxes was unchanged by treatment with valinomycin plus DIDS. In this
case, [K1]o was varied such that the membrane potential
of valinomycin-treated cells was expected to vary between 28 and 2129 mV, whereas for the untreated cells,
the membrane potential would remain essentially equal to
the ECl, 229 mV. Although these studies contained no
direct measurements of membrane potential, the RBC
literature contains considerable support for the view that
treatment with valinomycin will make the RBC membrane
a reasonably good “K1 electrode” (121). Thus the fact that
treatment with valinomycin had no effect on the furosemide-sensitive net flux of Na1 caused by variations of
either [Cl2]o or [K1]o strongly argues that NKCC in the
duck RBC is insensitive to changes of Vm.
McRoberts et al. (229) used a very similar approach
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tubule lumen. There was no effect on NKCC activity
(measured as short-circuit current) when these compounds were applied to the basolateral membrane or the
bath side of the preparation. However, when applied to
the luminal side, all three agents inhibited the NKCC with
the following half-inhibitory constants: Iso-Pir 5 3 3 1026
M; Pir-Dex 5 6 3 1026 M; Pir-PEG 5 2 3 1026 M. In an
earlier work, these investigators used piretanide itself and
found the half-inhibitory constant to be 1 3 1026 M (313).
Since there is a great deal of evidence that these agents
bind directly to the NKCC protein itself (see sect. VIIB),
these results are consistent with the site of inhibitory
action being on a portion of the NKCC molecule accessible from the external face of the plasmalemma.
In keeping with the above interpretation, Haas (110)
has tentatively suggested that bumetanide binds exclusively to the outwardly facing cotransporter. Unfortunately, this suggestion is now being cited as proof (e.g.,
Ref. 183). Nevertheless, at present, there is absolutely no
evidence excluding the possibility that bumetanide may
also bind to the inward-facing cotransporter. Indeed, if
bumetanide acts by competing with Cl2 for the second
anion transport site as has been suggested (116, 117; but
see Ref. 149), then one might reasonably expect inhibition
from an internal site as well.
225
226
JOHN M. RUSSELL
Volume 80
in Madin-Darby canine kidney (MDCK) cells. They varied
[K1]o in the presence and absence of 20 mM valinomycin
while measuring 22Na uptake. They found that valinomycin had no effect on the [K1]o-stimulated 22Na uptake in
Na1-depleted cells. Hannafin and Kinne (125) found a
similar lack of valinomycin effect on bumetanide-sensitive 36Cl uptake by membrane vesicles prepared from the
thick ascending limb of rabbit loop of Henle. It should be
noted that neither of these studies provided evidence that
varying [K1]o in the presence of valinomycin actually
affected Vm.
The question of electrical silence could be more
directly addressed in the internally dialyzed squid giant
axon because membrane potential and influx could be
directly measured in the same axon (307). Membrane
potential was measured with a microelectrode and was
varied using veratridine, an agent which opens Na1
channels in the squid axon, resulting in the depolarization of the Vm. The action of veratridine was reversed
by the blocker of Na1 channels tetrodotoxin. As seen in
Figure 9, depolarization of the Vm (in this case, by
almost 30 mV) caused Cl2 influx to increase slightly,
but this increase was insensitive to bumetanide and
therefore probably occurred via voltage-sensitive Cl2
channels (146). The bumetanide-sensitive, steady-state
influx in the presence of veratridine (Vm depolarized by
an average of 28 mV) averaged 19.8 6 2 pmolzcm22zs21
(n 5 4). This value of bumetanide-sensitive Cl2 influx is
the same as that reported for the squid NKCC under
similar conditions of external ion concentrations and of
normal membrane potential (306). It is important to
recognize that at 17°C, a 26-mV change in membrane
potential should result in an e-fold (2.73) change in the
influx if the transporter carried one net charge. Obvi-
FIG. 9. Effect of membrane potential depolarization on bumetanidesensitive and -insensitive Cl2 influx into internally dialyzed squid giant
axon. Cl2 influx was measured using 36Cl. Axon was dialyzed with a
Cl2-free fluid to prevent contamination of unidirectional Cl2 flux data
with possible Cl2/Cl2 exchange fluxes. Transmembrane resting potential was measured with a 0.5 M KCl-filled micropipette inserted longitudinally alongside intracellular dialysis capillary. When membrane potential of axon was depolarized ;30 mV by application of veratridine, Cl2
influx increased by ;7 pmolzcm22zs21 (a). Repolarization of axon membrane potential (Vm) was accomplished by treating axon with tetrodotoxin (TTX). Upon repolarization, Cl2 influx decreased by ;6
pmolzcm22zs21 (b) even though axon was treated with 1 mM bumetanide,
a concentration which inhibits .90% of Na1-K1-Cl2 cotransport flux in
this preparation. Thus voltage-sensitive Cl2 influx was insensitive to
bumetanide and therefore presumably not occurring through Na1-K1Cl2 cotransporter. [From Russell (307).]
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FIG. 8. Lack of effect of membrane potential on
Cl2-dependent net Na1 fluxes. Initial intracellular
concentrations of K1, Na1, and Cl2 are shown on
bottom right. Net Na1 uptake was measured at 41°C
from a solution containing 50 mM Na1, 100 mM K1,
1025 M DIDS, 1024 M ouabain, 1026 M norepinephrine, 62 3 1026 M valinomycin, and 61 mM furosemide. Changes in [Cl2]o were made by substituting
methylsulfate to maintain a constant extracellular
osmolality. In presence of valinomycin, authors estimated PK/PCl 5 7.5. With the use of this permeability
ratio and with the assumption of a constant field,
calculated membrane potential for valinomycintreated cells was 27.9 mV over entire range of [Cl2]o
used. In absence of valinomycin, it was assumed that
PCl ... PK, PNa and that PMeSo4 5 0.5PCl. Again,
with the assumption of a constant field, membrane
potential was calculated to vary between 20.4 and
218.3 mV. [From Haas et al. (121).]
January 2000
Na1-K1-Cl2 COTRANSPORT
VI. STOICHIOMETRY/THERMODYNAMICS
A. Stoichiometry of the Transport Process
Transport via the NKCC requires that the three ions
bind to the cis-facing side of the NKCC protein (see sect.
VII) and then be translocated to the trans-side of the
membrane. Because the number of ions that must bind to
the NKCC need not necessarily equal the number of ions
that are actually translocated, it follows that there are at
least two processes the stoichiometries of which are of
interest: 1) ion binding and 2) ion translocation. In the
consideration of this topic, it is important to limit ourselves only to those studies that explicitly examined ion
transport. The ion-dependent stoichiometry of bumetanide binding will be considered in a later section.
Conceptually, the ideal way to measure the transport
stoichiometry of the cotransporter would be to isolate the
cotransporter from all other ion transport mechanisms,
then directly measure the unidirectional fluxes of Na1,
K1, and Cl2 simultaneously under conditions in which
there was no Na1, K1, or Cl2 present on the opposite side
of the membrane (0-trans condition). In this way, three
kinds of confounding errors could be avoided: 1) there
would be no contamination of the NKCC fluxes by fluxes
mediated by other mechanisms, 2) there would be no
possibility of exchange fluxes mediated by the NKCC, and
3) the effects of trans-side ions on the NKCC would be
prevented (e.g., see sect. IXB). Thus all the Na1, K1, and
Cl2 crossing the membrane would do so via the cotransporter, and ion transport would occur in only one direction. This would give an unambiguous measure of the
three simultaneous unidirectional fluxes via the cotransporter. Unfortunately, such an ideal situation has not
been, and probably cannot be, achieved.
In general, three kinds of approaches have been used
to address the question of transport stoichiometry of the
NKCC. One is to measure the furosemide- or bumetanidesensitive changes in intracellular concentrations of Na1,
K1, and Cl2 caused by rapid changes of the extracellular
concentration of one of these ions. The use of such nonsteady-state, net fluxes to determine the stoichiometry of
a single turnover of the cotransporter requires that potential “shunting” and/or “additive” pathways for each of the
ions be either blocked or quantitatively accounted for.
This is usually accomplished by looking at only that portion of the net flux that is furosemide or bumetanide
sensitive. Neither of these agents, when used in appropriate concentrations to identify the NKCC, would be expected to block potential parallel fluxes (those occurring
in the same direction as the NKCC fluxes) or backleak
flux pathways. Removal of at least two of the three ions
from the side of the membrane toward which isotopically
measured cotransport is occurring (i.e., 0-trans condi-
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ously, no such change of bumetanide-sensitive influx
was observed. Therefore, these data support the view
that the steady-state, unidirectional influx via the NKCC
in the squid giant axon is insensitive to changes of the
membrane potential.
Kracke and Dunham (181) further examined the
question of voltage sensitivity of the NKCC by measuring 22Na uptake (unidirectional influx) into human
RBC. Like Haas et al. (121), they also used valinomycin
and DIDS to voltage-clamp the Vm to the K1 equilibrium
potential. Over a membrane potential range calculated
to vary between 242 and 2118 mV, these workers
demonstrated that there was no significant change in
furosemide-sensitive 22Na influx. If the NKCC mediated
the movement of a single charge per transport cycle,
the constant field equation would predict a 37-fold
change in 22Na efflux over such a membrane potential
range. The authors controlled for the possibility that
much of the 22Na influx might be via an Na1/Na1 exchange mode of the cotransporter by measuring the
furosemide-inhibitable 22Na influx in cells at three different [Na1]i values. Here they found that, if anything,
furosemide-inhibitable 22Na influx was reduced by increased [Na1]i (see sect. IXB2A). Thus, under the conditions of their experiments, there was no evidence of
Na1/Na1 exchange occurring via the NKCC. The overall
conclusion once again is that the cotransport mechanism is unaffected by membrane potential changes, in
this case, rather large changes.
Alvarez-Leefmans et al. (11–13) have shown that
frog dorsal root ganglion cells maintain a higher than
equilibrium [Cl2]i by means of an external Na1- and
K1-dependent process. Removal of external Cl2 resulted in a decrease of [Cl2]i from a value of 25–30 to
,2– 4 mM (as measured with Cl2-selective microelectrodes). When the external Cl2 was returned, the [Cl2]i
recovered by a mechanism that required the presence
of extracellular Na1 and extracellular K1, and which
could be greatly inhibited by either furosemide (1 mM)
or bumetanide (10 mM). The recovery of [Cl2]i was not
accompanied by any significant change of the resting
membrane potential. These results of direct measurements of [Cl2]i and resting membrane potential offer
further strong proof of the electrically silent nature of
the operation of the NKCC.
Thus the finding that the overall transport process is
electrically silent provides strong evidence that the cotransport stoichiometry is such that the sum of the cations transported during one cotransport cycle equals the number of
Cl2 cotransported during that same cycle. However, it
should be pointed out that nothing in the preceding analysis
precludes the possibility that ion binding and/or transport
might be influenced by the membrane potential or the membrane electrical field.
227
228
JOHN M. RUSSELL
1. Evidence for a Na1:K1:Cl2 stoichiometry of 1:1:2
Because the overall transport of the ions is an electroneutral process (see sect. VC), it follows that during a
single turnover of the cotransporter the sum of the cations (Na1 1 K1) crossing the membrane will be equal to
the sum of the Cl2 crossing the membrane. Therefore, the
simplest possible transport stoichiometry is 1 Na1:1 K1:2
Cl2. Indeed, this was the stoichiometry first reported by
Geck et al. (90). Using K1-depleted (and probably Cl2depleted, as well), Na1-enriched, Ehrlich ascites tumor
cells, these workers measured the net, furosemide-sensitive uptake of each of the three ions as a function of each
ion’s extracellular concentration. For example, when the
net uptakes of Na1 and Cl2 were measured as a function
of [K1]o, the ratio of Cl2 uptake to K1 uptake was 2.0, and
the ratio of Na1 uptake to K1 uptake was 0.8 (see Fig. 6,
right). Identical studies varying [Na1]o or [Cl2]o yielded
the same relative flux ratios. These results led to their
conclusion that the NKCC transported ions into the cell
with a stoichiometry of 1 Na1:1 K1:2 Cl2 and was the first
report of the cotransporter’s stoichiometry of ion transport.
Haas et al. (121) studied the simultaneous net effluxes of K1 and Na1 from duck RBC. The net effluxes
were measured into an external fluid that contained no
Na1 or K1. This design served two purposes: 1) to maximize the gradient down which these fluxes occur,
thereby enhancing their magnitude, and 2) to prevent any
possibility of a backflux of either Na1 or K1. Figure 10
shows that the ratio of the net losses of Na1 to K1
(virtually all of which were furosemide sensitive) was
unaffected by changes in intracellular concentrations of
either K1 or Na1. In addition, they reported (but did not
show) that the net loss of Cl2 in these two studies was
equal to the sum of the Na1 plus K1 losses. This result
supports both the concept of electroneutral cotransport
of the three ions as well as a stoichiometry of 1 Na1:1
K1:2 Cl2. However, unless some other ion is moving
(highly unlikely in this case given the design of the study
and the size of the fluxes), the sum of the net K1 plus Na1
movements has to equal the sum of the net Cl2 movement
to maintain macroscopic electroneutrality. Because it is
possible that the net loss of cations created an outwardly
directed electrochemical driving force on Cl2, which exited the cells by an alternate pathway (e.g., a channel),
these results alone cannot prove the stoichiometry of the
actual coupled cotransport event. However, the combination of these results with those discussed previously (see
Fig. 8), in which these same workers demonstrated that
the Cl2 movements were not voltage driven, makes a
powerful argument for the 1:1:2 stoichiometry. Notice
that the net losses (effluxes) depended entirely on the
simultaneous presence of all three ions intracellularly;
removal of either intracellular Na1 or K1 completely
prevented the net loss.
Among the earliest use of the kinetic analysis approach to the determination of the stoichiometry of cotransport by the NKCC was a study by McRoberts et al.
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
tions) reduces the possibility of serious, unaccounted for
backleaks as well as any effects of these intracellular ions
on the NKCC itself (e.g., see sect. IXB). Such a design
effectively means that net fluxes are the same as unidirectional fluxes.
An approach that avoids the use of net flux measurements has been to measure the initial rate of furosemide/
bumetanide-sensitive isotopic uptake flux of one of the
three cotransported ions (usually K1) while varying the
extracellular concentration of each of the three cotransported ions. The resulting fluxes are plotted as a function
of the ion concentration and fitted with the Hill equation
to determine the Hill coupling coefficient. The Hill coefficient is then taken as the stoichiometric coupling coefficient of cotransport for the ion whose external concentration has been varied. For this approach to yield the
stoichiometry of ion cotransport, it is necessary that the
binding of each of the ions to the cotransporter protein be
ordered and highly cooperative; otherwise, it can only
yield a lower limit to the actual stoichiometry of cotransport. There is good evidence that such order and cooperativity exists (see sect. VIIA).
As stated previously (see sect. IVA), there is evidence
(at least in some RBC) that the NKCC can mediate isotopic self-exchange fluxes. This possibility creates a problem when the intracellular concentrations of the cotransported ions are relatively high, a condition which, in small
intact cells, is difficult to avoid for all three ions. This
condition favors isotopic self-exchange fluxes via “partial
reactions” of the NKCC (see sect. VII) which would result
in erroneous estimates of stoichiometry. In fact, this type
of error has occurred (see sect. VI) and probably accounts
for reports of variable stoichiometry. Problems with isotopic self-exchange fluxes can be avoided by depleting the
cell of the ion whose isotopic flux is being measured
before the fluxes are determined.
A third approach involves measuring furosemide/bumetanide-sensitive unidirectional fluxes under steadystate conditions and 0-trans conditions. This has been
possible in the internally dialyzed squid giant axon. By
continuous intracellular dialysis, one can establish and
study a set of steady-state conditions not possible for
small, intact cells (e.g., total depletion of intracellular
Na1, Cl2, or K1 with normal or above normal extracellular concentrations of these ions). In addition, in the squid
axon under these conditions, the NKCC is the major
pathway for transmembrane Cl2 movements, making 36Cl
fluxes a particularly effective monitor of NKCC flux activity. Therefore, this approach may come closest to
achieving the ideal situation described above.
Volume 80
January 2000
Na1-K1-Cl2 COTRANSPORT
229
(230). Working on confluent monolayers of cultured
MDCK cells, they measured bumetanide-sensitive 86Rb
and 22Na uptake as a function of the extracellular concentrations of Na1, K1, and Cl2. They found that the 86Rb
or 22Na uptakes were hyperbolic functions of either [K1]o
or [Na1]o, suggesting only a single K1 and Na1 were
required to activate the cotransport process. In contrast,
the plot of bumetanide-sensitive 22Na uptake as a function
of [Cl2]o was fit by a sigmoidal curve.
The data could be linearized by plotting the bumetanide-sensitive flux (DV) against DV/D[Cl2]on, where n was
between 1.4 and 2.1. This result suggested a degree of
cooperativity between cotransport flux and [Cl2]o. Because the n was greater than 1 for both Na1-stimulated
86
Rb uptake and K1-stimulated 22Na uptake, at least two
Cl2 would be required for each Na1 plus K1 that was
transported. This means that, theoretically, if the cooperativity between Cl2 binding and 86Rb and 22Na uptake is
very high, then a minimum of two Cl2 must bind to effect
cotransport. To the degree that the cooperativity is less,
the stoichiometry obtained from this analysis will be an
underestimate. This general approach has been used by a
large number of workers in the field (e.g., Refs. 169, 260,
346), and all derived the same stoichiometry, 1 K1:1 Na1:2
Cl2. Unfortunately, none has measured the fluxes of all
three ions under the same circumstances. Most have measured only one flux, usually 86Rb, so the possibility exists
that the actual ion transport stoichiometry might differ
from the stoichiometry of ion binding to the cotransporter.
2. Evidence for a Na1:K1:Cl2 stoichiometry of 2:1:3
This unusual stoichiometry has been reported for
two preparations, the squid axon and the ferret RBC.
Evidence from the squid giant axon rests on the results of
two kinds of experiments. The initial indication that the
stoichiometry in the squid axon might be different from
that reported in other preparations came from collating
the results of experiments that were originally designed to
determine 1) the dependence of the influx of each ion on
the presence of the other two ions in the extracellular
fluid or 2) the effect of raising the [Cl2]i on NKCC-mediated influxes of all three cotransported ions, or 3) the
effect of furosemide on the influxes of all three ions
(303–306). In all these experiments, the steady-state, unidirectional influxes of either 42K, 22Na, or 36Cl had been
measured in axons whose intracellular Na1 and Cl2 were
set at 0 mM by intracellular dialysis. This condition prevents isotopic self-exchange fluxes of these two ions.
[Because increasing [Cl2]i and [Na1]i both inhibit NKCC
influx in the squid axon (see sect. IXB1), there is no
evidence that either Cl2/Cl2 or Na1/Na1 exchange occurs
in the squid axon.] Collating the results of experiments
carried out over several years (303, 305, 306) and which
examined the effects of several different perturbations on
the individual influxes of each of the cotransported ions
strongly suggested that a stoichiometry of 1:1:2 might not
apply to the squid axon. These results suggested that the
ratio might be closer to 2 Na1:1 K1:3 Cl2, but because the
experiments had not been designed specifically to address the stoichiometry issue, no firm conclusions could
be reached.
To directly address the issue of stoichiometry of
transport, steady-state, unidirectional influxes of ion pairs
were measured simultaneously. For each pair, one member was always K1 so that all stoichiometric ratios were
determined relative to the K1 influx. Thus either 42K and
22
Na or 42K and 36Cl influxes were measured simulta-
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
1
FIG. 10. Effect of internal K (left) and
internal Na1 (right) on net Na1 1 K1
efflux from duck red blood cells. Cotransport was stimulated by adding 1026 M norepinephrine; furosemide concentration
was 1 mM. Co-ion gradients were maximized by incubating cells in media free of
Na1 and K1 (choline chloride replaced
cations); this also prevented any possibility of back-fluxes of the two cations (but
not of Cl2). All external media contained
0.1 mM ouabain. Left: with the use of nystatin technique, cells were preloaded to
contain [Na1]i of 60.5 mM. [K1] was varied
in nystatin loading solutions to yield different levels of cytosolic K1 ([K1]c) noted
on abscissa. Right: cells were preloaded
with nystatin technique to contain [K1]i of
50.2 mM. [Na1] was varied in nystatinloading solutions to yield different levels
of [Na1]c noted on abscissa. [From Haas et
al. (121).]
230
JOHN M. RUSSELL
TABLE
Cl2 with a stoichiometry of 1 Na1:1 K1:2 Cl2. It is possible that the squid possesses an isoform of the NKCC that
is different from those characterized to this point.
B. Thermodynamics of the Cotransport Process
The NKCC is believed to be a secondary active transport process despite its requirement for cellular ATP (see
sect. IXA). As such, it would be expected to be reversible,
that is, to mediate ion fluxes both into and out of the cell,
the net direction of cotransport depending on the direction of overall net free energy resident in the three ion
gradients. To determine the overall net free energy of any
particular system, one must know 1) the intra- and extracellular concentrations of Na1, K1, and Cl2 as well as 2)
the stoichiometry of the transport process (included in
this is whether the process is electroneutral or electrogenic). As discussed in section VC, the overall NKCC
cotransport process is electroneutral. For most cells, Na1
and K1 gradients are established by the Na1 pump such
that the outwardly directed concentration gradient for K1
somewhat exceeds the inwardly directed concentration
gradient for Na1. Thus, ultimately, it is the transmembrane chemical gradient for Cl2 that will determine
whether the net free energy available to the NKCC of any
particular cell will favor net influx or efflux.
Unlike the situation for [Na1]i and [K1]i, [Cl2]i can
vary over a fairly wide range depending on the particular
cell and the activity of other Cl2 transporting mechanisms
1
(e.g., Cl2 channels; Cl2/HCO2
3 exchanger; Na -dependent
2
2
Cl /HCO3 exchanger). In mammalian cells, [Cl2]i can
vary from as low as a few millimolar to as high as 50 – 60
mM. Table 3 collates data from eight representative cells
and shows the range of ion concentrations and the calculated net free energies available to the NKCC using both
stoichiometries. It can be seen from these data that the
net energy available to the NKCC of different cells can
vary from strongly favoring net uptake (i.e., DG being
quite negative) to being at, or very near, thermodynamic
3. Net free energy in the combined Na1 1 K1 1 Cl2 chemical gradients in several representative cells
Cell Type
Ehrlich ascites tumor cells
Heart muscle
Shark rectal gland
Squid giant axon
UMR-106-01 osteosarcoma
cells
Bovine aortic endothelial cells
Frog retinal pigment epithelial
cells
Frog dorsal root ganglion cells
[K1]i/[K1]o, mM
[Na1]i/[Na1]o, mM
[Cl2]i/[Cl2]o, mM
Stoichiometry
(1:1:2) DG, kJ/mol
Stoichiometry
(2:1:3) DG, kJ/mol
156/15 (202)
134.5/2.5 (†)
100/4.5 (54)
154*/4.4 (104)
350/18 (307)
142/5 (98)
32/130 (202)
12.5/140 (†)
8.4/117 (54)
13.8*/278 (104)
50/420 (307)
15/140 (98)
75/145 (202)
36.3/110 (†)
17.9/126 (54)
70–116/250 (216)
120/480 (307)
31.8/149 (98)
20.98
21.67
28.86
22.40 to 24.87
24.32
25.10
26.29
210.76
220.68
211.61 to 215.31
213.31
214.84
130/5.4 (32)
110/2 (2)
16/116 (32)
14/110 (2)
55/125 (32)
27/90 (2)
20.71
21.13
27.94
29.10
105/2.5 (11)
25/115 (11)
31/122 (11)
21.29
1 m
i
1
28.36
2 n
i
1 m
o
Numbers in parentheses indicate reference from which values were obtained.
DG 5 RT ln [Na ] [K ]i[Cl ] /[Na ] [K1]o[Cl2]no , where
R 5 8.314 J/mol z degree and T 5 °K.
* Original measurement is with ion-sensitive microelectrodes. Values in table were calculated using
reported ion activities and assuming an activity coefficient of 0.8.
† M. Lieberman, personal communication.
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
neously (306). Again, the control internal ionic conditions
were set to 0 mM [Cl2]i and 0 mM [Na1]i to prevent the
possibility of isotopic self-exchange for Na1 and Cl2. The
effects of two perturbations on the ratio of the flux pairs
were examined. One perturbation was to increase [Cl2]i
from 0 to 150 mM. This is a treatment known to inhibit
flux through the NKCC (see sect. IXB1). This protocol
yielded an apparent stoichiometry of 1.8 Na1:1 K1:3.3
Cl2. The second perturbation was to measure the ion-pair
influxes in the absence and presence of furosemide. The
furosemide-sensitive influxes had an apparent stoichiometry of 2.2 Na1:1 K1:3.1 Cl2. Thus both of these experiments yielded a stoichiometry of ;2 Na1:1 K1:3 Cl2
which suggests an “extra” Na1 and Cl2 are transported
per cotransporter turnover. It is noteworthy that the results of Figure 7 (which show the external ion dependence of the three ions) also support the notion that, in
the squid axon, the transport stoichiometry is 2 Na1:1
K1:3 Cl2.
A similar conclusion regarding the cotransport stoichiometry was reached by Hall and Ellory (122). They
measured isotopic uptake of 86Rb (for K1), 22Na, and 36Cl
into ferret RBC. The ferret RBC contains very high concentrations of Na1 (e.g., Refs. 69, 122) and is able to
engage in Na1/Na1 and Cl2/Cl2 self-exchange fluxes via a
partial reaction of its NKCC (215, 219). This raised the
possibility that some of the isotopic uptake fluxes of 22Na
and 36Cl measured by Hall and Ellory (122) did not represent a full turnover of the NKCC. Flatman (69) tested
for this possibility by measuring bumetanide-sensitive net
effluxes of Na1 and K1 into Na1- and K1-free solution.
With this experimental design, which prevented self-exchange fluxes, Flatman (69) showed that the ratio of
bumetanide-sensitive Na1-to-K1 efflux was about 1:1 in
the ferret RBC. With the assumption that the NKCC is
electroneutral in ferret RBC as has been shown for other
cells (see sect. VC), this implies an overall transport stoichiometry of 1:1:2 (Na1:K1:Cl2).
Thus, at the present time, every cell examined except
the squid giant axon appears to transport Na1, K1, and
Volume 80
January 2000
Na1-K1-Cl2 COTRANSPORT
VII. TRANSPORT MODEL OF THE SODIUMPOTASSIUM-CHLORIDE COTRANSPORTER
A. Evidence for Cooperative and Ordered Ion
Binding to the NKCC
Normal operation of the NKCC requires that 1 (or 2)
Na1,1 K1, and 2 (or 3) Cl2 must all bind to the NKCC on
the same side of the membrane before cotransport across
the cell membrane will occur (see sect. VA). Is the order of
binding of these cotransported ions random? Or is there a
preferred order of binding? A preferred order of binding
would occur from an allosteric effect when the binding of
one ion increases the apparent affinity of a binding site for
the next ion. This is a special form of cooperativity.
Experimental evidence for ordered binding can be obtained by performing activation studies for any one of the
cotransported ions, e.g., Cl2, at various concentrations of
the other two co-ions, e.g., Na1 and K1. Several such
studies have been reported for cotransport influx. In all
cases, the apparent external affinity for each of the cotransported ions increases with increased extracellular
concentrations of the co-ions (e.g., Refs. 33, 58, 125, 234,
295).
If, as seems very likely, the cotransported ions bind
in an ordered fashion, what is that order? Although the
question is simple, getting an answer has proven difficult.
Three groups have addressed this issue, and the result is
three somewhat different models.
The best-tested model to date resulted from an insightful use of the ion dependencies of self-exchange
fluxes mediated by the cotransporter in duck RBC (200,
217–219) and in human RBC (58). Briefly, these authors
made two key observations regarding the ionic requirements necessary to get either K1/K1 self-exchange or
Na1/Na1 self-exchange via the NKCC. They showed that
the NKCC of the duck RBC mediated K1/K1 exchange
when the extracellular medium contained only K1 and
Cl2 and the cytoplasm contained K1 at a high concentration as well as lesser, but finite concentrations of Na1 and
Cl2. On the other hand, Na1/Na1 exchange was favored
by a different set of internal and external ion compositions: the extracellular fluid needed to contain all three
co-ions while the internal fluid needed to contain only
Na1.
From this pattern of ionic requirements for NKCCmediated self-exchange fluxes, the binding order model
seen in Figure 11 was postulated by McManus and coworkers (217–219). The model assumes that only the fully
loaded and the completely unloaded form can “cross” the
membrane and that fully loaded “carriers” oscillate between inwardly and outwardly facing conformations
faster than do completely unloaded carriers. It assumes
that the NKCC can mediate either net influx or efflux of all
three co-ions, depending on the thermodynamic gradient
(see sect. VIB). It further assumes glide symmetry, which
means the first ion to bind on one side will be the first ion
to be released on the other. Net cotransport influx would
require that the ions bind to the cotransporter in the order
shown as steps 1– 4. Step 5 represents the reorientation of
the NKCC molecule such that the binding sites are now
accessible from the intracellular compartment, and steps
6 –9 represent the ordered release of the ions. Step 10 is
the reorientation of the completely unloaded cotransporter to an outwardly facing conformation. According to
this model, Na1/Na1 exchange involves only steps 1– 6,
followed by steps 6 –1. The high [Na1]i serves to prevent
net Na1 unloading at the internal site (only Na1/Na1
self-exchange can occur). On the other hand, K1/K1 exchange involves steps 3– 8 followed by steps 8 –3. The
high [K1]i serves to prevent the release of the second Cl2.
Duhm (58) also reported data obtained from human RBC,
most of which could be well-fit by this clever model.
Recently, this model has been quantitatively tested
using simulations (24). Flux data from duck RBC, HeLa
cells, and epithelial cells were fitted with differential
equations developed based on the Lytle-Haas-McManus
model. The experimental data were well fit by these equations.
However, there are nagging problems that suggest
that this model may not be the absolute final word on the
mechanism of ion binding by the NKCC. The model was
initially derived from, and explicitly predicts, exchange
fluxes for all the co-ions. Although these appear to be
present and prominent in duck RBC and in ferret RBC
(69), they are not universally described. For instance,
Kracke et al. (180) were unable to demonstrate K1/K1
exchange in human RBC. Kaji (160) also failed to demonstrate K1/K1 exchange in cultured mouse medullary thick
ascending limb cells. As already mentioned, there is no
evidence for Cl2/Cl2 or Na1/Na1 exchange in squid axon
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equilibrium. In a “typical” mammalian cell (e.g., [K1]i
5 140 mM, [K1]o 5 4.5 mM, [Na1]i 5 15 mM, [Na1]o
5 140 mM, [Cl2]o 5 110 mM), the thermodynamic equilbrium for the NKCC (with a stoichiometry of 1 K1:1 Na1:2
Cl2) would be with a [Cl2]i of ;60 mM. The 2:1:3 stoichiometry derives much more energy from the same ionic
gradients to pump Cl2 into the cell. In the case of the
squid giant axon, this stoichiometry yields an equilibrium
[Cl2]i of nearly 750 mM.
Consideration of the information in Table 3 raises the
question of why all cells that express the NKCC do not
have a [Cl2]i at or very near 60 mM. What prevents the
NKCC from achieving thermodynamic equilbrium? What
serves as the physiological “brake” on the NKCC in these
cells? Considerable evidence points to the phosphorylation state of the cotransporter protein and/or the level of
[Cl2]i (see sect. IX).
231
232
JOHN M. RUSSELL
Volume 80
(see sect. VIB). Other qualitative predictions of this model
have also not been verified. Milanick (232) pointed out
that the Lytle-McManus-Haas model predicts that high
[K1]o or [Cl2]o would have inhibitory effects on net efflux
via the NKCC, as well as on Na1/Na1 exchange fluxes. No
such effects of extracellular Na1, K1, or Cl2 have been
reported. Although increases of [Cl2]i have profound inhibitory effects on NKCC-mediated influxes (and effluxes;
see sect. IXB), the complete removal of extracellular Cl2
has only a modest stimulatory effect on NKCC-mediated
effluxes in the squid giant axon (8, 29). Of course, it is
very possible that different isoforms of the NKCC may
have somewhat different ion binding mechanisms or patterns.
If the glide symmetry model is correct, then the
simplest structural correlate would be that the Na1 binding site would be found deeper in the membrane than the
K1 site. Isenring et al. (149) have recently shown by
means of mutagenesis studies that the transmembrane
region apparently conferring Na1 binding properties is
located more superficially within the plane of the bilayer
than is the putative K1 binding site. Of course, this interpretation depends on the correctness of the topological
model (Fig. 1). It is of interest that this structural information suggests that the first ion to bind ought to be the
K1, and a recent squid axon flux study arrived at that
same conclusion (7).
Miyamoto et al. (234) have developed a different
ion-binding model using flux results obtained from studies on HeLa cells. In agreement with Lytle and McManus
(218), they postulate that Na1 binds before K1. However,
their model suggests that one Cl2 binds simultaneously
with the Na1 at a “low Cl2 affinity” site, and the second
Cl2 binds simultaneously with K1 at a “high Cl2 affinity”
site. In addition, they postulate mirror symmetry, i.e., that
debinding at the intracellular face occurs in the opposite
order as binding on the extracellular face. This model
does not account for Na1/Na1 or K1/K1 exchange (but
see above). Finally, Benjamin and Johnson (24) show that
the data of Miyamoto et al. (234) could be fit rather well
with the Lytle-McManus-Haas model.
McRoberts et al. (229) were able to describe much of
the kinetic properties of the NKCC found in the MDCK
cells (a canine kidney cell line) beginning with the assumption of random but cooperative binding of the ions
to the NKCC. This model predicts mutual dependence of
each ion’s apparent affinity on the concentrations of the
other two co-ions. At the same time, it predicts that the
maximum velocity (Vmax) values obtained would be unaffected by the co-ion concentrations. These predictions
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
FIG. 11. Hypothetical model of ordered ion binding with glide (“first on/first off”) symmetry. Model assumes that
cotransport is completely reversible and that only fully loaded and completely unloaded forms of cotransporter protein
are capable of changing orientation from inward-to-outward (or vice versa) facing. Numbers refer to order of binding,
reorientation, and release for one complete influx cycle. Because both binding and release are ordered, a high
concentration of substrate on release side can hinder release of subsequent ions. Thus a rise in [Na1]i would be expected
to prevent subsequent release of Cl2 and K1 by preventing step 7. This could be manifested as a reduction of net influx
or as an increase in Na1/Na1 exchange depending on exact ionic conditions. [Modified from Lytle et al. (219).]
January 2000
Na1-K1-Cl2 COTRANSPORT
were verified by the results of Rindler et al. (295) for
MDCK cells as well as by Miyamoto et al (234) for HeLa
cells.
Clearly, the issue of whether the NKCC binds ions in
a preferred order or simply displays cooperativity of binding is still far from resolved. Whether the differences
described above are the results of unsuspected experimental complications or are the result of different isoforms of the NKCC occurring in the different cells being
examined remains to be resolved.
B. Evidence That Cl2 Binding Sites
Are Nonequivalent
22
mM NO2
Na influx 40 – 60% as much as
3 increased the
2
adding 90 –100 mM Cl . Similarly, Kinne et al. (174)
using vesicles from the rabbit kidney outer medulla
2
showed that NO2
3 and SCN , by themselves, could only
22
support ;26% as much Na influx as an equal [Cl2]
when they were the only anions, but in the presence of
10 mM Cl2, 90 mM SCN2 was 73% as effective as 90 mM
Cl2 and NO2
3 ;55% as effective. These results have
been interpreted to mean that, in the presence of small
concentrations of Cl2, the NKCC can bind and transport other anions. This suggests that the sequence of
binding of the cotransported ions is Na1:Cl2:K1:A2,
2
where A2 is NO2
3 or SCN . If this interpretation is
correct, then there may be two distinct anion binding
sites whose binding characteristics differ (see sect.
VIIIB1 for more evidence of two distinct sites).
It is important to point out that not all workers have
2
been able to demonstrate such effects for NO2
3 and SCN .
Hegde and Palfrey (130), working with intact duck RBC,
found that neither of these anions could support NKCCmediated 86Rb influx in the presence of 10 mM Cl2. Indeed,
these workers showed both anions further inhibited the
bumetanide-sensitive 86Rb influx. Miyamoto et al. (234) reported that, in the complete absence of extracellular Cl2,
1
NO2
3 supported a small fraction of furosemide-sensitive Rb
2
2
uptake, but in the presence of Cl , NO3 inhibited NKCCmediated Rb1 uptake. These latter workers reported that
SCN2, even in the complete absence of Cl2, did not support
NKCC-mediated Rb1 uptake, and in the presence of Cl2,
SCN2 was inhibitory. Given what we are beginning to learn
about structure-function properties of the two isoforms, and
the existence of splice variants, it seems highly likely that at
least some of these functional differences will turn out to
reflect some structural differences.
There is additional evidence for the two Cl2 binding/
transport sites that may have quite different apparent
affinities. Brown and Murer (33) plotted their [Cl2]o (gluconate replaced Cl2) versus 86Rb uptake data with an
Eadie-Scatchard plot (used to evaluate kinetic parameters
of two enzymes using the same substrate, Ref. 317). This
plot suggested there were two Cl2 binding sites, one with
an apparent Km of 5.1 mM, and the other with an apparent
Km of 55.3 mM. They suggest the lower affinity site is the
2
one that NO2
3 binds to in the presence of Cl . Miyamoto
et al. (234), using intact HeLa cells, have presented data
that could be fit with a two-site model. The two sites had
significantly different affinities (24 and 103 mM).
Taken together, these results strongly suggest that at
least some of the isoforms of the NKCC have two distinct
Cl2 binding/transport sites, and they have rather different
binding properties. This implies a rather different structure for each of these two Cl2 sites. Verification of this
hypothesis awaits future structural studies.
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When all the external Cl2 is replaced by another
anion, only Br2 has been found to be about as effective at
supporting NKCC function [usually measured as bumetanide-sensitive K1(Rb1) uptake] as is Cl2. Gluconate and
sulfamate appear to be uniformly ineffective at supporting cotransport fluxes. For other anion replacements, the
story gets complicated. As discussed above, there is evidence that the NKCC binds the cotransported ions in the
order Na1:Cl2:K1:Cl2 and that the binding of each ion in
this sequence enhances the binding of the ion following it
in the sequence. This implies that the two Cl2 binding
sites might have different binding properties. Perhaps the
most compelling evidence for the Cl2 binding sites having
different properties is that some anions cannot support
NKCC fluxes when they are the sole anion available but
will stimulate NKCC-mediated fluxes in the presence of
low concentrations of Cl2, presumably by binding to one
of the Cl2 binding/transport sites.
One such anion is NO2
3 . Numerous studies have
shown that NO2
3 , when present as the only anion, will
not support NKCC ion transport (e.g., Refs. 86, 130,174,
311). However, several studies have demonstrated that,
in the presence of low, nonsaturating [Cl2], NO2
3 will
support some NKCC-mediated ion transport (33, 174,
234, 346). Brown and Murer (33) used vesicles from
apical membranes of LLC-PK1 cells and showed that
when external Cl2 was completely replaced by NO2
3 ,
there was no bumetanide-sensitive 86Rb uptake. However, in the presence of small concentrations of extracellular Cl2, NO2
3 was quite effective at supporting
NKCC cotransport. This manifested itself as a hyperbolic relation between [Cl2]o and NKCC-mediated 86Rb
2
uptake when NO2
substitute. In contrast,
3 was the Cl
when gluconate was used as the Cl2 substitute, the
relation between [Cl2]o and NKCC-mediated 86Rb uptake was sigmoidal. Using vesicles from rabbit parotid
glands, Turner and George (345) showed that NO2
3
alone could support NKCC-mediated 22Na influx ;10 –
25% as well as Cl2. However, in the presence of a
subsaturating concentration of Cl2, addition of 90 –100
233
234
JOHN M. RUSSELL
C. Cation Specificity
VIII. BUMETANIDE BINDING STUDIES
As discussed in section VB, inhibition of ion transport
by bumetanide or its congeners is a critical piece of evidence
that the responsible mechanism is the NKCC. Another important use of the loop diuretics was as a probe for the
NKCC protein. Forbush’s group (358) was ultimately able to
clone the NKCC from shark rectal gland as a result of having
first labeled a membrane protein with [3H]bumetanide. They
developed antibodies to this protein and then used the antibodies to screen an expression library for the bumetanidebinding protein. Their strategy started with the assumption
that [3H]bumetanide bound uniquely to the NKCC protein.
This assumption is based on findings that the binding of
bumetanide (and its congeners) to membranes have, in
many (but not all) cases, three properties. These properties
are consistent with the view that this binding is specifically
to one of the Cl2 binding sites of the NKCC involved in Cl2
transport across the membrane (e.g., Refs. 117, 173). They
are as follows: 1) bound molecules are displaced by other
members of the loop diuretic family with apparent affinities
that match their potency as inhibitors of cotransport fluxes;
2) Na1, K1, and Cl2 all must be present in the binding
medium in order for specific binding to occur; and 3) raising
[Cl2] in the binding medium above the optimal level necessary for binding results in a reduction of the specific binding.
A. Functional Evidence for Effects of External
Ions on Bumetanide Binding
1. Effects of external Cl2
Even before the recognition of a coupled NKCC
mechanism, some early workers examining the effects of
the loop diuretics in isolated perfused nephrons had sug-
gested that the loop diuretic furosemide, which carries a
net negative charge at physiological pH, might act by
competing with Cl2 for a transport site on the apical
membrane of kidney thick ascending limb cells. The results of the initial experiments designed to test this idea
were inconclusive (35). However, a little later, Ludens
(211) using the toad cornea as a model for renal thick
ascending limb cells showed that the ability of furosemide
to block the short-circuit current (which depends on an
apical membrane NKCC) was reduced as the [Cl2]o was
increased. When these results were plotted as a doublereciprocal plot, the characteristic relationship of a competitive inhibitor was revealed. The fitted Km for Cl2 in
the absence of furosemide was 53 mM; in the presence of
22 mM furosemide, the apparent Km for external Cl2 rose
to 145 mM.
Haas and McManus (117) reexamined this issue by
measuring norepinephrine-stimulated net uptake of Rb1
(as a K1 surrogate) and Li1 (as a Na1 surrogate) into
duck RBC as a function of [Cl2]o. Just as Ludens (211)
had shown, they found that increasing [Cl2]o had the
effect of reducing the apparent affinity for bumetanide
(see Fig. 12). Similar results showing apparent competitive interaction between loop diuretics and Cl2 have been
reported for several other preparations (e.g., shark rectal
gland plasma membranes, Ref. 173; chick cardiac cells,
Ref. 77). It should be pointed out that these data cannot
be fit by a simple Michaelis-Menten relationship. However, if these data are plotted as percent inhibition versus
bumetanide concentration, they can be fitted with a Hill
equation. In this case, the apparent Hill coefficients are
considerably ,1 (for [Cl2] 5 20 mM, the Hill n 5 0.46; for
[Cl2] 5 100 mM, n 5 0.55). This implies a more complex
(and less direct) interaction between bumetanide and the
NKCC protein than a one-to-one binding competition.
Thus there may be allosteric interactions that account for
the observed effects. From a standpoint of the practical
use of these agents, it should be noted that at normal
[Cl2]o values, concentrations of bumetanide above 1–10
mM are likely to be fully inhibitory. Interestingly, this
concentration of bumetanide is also fully inhibitory for
the squid NKCC, where [Cl2]o is 560 mM.
2. External Na1 and K1 effects
Palfrey et al. (264) examined the effects of reducing the
extracellular concentrations of Na1 or K1 on cAMP-stimulated 86Rb influx into turkey RBC. They reported that such
reductions actually shifted the bumetanide dose-response
curves to the right, i.e., reduced the apparent affinity for the
inhibitor. Kracke et al. (180) also demonstrated a clear dependence of the degree of furosemide inhibition of the human RBC NKCC on the concentration of extracellular K1.
These results suggest that the cotransported cations are
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It has been shown in several preparations that Rb1 is
as effective or nearly as effective as K1 in supporting
fluxes via the NKCC (e.g., Refs. 33, 130). It has also been
reported that Cs1 can partially substitute for K1 (e.g.,
Refs. 86, 130) and that Tl1 may actually be more effective
than K1 at supporting the cotransport fluxes (86). Among
physiologically relevant cations, NH1
4 may substitute at
the K1 site (duck RBC, Ref. 130) particularly for the
NKCC2 isoform in the thick ascending limb of the kidney
(15, 96, 174). This may play an important role in the
nephron for NH1
4 recycling, which is important for excretion of excess acid. Sodium is completely ineffective at
replacing K1 on the cotransporter. The Na1 site may
accept Li1 to a certain degree (e.g., Refs. 117, 130, 311).
No other cation has been reported to substitute for Na1
on the NKCC.
Volume 80
January 2000
Na1-K1-Cl2 COTRANSPORT
235
necessary for loop diuretic inhibition and, presumably, for
binding of the diuretics to the NKCC.
The fact that high [Cl2] seemed to reduce the apparent affinity for bumetanide, whereas Na1 and K1 seemed
to increase it, has been interpreted in light of the ordered
ion-binding hypothesis (see sect. VIIA). This led to the
view that bumetanide competes for the second of two Cl2
transport binding sites. This site is also believed to have a
lower Cl2 affinity and to be less selective among anions
than the “first” site (see sect. VIIB; but also see Ref. 149).
Thus the thought is that bumetanide and Cl2 compete for
the second Cl2 binding site following the binding of Na1,
the first Cl2 and then a K1. When bumetanide binds to the
“second” Cl2 site, cotransport is blocked. Alternatively,
these results are consistent with a mechanism in which
the loop diuretics bind to some other anion binding site
on the NKCC whose affinity is affected (allosterically) by
the presence of Na1, K1, and Cl2.
B. [3H]Bumetanide Binding Studies
Forbush and Palfrey (73) synthesized and studied in
detail [3H]bumetanide and [3H]benzmetanide binding to
membranes isolated from the outer medulla of the dog
kidney. Given the region of the kidney from which the
membranes were prepared and the properties of the membranes to which the loop diuretics preferentially bound,
there is good reason to believe they were studying the
binding properties of the apical membrane of thick as-
cending limb cells. Northern and in situ hybridization
experiments have subsequently shown that this region of
the kidney contains the absorptive or NKCC2 isoform of
the cotransporter (79, 143, 280).
They demonstrated a saturable component of reversible [3H]bumetanide binding to these membranes.
A Scatchard analysis of the binding data was consistent
with one bumetanide molecule per binding site. They
showed that, at 22°C, binding had a half-time of ;5 min
and a dissociation half-time of ;10 min. (At 0°C, dissociation had a half-time of .120 min.) From the association and dissociation rates they calculated an equilibrium binding constant for bumetanide of 4.9 3 1028
M. These determinations were performed in a medium
that contained 30 mM each of Na1, K1, and Cl2. Further evidence that they were studying binding to the
site that effects transport inhibition came from results
of displacement studies using other members of the
sulfamoylbenzoic acid loop diuretic family (e.g., furosemide, benzmetanide). These agents displaced [3H]bumetanide binding with apparent affinities that were in
the same rank order as those reported to effect inhibition of transport in other preparations such as avian
RBC (probably the NKCC1 isoform) or rabbit kidney
thick ascending limb of the loop of Henle (probably the
NKCC2 isoform). The rank order of displacement was
benzmetanide . bumetanide . furosemide . p-methoxybenzmetanide . 3-amino-4-phenoxy-5-sulfamoylbenzoic acid.
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2
FIG. 12. Effect of varying external [Cl ]
([Cl2]o) on bumetanide inhibition of Cl2-dependent uptake of Rb1 and Li1 in duck red
blood cells stimulated with norepinephrine.
Lines were drawn by a curve-fitting program
that also calculated IC50 values shown. Difference in IC50 for 2 curves was statistically significant (P , 0.001). [From Haas and McManus (117).]
236
JOHN M. RUSSELL
Volume 80
1. Ion requirements for bumetanide binding
In view of the functional evidence cited previously
for interaction among the three cotransported ions and
inhibition of the NKCC by the loop diuretics, Forbush and
Palfrey (73) performed a series of studies that examined
the effect of independently varying each transported ion
on saturable [3H]bumetanide binding. Figure 13 shows the
results. Several important points emerged from this study.
First, in the absence of any one of the three cotransported
ions, [3H]bumetanide binding was reduced to no more
than 10 –20% of maximal binding levels (i.e., binding measured in the presence of all 3 ions). Second, both Na1 and
K1 activate [3H]bumetanide binding, with the relationship
between [3H]bumetanide binding and both [K1] and [Na1]
being monotonic, saturating functions. This result has
been cited as further support for a 1:1 Na1:K1 stoichiometry of NKCC cotransport (see sect. VIA1). Third, the
effects of Cl2 on [3H]bumetanide binding were biphasic.
Very low concentrations of Cl2 were necessary for maximal binding, but increasing [Cl2] above 4 mM resulted in
a significant reduction of diuretic binding. Physiological
levels of [Cl2] reduced the binding by .50% when the
membranes were exposed to a subsaturating concentration (2.3 3 1027 M) of bumetanide. This result was generally interpreted to mean that there are two Cl2 sites; the
first site must bind a Cl2, and the second site is where Cl2
and bumetanide compete for binding.
The general pattern of overall ion effects on [3H]bumetanide binding to the putative NKCC protein has been
reported by several investigators for a variety of cells
(e.g., Refs. 106, 111, 126, 130, 160, 255, 256, 287, 345). Only
one significant deviation from this overall pattern of iondependent [3H]bumetanide binding to putative NKCC proteins has been reported. Altamirano et al. (9) studied
binding to squid optic lobe microsomes and were unable
to demonstrate any Na1 requirement for binding, although the effects of K1 and Cl2 were qualitatively as
reported for all other preparations. Whether this constitutes additional evidence that the squid NKCC is a different isoform (squid NKCC has a different transport stoichiometry also; see sect. VIA2) remains to be determined.
The forgoing results established ion-dependent
[3H]bumetanide labeling as a potentially important tool
for studying the NKCC. There have been a large number
of studies using labeled loop diuretics (mainly [3H]bumetanide) in a variety of tissues examining what we now
know to be both of the currently identified isoforms of the
NKCC. Virtually all the results agree that specific [3H]bumetanide binding is enhanced by the presence of all three
cotransported ions and can be competed off by other
members of the loop diuretic family with a characteristic
relative potency sequence (e.g., benzmetanide . bumetanide . piretanide . furosemide, Ref. 269). However,
there are numerous differences in other details regarding
the properties of diuretic binding. Some of these differences undoubtedly relate to true differences in properties
of different isoforms; others may relate to differences in
tissue or membrane purity or unidentified differences in
experimental conditions.
Another possible reason for some of the aforementioned differences may have to do with the assumed
near-absolute specificity of the binding. As a result of the
forgoing studies, it has been widely accepted that the
three key fingerprints for the specificity of diuretic binding to the NKCC protein are 1) demonstration that the
putative site binds the diuretics with relative binding affinities of benzmetanide . bumetanide . piretanide . furosemide; 2) binding to the putative NKCC site must
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3
FIG. 13. [ H]bumetanide binding is a
function of [Na1], [K1], and [Cl2]. Membranes prepared from dark red outer medulla of dog kidneys were incubated with
[3H]bumetanide (0.25 mM) for 20 min in
presence of various salts. Unless otherwise
indicated, ion concentrations were 128 mM
Na1, 64 mM K1, 128 mM Cl2, and 32 mM
SO22
4 . In each experiment, concentration
of only one ion was varied. f, K1 substituted with choline; M, Na1 substituted with
choline; ‚, Cl2 substituted with SO22
4 .
Each curve is a separate experiment and
has been normalized to maximal binding in
that experiment. In the cases of both Na1
and Cl2 studies, ion concentration was increased to 128 mM, as shown by 2 data
points at extreme right of figure. ‚, Cl2
substitution data from a separate experiment in which membranes were isolated in
a Cl2-free medium (i.e., at 0 on abscissa).
1
M, Na replacement in an experiment with
10 mM K1 and 15 mM Cl2 instead of 64
mM K1 and 128 mM Cl2 (i.e., at 0 on abscissa). [From Forbush and Palfrey (73).]
January 2000
Na1-K1-Cl2 COTRANSPORT
2. Use of [3H]bumetanide binding to identify the
NKCC protein
Having identified a class of compounds that displayed reasonable selectivity for inhibiting NKCC function, and whose binding to cell membranes has, for the
most part, exhibited the three properties consistent with
binding to the NKCC protein, the obvious next step was to
use these agents to label and identify the diuretic-binding
protein. The problem was that bumetanide and its congeners do not bind covalently so that the harsh procedures
necessary to isolate and identify membrane proteins
would result in release of the reversibly bound tritiated
ligand. The solution to this problem of reversible binding
was to cause the tritiated diuretics to bind covalently by
irradiating the membranes with ultraviolet light while
they were in the presence of the labeled bumetanide
congener.
Several studies have shown that this treatment results in a low level of covalent binding of the tritiated
diuretic to membrane proteins. Because it is entirely possible that the covalent reaction resulting in the irreversible nature of the binding could occur at a site quite
different from that at which reversible binding occurs, it
follows that it is important to demonstrate that irreversible binding is accompanied by irreversible inhibition of
cotransport fluxes. Even this does not guarantee that the
covalent binding is to the NKCC protein. For example, it
could bind a neighboring protein but be held in position to
maintain inhibition of the NKCC. However, if irreversible
binding does not result in irreversible inhibition, this
would lead to serious doubts as to the location of the
irreversible binding.
The first convincing demonstration that near-ultraviolet (UV) irradiation (l 300 – 400 nm) would result in an
irreversible inhibition of NKCC-mediated flux was made
by Amsler and Kinne (17). They exposed LLC-PK1 cells (a
cell line derived from pig kidney) bathed in bumetanidecontaining solution to near-UV light. This treatment
caused a selective inhibition of the NKCC-mediated 86Rb
influx with little or no effect on sodium pump-mediated
influx or on the ouabain- and bumetanide-insensitive influx as long as the bumetanide concentration at the time
of UV exposure was ,5 mM. Exposure of the cells to the
same near-UV irradiation in the absence of bumetanide
was without effect. Thus this result shows that photoactivation of bumetanide will result in an irreversible inhibition of the cotransporter functionally without obvious
effects on other transport processes.
The definitive study using irreversible loop diuretic
binding was that of Haas and Forbush (112). In that study,
the authors made use of the fact that one congener of
bumetanide, 4-benzoyl-5-sulfamoyl-3-(3-phenyloxy)benzoic acid (BSTBA), contains a photoactivatable group,
benzophenone. Their study used membranes prepared
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depend on Na1, K1, and Cl2 as described above; and 3)
raising [Cl2] above a critical level must result in a reduction of specific [3H]bumetanide binding.
However, the literature of this area provides at least
one clear example that those three criteria alone are
inadequate to guarantee the identity of the diuretic binding protein. The Tamm-Horsfall protein (338) is found in
the urine of many mammals (312). Immunohistochemical
studies showed that it is expressed in the distal regions of
the nephron, especially in the luminal membrane of the
thick ascending limb of the loop of Henle, which, not
coincidentally, is the site of the NKCC2 isoform. It is a
highly glycosylated protein with a molecular weight of
;100,000. Soon after Forbush and Palfrey (73) reported
the properties of [3H]bumetanide binding to dog kidney
membranes prepared from predominantly thick ascending limb of the loop of Henle cells, a report appeared on
a study of labeled furosemide binding to purified TammHorsfall protein (105). There was a remarkable similarity
between the key properties of the [3H]bumetanide binding
reported by Forbush and Palfrey (73) and those of furosemide binding to the Tamm-Horsfall protein. Thus
[14C]furosemide binding was greatly enhanced by the
presence of Na1, K1, and Cl2 to the binding media.
Raising the [Cl2] in the binding medium above a critical
level led to reduced furosemide binding. Also, it was
reported (but not shown) that other furosemide congeners displaced the [14C]furosemide. Thus, qualitatively,
this binding met the general criteria for binding to the
NKCC. Quantitatively, there were some differences from
those reported by Forbush and Palfrey (73). For instance,
the apparent mean affinity constant (K0.5) for stimulation
of diuretic binding is lower for Na1 than for K1, an order
opposite to that usually observed, and the inhibitory effect of Cl2 was not observed until [Cl2] was greater than
100 mM, a much higher value than is usually reported.
Finally, Santoso et al. (312) using immunohistochemical
techniques showed a lack of correlation between the
location of the Tamm-Horsfall protein and known locations of transport via the NKCC. The recent success of
cloning and expressing the NKCC has clearly shown that
the Tamm-Horsfall protein is not the NKCC, yet the uncanny similarity of the diuretic binding properties of this
protein to those of the NKCC should serve as another
warning against the use of reversible ligands for the unequivocal identification of proteins.
Nevertheless, the use of labeled loop diuretic congeners has led to important insights about three aspects of
NKCC structure/function: 1) to identify the NKCC protein,
which, in turn, led to one of the successful strategies for
cloning the NKCC; 2) to identify the activated state of the
NKCC protein; and 3) to study the site of loop diuretic
binding.
237
238
JOHN M. RUSSELL
3
FIG. 14. Evidence of photoactivated, irreversible binding of Hlabeled 4-benzoyl-5-sulfamoyl-3-(3-phenyloxy)benzoic acid (BSTBA)
binding to membranes prepared from dog kidney cortex. Top: a densitometric scan of SDS-polyacrylamide gel of [3H]BSTBA-photolabeled
membranes just before cutting gel into slices for scintillation counting.
Arrows indicate position of molecular mass standards (from left to right,
in kDa) of 200, 116, 92, and 66, and last arrow is tracking dye. Bottom:
distribution of [3H]BSTBA in gels of membranes incubated in 0.27 mM
[3H]BSTBA and exposed to UV light. Solid symbols and heavy lines refer
to membranes in absence of 10 mM bumetanide, and open symbols and
thin lines represent data from membranes prepared in presence of
bumetanide. Gels were cut into 4-mm slices, digested, and counted.
[From Haas and Forbush (112).]
3. Use of [3H]bumetanide binding to identify the
activated state of the NKCC
An underlying hypothesis behind much of the published work on specific binding of the loop diuretics is
that the sulfamoylbenzoic acid diuretics act by binding to
a Cl2 transport site on the NKCC protein. In addition, it is
postulated that the ion transport sites are only available
for binding when they are in an activated state (presumably phosphorylated; see sect. IXA2A). A useful corollary
of this hypothesis is that [3H]bumetanide binding studies
could be used to measure the levels of activated NKCC
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from the cortex of dog kidneys (a site of NKCC1), either
a crude membrane preparation or fractions of membrane
prepared by centrifugation through a sucrose gradient.
They demonstrated that BSTBA could competitively displace [3H]bumetanide from these membranes with an
apparent affinity nearly identical to that of bumetanide
itself. They exposed these membranes bathed in a solution containing 0.27 mM [3H]BSTBA plus Na1, K1, and Cl2
to UV light (l 5 366 nm), in the absence and presence of
10 mM unlabeled bumetanide. They then separated the
proteins in these membranes using polyacrylamide gel
electrophoresis as seen in Figure 14. Of the three peaks of
[3H]BSTBA labeling observed, only one (the peak centering around gel slice number 15) is blocked by the unlabeled bumetanide. The molecular mass of the protein
centered in this region was ;150 kDa. They did observe
another peak of labeling in the 50- to 60-kDa region of the
gel (gel slice number 30). Several observations argue
against this peak being the NKCC protein. First, cold
bumetanide did not compete it off. Second, binding to this
peak was linear up to 1.2 mM, whereas the binding to the
150-kDa region saturated with an apparent half-saturation
constant of 0.094 mM. Third, binding to the 50- to 60-kDa
protein was largely unaffected by omission of K1 and Cl2
(but not Na1) from the media. Thus not all three requirements were met in the case of the 50- to 60-kDa protein.
Could it be a proteolytic fragment of the NKCC? That also
seems unlikely since the authors showed that the 50- to
60-kDa protein binding came predominantly from much
lighter membrane fragments than did the 150-kDa protein.
Interestingly enough, the authors mention that when they
photolabeled membranes using [3H]bumetanide, they, like
Jørgensen et al. (159), found labeling only in the lowmolecular-weight region. No functional evidence of irreversible inhibition caused by UV irradiation was given for
either the BSTBA or the bumetanide.
Forbush and co-workers have used photoactivated,
irreversible binding of [3H]BSTBA to study the NKCC of
two other preparations, duck RBC (113), and the shark
rectal gland (72, 213–215). The results of these later studies agreed well with the original study in terms of providing several lines of evidence that the irreversible binding
was to a protein that represented at least a part of the
NKCC protein. In addition, two new pieces of information
were obtained. First, Haas and Forbush (113) demonstrated that BSTBA would irreversibly block NKCC-mediated fluxes in parallel with its binding to the 150-kDa
protein. Second, in the shark rectal gland, they identified
the binding protein as a 195-kDa protein that was present
in great abundance. This permitted Forbush and co-workers (213, 221) to partially purify the protein and use it to
produce several monoclonal antibodies. These antibodies
were, in turn, used to screen a shark rectal gland cDNA
library and isolate the shark rectal gland NKCC cDNA
(358).
Volume 80
January 2000
Na1-K1-Cl2 COTRANSPORT
by treatment with phalloidin without any effect on the
level of [3H]bumetanide binding. Hegde and Palfrey (130)
showed that both [3H]bumetanide binding and 86Rb influx
into duck RBC were stimulated by raising the pH of the
external medium from 6 to 8. However, the degree of
stimulation of the two properties was quite different. To
further illustrate this, the results of Haas and McManus
(118) and Haas and Forbush (111) have been combined in
Figure 15. These two papers reported the effects of
shrinking on duck RBC in the absence or presence of 1026
M norepinephrine. Figure 15 shows that although norepinephrine increases both ion fluxes and [3H]bumetanide
binding, the changes are not proportional.
These observations may suggest some error in the
hypothesis (e.g., bumetanide does not bind to a Cl2 cotransport site, but to some other anion binding site on the
NKCC) or faulty experimental design. In many cases, the
measurements of flux and bumetanide binding were made
in separate studies and were not designed to test the
hypothesis per se.
Thus, as pointed out by Haas and McBrayer (115), the
use of [3H]bumetanide binding to monitor NKCC activity
is not fully proven. Nevertheless, it has been a useful tool
to demonstrate that a number of agents that either stimulate or inhibit cotransport activity also similarly affect
diuretic binding.
4. Use of [3H]bumetanide binding to study the
interaction between Cl2 and bumetanide
3
1
FIG. 15. Comparison of [ H]bumetanide binding and Na flux in
duck red blood cells exposed to a range of external osmolalities either
in presence or absence of 1026 M norepinephrine (N-E). Exposure to
different osmolalities caused cell volume (cell water) to vary on either
side of normal value of 1.49 kgH2O/kg cell solids. All data were normalized to value obtained at normal cell volume. In absence of N-E, relative
changes of [3H]bumetanide binding (F) and Na1 flux (‚) increase caused
by cell shrinkage were parallel. However, in presence of N-E, [3H]bumetanide binding (f) is enhanced much more than Na1 flux ({). [Flux
data from Haas and McManus (118); binding data from Haas and Forbush (111).]
As discussed in section VIIIA1, it is postulated that
bumetanide binding requires the first Cl2 to bind to a
transport site and then bumetanide competes with Cl2 for
the second Cl2 transport site (e.g., Refs. 110, 117, 173).
Thus it has been shown that Na1 and K1 and low concentrations of Cl2 increase the affinity of the NKCC for
both Cl2 binding and bumetanide binding. However, Griffiths and Simmons (106), working with rabbit renal cortical membranes, showed that although inclusion of all
three ions increases the total specific [3H]bumetanide
binding, it does not increase the apparent affinity for such
binding.
Turner’s group (237, 345) has specifically addressed
the issue of whether bumetanide competes with Cl2 for a
transport site or whether bumetanide binds to another
site (or sites) for which Cl2 may have an affinity. In the
first of these studies, Turner and George (345) directly
compared the effects of a group of anions on both NKCCmediated ion fluxes and [3H]bumetanide binding. Using
basolateral membrane vesicles prepared from rabbit parotid gland, they characterized the ionic requirements for
[3H]bumetanide binding and found, as others have (see
above), that Na1, K1, and Cl2 must all be present for
specific bumetanide binding to occur. Furthermore, they
showed that binding was maximal when [Cl2] is 5 mM; an
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under a variety of conditions. Convincing evidence for
this hypothesis requires, as a minimum, the demonstration that the Vmax for NKCC ion transport activity and the
maximum binding (Bmax) for [3H]bumetanide binding
both change in the same direction and by a proportional
amount in response to a given stimulus. There are very
few studies in which both measurements have been made
under identical conditions.
Nevertheless, there is considerable qualitative evidence showing that stimuli that increase cotransport flux
often increase the level of [3H]bumetanide binding (e.g.,
Refs. 76, 111, 118, 162, 257), although not always proportionately. There are also notable exceptions to this generalization. For example, two groups (175, 249, 250) have
shown that although bradykinin significantly enhances
cotransport fluxes in vascular endothelial cells, it has no
effect on [3H]bumetanide binding. Slotki et al. (327) have
shown that an 8-h incubation of HT-29 cells with forskolin
increases the bumetanide-sensitive 86Rb influx by two- to
fourfold without changing the Bmax of [3H]bumetanide
binding. In T84 cells, Mathews et al. (228) showed that
cAMP-stimulated NKCC flux activity could be attenuated
239
240
JOHN M. RUSSELL
4. Comparison of the effects of a series of anions
on supporting cotransport fluxes and inhibiting
[3H]bumetanide binding
TABLE
Anion
Relative BumetanideSensitive Flux in
Presence of 50 mM
Anion
Relative Inhibition of
[3H]bumetanide Binding in
Presence of 5 mM Anion
Br2
Cl2
NO2
3
F2
Formate2
Acetate2
SO22
4
Gluconate2
1.26
1.0
0.39
0.10
0.009
0.0
0.0
0.0
1.04
1.0
0.8
0.0
0.01
0.09
0.72
0.12
additional increase of [Cl2] resulted in a reduction of
specific binding. They next examined the effect on [3H]bumetanide binding of having 5 mM [Cl2] present plus 95
mM of the test anions. In a parallel study, they examined
bumetanide-sensitive 22Na uptake from a media containing 50 mM Cl2 plus 100 mM of the test anions. Table 4
compares the relative effectiveness of this series of anions at either supporting cotransport in the presence of a
subsaturating [Cl2] for transport or at inhibiting [3H]bumetanide binding in the presence of an optimal [Cl2] for
binding. For four of the eight anions, Br2, Cl2, NO2
3 , and
formate, the relative order of effectiveness for the two
functions is the same, although there are quantitative
differences. However, for the other four anions, the differences are extreme. Sulfate, acetate, and gluconate do
not support cotransport either alone (not shown) or in the
presence of 50 mM Cl2, yet they displace [3H]bumetanide.
In particular, sulfate is nearly as effective as Cl2 at displacing [3H]bumetanide from its binding sites. Conversely, fluoride with a small, but finite ability to support
cotransport fluxes has no effect on diuretic binding. This
suggests that the second anion binding site for cotransport may have very different properties from the [3H]bumetanide binding site, a finding which casts doubt on the
view that they are one and the same site.
Turner and co-workers (237) followed up this study
by showing that whereas formate alone could monotonically stimulate [3H]bumetanide binding, SO22
4 could only
inhibit such binding. Furthermore, when 50 mM SO22
4 was
added to 5 mM Cl2, [3H]bumetanide binding was considerably reduced. On the other hand, 50 mM formate in the
presence of 5 mM Cl2 greatly stimulated [3H]bumetanide
binding. Neither of these anions had any effect on [3H]bumetanide dissociation. They interpret their findings as
supporting a model in which there are two intracellular
anion sites (separate from the cotransport sites) that
influence bumetanide binding. One is a relatively highaffinity site that stimulates bumetanide binding, hence the
need for low concentrations of Cl2 for [3H]bumetanide
binding. The second is a relatively low-affinity site that
inhibits [3H]bumetanide binding. The authors postulate
that this anion site that inhibits [3H]bumetanide binding
may be the same as that which inhibits NKCC flux. In this
regard, it should be noted that intracellular SO22
4 had only
modest inhibitory potency against NKCC influx. Regardless of whether the bumetanide-binding inhibitory site is
the same as the NKCC flux inhibitory site, it seems clear
that the effect of Cl2 to inhibit [3H]bumetanide binding is
unlikely to be mediated through competition with Cl2 for
a cotransport site.
Further evidence suggesting that the bumetanide binding site is likely to be different from the Cl2 binding/transport sites comes from work of Hegde and Palfrey (130).
They studied [3H]bumetanide binding to duck RBC membranes as a function of [Cl2]. They found, as many others
have found, that increasing [Cl2] above a certain level (15
mM in this case) results in a reduction of the diuretic binding. However, when they plotted these data several ways,
they found that raising [Cl2] caused a reduction in the Bmax
with no change of KD. This is indicative of noncompetitive
inhibition, which also suggests that bumetanide does not
bind to a Cl2 binding/transport site, but rather to another
site (cf. Refs. 148, 149). In addition, they point out that the
second Cl2 binding/transport site has a halide selectivity
sequence (I2 . Br2 . Cl2) that is similar to anion selectivity sequence 1 (357). Because this is a common sequence for
many anion binding proteins in biological systems, it could
not account for the relatively high selectivity bumetanide
has for the NKCC.
Recently, Isenring and co-workers (148, 149) have
presented structural evidence showing that different regions of the NKCC1 molecule are responsible for binding
of bumetanide and Cl2. They studied effects on ion and
bumetanide binding caused by making chimeras and
point mutations of the putative transmembrane regions of
shark and human NKCC. They showed that TM2 is critical
for both Na1 and K1 binding and cotransport, TM4 is
critical for K1 and Cl2 binding and cotransport, whereas
TM7 is critical for all three ions. On the other hand,
bumetanide binding is critically dependent on TM2, TM11,
and TM12. These results are consistent with the view that
the bumetanide binding site(s) is not the same as those
for Cl2 transport.
IX. REGULATION/MODULATION
OF COTRANSPORTER ACTIVITY
A. Role of ATP
The NKCC is an example of secondary active transport, i.e., a membrane protein-mediated solute transport
mechanism that derives its energy from the combined
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Data from Turner and George (345).
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Na1-K1-Cl2 COTRANSPORT
chemical gradients of the transported ions. Nevertheless,
as we shall see below, ATP plays a fundamental role in the
operation of the NKCC.
1. Evidence for a direct role of ATP
nally dialyzed squid giant axon (303). In this preparation,
ATP concentration can be varied while maintaining normal ion gradients, intracellular pH (pHi), and cell volume,
something not possible in other preparations. In a series
of studies it was shown that reduction of ATP concentration to ,10 mM resulted in 1) complete inhibition of
furosemide/bumetanide-sensitive, external Na1-, external
K1-dependent Cl2 influx (5, 303, 306); 2) complete inhibition of furosemide/bumetanide-sensitive, external K1and external Cl2-dependent, Na1 influx (303, 304, 306); 3)
complete inhibition of furosemide/bumetanide-sensitive,
external Na1- and external Cl2-dependent K1 influx (304,
306); 4) complete inhibition of bumetanide-sensitive, internal K1- and internal Na1-sensitive Cl2 efflux (8); 5)
complete inhibition of bumetanide-sensitive, internal K1and internal Cl2-dependent, Na1 efflux (8). The foregoing
results clearly demonstrate the obligatory nature of the
ATP requirement for unidirectional fluxes via the reversible NKCC found in the internally dialyzed squid giant
axon. The relationship between ATP concentration and
bumetanide-sensitive 36Cl influx was hyperbolic with an
apparent K0.5 for ATP of ;90 mM (5). It is important to
note that the inhibitory effect of ATP deprivation develops as rapidly as intracellular ATP is washed out. Thus
either a “very long-lived” phosphorylated species is not
involved or a protein phosphatase rapidly dephosphorylates the active species. The latter would imply a phosphorylated system with a relatively rapid turnover. Finally, the inhibition by ATP depletion is readily and fully
reversible.
Experiments with the, b,g-methylene analog of ATP
(a “nonhydrolyzable” analog) showed that this analog
would not support NKCC but could apparently compete
with normal ATP for the activation of the cotransporter
(304). In the presence of cellular ATP, intracellular vanadate (0.3 mM) had no effect on furosemide-sensitive K1 or
Cl2 influx (306). Thus, although the cotransporter requires ATP, it seems highly unlikely that ATP activates
NKCC via an E1-E2-type ATPase.
Further early evidence for a role for ATP in the
operation of the cotransporter was provided by Saier’s
group. They demonstrated that MDCK cultured cells possess an NKCC and that reducing cellular ATP concentration to ;3% of control (probably ;30 – 60 mM) was sufficient to abolish the K1-dependent 22Na and the Na1dependent 86Rb uptake (229, 295). Subsequently, ATP
requirements have been demonstrated for NKCC in human RBC (3, 48), ferret RBC (70, 122), turkey erythrocytes
(268, 347), and human cultured colonic cells (169). In the
case of the human RBC, inhibition of furosemide-sensitive
(1 mM) Na1 and K1 fluxes was incomplete. The reported
ATP levels were ,100 and 30 –50 mM, respectively.
There is now widespread agreement that ATP is necessary for cotransport. It is therefore somewhat surprising that there are several reports of NKCC-mediated ion
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In their seminal paper, Geck et al. (90) struggled with
the possible role of ATP. They had reported earlier (131)
that metabolic inhibition (treatment with antimycin A)
prevented the kind of ion movements that they were now
showing to be via the cotransporter. In their 1980 study,
they attempted to distinguish between primary and secondary active transport by plotting furosemide-sensitive
water flow into the Ehrlich cells as a function of the
calculated free energy in the chemical gradients of Na1,
K1, and Cl2, using a stoichiometry of 1 Na1:1 K1:2 Cl2.
This analysis appeared to show that insufficient energy
resided within the chemical gradients of the three cotransported ions to account for the observed net transport. They pointed out that their analysis rested on two
unverified assumptions: 1) that the ionic activity coefficients in the cytoplasm and the incubation medium were
the same and 2) that the ions were not compartmentalized
within the cell. The actual raw ion concentration data
used by Geck et al. (90) to calculate the relationship were
not given in the paper. It therefore becomes a moot point
that there is some evidence from studies with ion-selective microelectrodes that the intracellular activity coefficients for Na1 and K1 are lower than their extracellular
activity coefficients (49). The data given simply do not
permit one to correct for low activity coefficients nor test
whether another Na1, K1, Cl2 stoichiometry would better
fit the data.
Geck et al. (90) tested for the stoichiometric utilization of ATP by NKCC by asking whether furosemidesensitive net fluxes caused ATP hydrolysis or stimulated
cellular glycolysis. First, they inhibited ATP production
from mitochondrial oxidative phosphorylation using antimycin A and measured the rate of decline of cellular ATP
content. Treatment with ouabain (an inhibitor of the
Na1-K1 pump) slowed down the rate of ATP depletion by
a factor of ;2. However, furosemide treatment had no
measurable ATP-sparing effect either with or without
ouabain. Because the magnitudes of the NKCC fluxes and
the Na1-K1 pump fluxes are comparable, the authors
concluded that ATP hydrolysis did not provide the “missing” energy for NKCC. In addition, they were unable to
demonstrate any furosemide-sensitive changes in glycolytic lactate production. Thus the role of ATP in the NKCC
mechanism of Ehrlich cells was unclear, although Dr.
Heinz stated during a discussion at a meeting that the
energy source for NKCC cotransport must be something
other than ATP (85).
The first direct demonstration that a cation-Cl2 cotransporter required cellular ATP was made in the inter-
241
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JOHN M. RUSSELL
fluxes using membrane vesicle preparations in which ATP
was not added to the intravesicular fluid (33, 80, 173, 174,
345, 353). It is possible that these vesicles contained
compartmentalized ATP (230). Alternatively, the process
of vesicle preparation may have “frozen” the NKCC in a
phosphorylated state, e.g., perhaps the protein phosphatase(s) responsible for dephosphorylation (see sect.
IXA2C) are cytoplasmic and lost in the process of vesicle
preparation.
2. Possible mechanisms of ATP activation
axon are examples of such processes. Perhaps even more
surprising is the finding that sugar uptake in RBC (a
facilitated diffusion process, e.g., no movement against a
gradient) is also modulated by cellular ATP (132). For the
Na1/Ca21 exchange and Na1-glutamate uptake systems in
squid axon and the glucose uptake mechanism in RBC,
the evidence available supports the concept that ATP
affects the affinity properties of the carriers such as to
permit them to be closer to saturation at physiological
substrate levels. In general, no effect on Vmax was noted
in these kinetic studies of the ATP effect. It is important
to note that in these instances ATP had a modulating
effect, that is, the transport processes were not abolished
in the absence of ATP. In contrast, ATP depletion completely abolishes NKCC cotransport activity (see above).
Only one attempt has been made to examine the kinetic
effects of ATP depletion on the transport behavior of the
NKCC. Ikehara et al. (145) treated HeLa cells with various
concentrations of glucose in the presence of the inhibitor of
oxidative phosphorylation, carbonyl cyanide m-chlorophenylhydrazone (CCCP). This treatment achieved stable intracellular ATP concentrations within 90–120 min. These cells
were then exposed to various concentrations of extracellular Rb1, and the furosemide-sensitive net uptake of this K1
replacement was measured. They showed that the apparent
K0.5 for the ATP was ;0.95 mM, and the Hill coefficient was
between 1.5 and 2. This K0.5 was ;10 times higher than was
reported for the squid giant axon (5). Whether this difference in ATP affinity is a real difference between the mammalian and the squid NKCC is unclear. The means used to
reduce ATP concentration in the HeLa cells would necessarily result in an increase of ADP concentration, and it is
possible that this nucleotide might engage in competitive
inhibition with ATP for the ATP-binding site and thus increase the apparent K0.5. Ikehara et al. (145) further showed
that as cellular ATP concentration was decreased, the apparent affinity of the NKCC for extracellular Rb1 also decreased with no effect on the Vmax of the cotransporter. This
result suggests that phosphorylation of the cotransporter
may activate cotransport by a multistep process rather than
a simple “all-or-none” mechanism. Although this seems consistent with Lytle’s (212) result showing increasing levels of
phosphorylation of the NKCC protein with increasing levels
of stimulation, it does not seem to fit with the observation
that ATP depletion will completely block NKCC transport
activity (see above).
Although the majority of available evidence supports
the present view that the effects of ATP on the NKCC are
mediated via a protein phosphorylation/dephosphorylation mechanism, other possibilities exist. We have already
discussed the evidence that the ability of loop diuretics
such as bumetanide to bind to the NKCC may be in some
cases a measure of the activation state of the NKCC
protein. To the extent this is true, it is of interest that the
maximal [3H]bumetanide binding (Bmax) to squid optic
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The results of the experiments utilizing turkey erythrocytes are particularly interesting in view of the fact that
two means of stimulating NKCC were tested for their ATP
sensitivity. One way of stimulating the avian cotransporter was to increase cellular levels of cAMP. Obviously,
one would expect ATP to be required for a cAMP-mediated response, both to generate cAMP in response to the
agonist (e.g., epinephrine) and to provide the necessary
phosphate group for the cAMP-activated protein kinase to
attach to protein(s). Satisfyingly enough, two groups (268,
347) found the cAMP-stimulated NKCC activity was reduced as ATP concentration was reduced. Another means
of activating NKCC activity in avian RBC was to cause the
cell to shrink. Apparently, this mode of activation is independent of the cAMP-stimulated mode. Again, both
groups showed that the enhanced cotransport activity
was inhibited by ATP depletion. However, a puzzling aspect of these findings arises from quantitative considerations. Both groups found that the cAMP-elicited NKCC
activity was more sensitive to ATP depletion than the cell
shrinkage-elicited activity. As mentioned above, the two
mechanisms are apparently mediated via separate pathways so a difference in the ATP requirement might not be
surprising. However, the relatively high K0.5 for ATP of
the cAMP-elicited activity is puzzling (1.7 mM, Ref. 347;
0.6 mM, Ref. 268). Membrane-bound protein kinase A
(PKA) from human RBC has been shown to have, like
most protein kinases, a high affinity for ATP (K0.5 5 10
mM; Refs. 97, 301). Whether these apparent discrepancies
reflect technical problems (e.g., ATP compartmentalization in intact cells that is not reflected in whole cell ATP
determinations) or fundamentally different experimental
conditions that might make hazardous the comparison of
biochemical measurements of enzyme activity in membrane fragments with rates of ion transport, is unknown
at the present time. Alternatively, cAMP-mediated stimulation may require higher ATP concentrations at an activation step that occurs after cAMP production and the
presumed protein phosphorylation.
Other transport processes believed to be secondary
active or gradient driven have been shown to be affected
by ATP depletion. Sodium/calcium exchange (19, 26, 56)
as well as Na1-glutamate cotransport (20) in squid giant
Volume 80
Na1-K1-Cl2 COTRANSPORT
January 2000
TABLE
degree of transport activation and the degree of phosphorylation caused by any given stimulus. At least three
studies have measured both cotransporter activity and 32P
incorporation under the same conditions and reported
that cotransport activity and NKCC protein phosphorylation increase in parallel. Haas et al. (119) measured the
relative change in transepithelial 36Cl flux across dog
tracheal epithelial cells (see Table 5) and the relative
change of 32P incorporation into the NKCC protein (identified using the T4 monoclonal antibody, Ref. 221). They
subjected the cells to a variety of stimuli including isoproterenol, UTP, hypertonic fluid, and a nystatin-induced
change of [Cl2]i (see sect. IXB1A). Each of these treatments gave very nearly identical relative changes of transport and 32P labeling. Similarly, O’Donnell et al. (254)
studied bumetanide-sensitive 86Rb uptake into endothelial
cells as the measure of NKCC cotransport activity and 32P
incorporation into protein identified using the T4 monoclonal antibody. They showed that a 10-min exposure to
hypertonic fluid (;1.33 normal osmolality) increased the
bumetanide-sensitive 86Rb uptake by 1.6-fold, whereas
phosphoimage analysis of Western blots showed a 1.9fold increase of 32P incorporation. Lytle (212) used duck
RBC to elegantly show that as cell volume is decreased by
exposure to hyperosmotic fluids, the degree of NKCC
transporter activity (measured as bumetanide-sensitive
86
Rb uptake) and the degree of phosphorylation of the
NKCC protein (isolated using NKCC-specific antibody)
increased in parallel (Fig. 16).
However, not all workers have been able to demonstrate a near one-for-one relationship between degree of
ATP phosphorylation and activation of the NKCC. For example, Lytle and Forbush (213) used the shark rectal gland
and measured NKCC transport activity indirectly, using specific labeling by [3H]benzmetanide. By varying the extracellular osmolality, they were able to change cell volume. They
measured the effects of a range of cell volumes on both the
specific [3H]benzmetanide binding and the content of 32P in
the 200-kDa band on an electrophoresis gel. Figure 17 shows
that there is good qualitative agreement between these two
variables. Quantitatively, however, there was a much greater
relative increase of [3H]benzmetanide binding (;15-fold)
than there was of total 32P content (;4-fold). These workers
also determined that the only phosphoamino acids they
could detect were phosphoserine and phosphothreonine; no
5. Correlation of NKCC cotransport in tracheal epithelial cells with “intracellular” [Cl2]
Reduced “Intracellular” [Cl2] (1Nystatin)
Transepithelial
36
Cl flux ratio
UTP
66 mM
49 mM
32 mM
2.64 6 0.65 (22)
1.45 6 0.18 (8)
2.21 6 0.40 (3)
2.78 6 0.62 (29)
Values are ratios 6 SD of number of stimulated/control levels of basolateral-to-apical (transepithelial) 36Cl flux, given in parentheses. [Adapted
from Haas et al. (119).]
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lobe microsomes could be increased as much as 50%
simply by including 100 mM ATP in the binding media (9).
No effect on the apparent binding affinity of the bumetanide was noted. This effect by ATP had an apparent
half-saturation constant of 0.9 mM, well under the halfsaturation value of ;90 mM reported to activate NKCC ion
fluxes in the internally dialyzed squid axon (5). Because
this effect was observed in the nominal absence of Mg21
(0 mM Mg21 and 5 mM EDTA in the binding medium), it
seems very unlikely to be the result of additional NKCC
phosphorylation. Further evidence against this effect being via protein phosphorylation comes from the fact that
a range of adenine nucleotides was also effective at increasing the saturable [3H]bumetanide binding, including
ADP, cAMP, a,b-methylene ATP, b,g-methylene ATP, and
adenosine 59-O-(3-thiotriphosphate). Even adenine (at 1
mM) caused a slight enhancement. However, thymine and
cytosine were completely without effect at 1 mM. It is
important to note that in the nominal absence of ATP,
specific [3H]bumetanide binding to the microsomes was
about one-half to two-thirds the value seen in the presence of ATP. Therefore, this effect of adenine nucleotides
may be in addition to the phosphorylation mechanism.
A) EVIDENCE FOR NKCC PHOSPHORYLATION AND COTRANSPORT
ACTIVATION. If ATP is not stoichiometrically consumed
during NKCC activation, then why is its intracellular presence an absolute requirement for cotransport? Given the
wide variety of protein-mediated biological activities that
are regulated by protein phosphorylation and dephosphorylation (e.g., Ref. 184), it has always seemed likely
that the NKCC protein must be phosphorylated to mediate
cotransport.
There are several reports showing that stimuli that
result in an increase of cotransport activity also cause an
increase in the degree of phosphorylation of the NKCC
protein (e.g., Refs. 119, 175, 212, 213, 254, 335, 341, 342).
In at least two studies a temporal correlation has been
demonstrated between transport activation and NKCC
protein phosphorylation (212, 213). (In all these studies,
the NKCC protein was identified using NKCC-specific antibodies.) These reports provide strong circumstantial evidence for the widely held view that the NKCC is activated
by being phosphorylated. If the NKCC protein is only able
to engage in ion transport when it is phosphorylated, then
there ought to be a quantitative relationship between the
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JOHN M. RUSSELL
Volume 80
FIG. 16. Cell volume changes affect both cotransport activity and
phosphorylation of NKCC protein in parallel. Duck red blood cells were
labeled with 32P and exposed to a range of osmolalites ranging from 220
to 420 mosmol/kgH2O. NKCC protein was isolated by immunoprecipitation (using T14 antibody) and SDS-PAGE. Its 32P content was analyzed
by Cerenkiv counting. Cell volume equals cell water content, and cotransport activity equals bumetanide-sensitive 86Rb influx. Normal cell
volume is denoted by shaded region. [From Lytle (212).]
phosphotyrosine was found. For stimulation both by 20 mM
forskolin or by exposure to an external fluid that was about
1.5 times normal osmolality, the phosphoserine and phosphothreonine content increased by ;3.5- and 5.7-fold, respectively. Given that [3H]benzmetanide binding is, at best,
an indirect measure of cotransport activity, it may be that
the degree of such binding and transport activity of the
NKCC are not quantitatively related. Alternatively, it may be
that measuring the phosphorylation of the 200-kDa protein
included other, non-NKCC proteins. However, it should be
noted that Lytle (212), using immunoblotting techniques,
found that only the NKCC protein exhibited an increase of
phosphorylation after cell shrinkage of duck RBC.
Finally, it is important to note that not all workers
have been able to show that all stimuli that increase
NKCC-mediated fluxes cause an increase in phosphorylation. Tanimura et al. (339), working with rat parotid acini,
were unable to demonstrate an increase of phosphorylation of the NKCC protein identified using an antibody
when they stimulated fluxes using calyculin A or hypertonic shrinkage of the cells. Interestingly, when they stimulated fluxes with agents that increase cAMP concentration, they were able to detect increased protein
phosphorylation. These authors suggested that there may
be multiple pathways by which the NKCC could be stimulated.
B) WHAT PROTEIN KINASE(S) MIGHT BE INVOLVED? Thus we
have seen that protein phosphorylation is crucial to the
activation of the NKCC1, and there is considerable evi-
3
FIG. 17. Effects of osmolality on both [ H]benzmetanide binding and
P labeling of NKCC protein. Osmolality of fluids bathing shark rectal
gland cells was varied to cause a gradation of stimulation of NKCC (solid
symbols). NKCC was maximally stimulated by applying 20 mM forskolin
(V). Normal cell volume was achieved at 915 mosM (see arrow on
abscissa). Cell shrinkage occurred when osmolality was increased by
adding sucrose. Cell swelling occurred when osmolality was reduced by
NaCl removal. An inverse relationship was observed between external
osmolality and cell water content [kg cell/kg cell solids 5 (3.237/mosM)
2 0.752]. Top: effect of osmotic changes on specific [3H]benzmetanide
binding. These data represent means 6 SE of 4 experiments. [Data
adapted from Lytle and Forbush (214).] Bottom: effect of osmotic
changes on 32P content of 195-kDa NKCC protein. Degree of phosphorylation was determined in 2 ways: scintillation counting of 195-kDa gel
band or by autoradiography and densitometry. These data are representative of 3 experiments. [From Lytle and Forbush (213).]
32
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dence that the NKCC1 protein itself is phosphorylated
when active (e.g., Refs. 212, 213, 288). Similar proof that
the NKCC2 isoform is phosphorylated is lacking; thus the
remainder of this discussion focuses on the NKCC1 isoform.
What protein kinase or kinases are involved in mediating this phosphorylation? As already discussed, the only
residues on the NKCC1 that Lytle and Forbush (213)
could identify as being phosphorylated were serine and
threonine. Recent successes in cloning the NKCC1 (e.g.,
Refs. 53, 79, 283, 358) have permitted workers to deduce
consensus phosphorylation sites from the predicted
amino acid sequence of the NKCC1 protein. There are as
many as 20 consensus sequences for kinases. Protein
January 2000
Na1-K1-Cl2 COTRANSPORT
FIG. 18. Four different stimuli all increase cotransporter activity and
NKCC protein phosphorylation, but their effects are not additive. Duck
red blood cells were exposed to the following stimuli (all at their
maximally effective doses): norepinephrine (NE), 10 mM; sodium fluoride (F2), 10 mM; sucrose (hyper), 100 mM; calyculin A (cal), 0.2 mM.
Cotransport activity (open bars) was measured as bumetanide-sensitive
86
Rb uptake. NKCC protein phosphorylation (hatched bars) was measured as amount of 32P content of immunoprecipitated NKCC protein.
All values were normalized to those of calyculin A. [From Lytle (212).]
complex and multifactorial. This should not be surprising.
The postulated functions of this cotransporter are quite
varied (see sect. X) requiring that the cotransporter receive input from a variety of stimuli. Only further experiments will tell us whether a final common kinase is
responsible for NKCC phosphorylation.
I) PKA? As previously mentioned, the mammalian
NKCC1 has consensus PKA sites (e.g., Refs. 53, 281),
whereas the shark NKCC1 does not (358). Nevertheless,
cAMP stimulates NKCC1 transport in the shark rectal
gland. It is now believed that cAMP stimulates the shark
NKCC1 indirectly, by increasing the electrodiffusive permeability of the apical membrane to Cl2 (104), thereby
causing a net Cl2 secretory efflux and reducing [Cl2]i. As
discussed in section IXB1, there is considerable evidence
that reducing [Cl2]i increases NKCC cotransport activity
and the degree of phosphorylation of the NKCC protein.
The activating effect of reduced [Cl2]i is almost certainly
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kinase C (PKC) and the protein kinase casein kinase II
(CK2) consensus phosphorylation sites are found in all
the transcripts so far isolated (53, 79, 283), whereas PKA
consensus sites have been identified in all isoforms except shark (358). Of these three candidates, functional
evidence has been presented to support claims for both
PKA and PKC as mediators of NKCC function. Almost no
functional evidence currently exists that addresses a possible role for protein kinase CK2.
Recently, Lytle (212) has argued that no matter what
the stimulus, a single kinase is responsible for activating
the NKCC via phosphorylation. Using duck RBC, he stimulated bumetanide-sensitive 86Rb fluxes and measured
phosphorylation in immunoprecipitated protein using a
specific monoclonal antibody against NKCC1. Four different stimuli (osmotic cell shrinkage, norepinephrine, fluoride, and calyculin A) were all capable of increasing 86Rb
fluxes by 10-fold. All four stimuli exclusively phosphorylated serine and threonine sites in agreement with the
results of Lytle and Forbush (213) mentioned above. As
seen in Figure 16, when the NKCC-mediated flux was
zero, there was still measurable phosphorylation. Phosphorylation increased with increasing stimulus in parallel
with the increase of NKCC-mediated 86Rb influx. Stoichiometrically, the baseline phosphorylation corresponded to
1 ATP/NKCC copy, and maximal stimulation corresponded to ;6 ATP/NKCC copy. Thus NKCC-mediated
influx increased as a function of the degree of phosphorylation of each individual cotransporter rather than as a
result of an increased number of cotransporters that were
phosphorylated. There was no additivity to the effects of
the four stimuli, and each of the cotransport stimuli
caused the same increase in phosphorylation of the immunoprecipitated protein (Fig. 18). No matter which stimulus was applied, the same eight major tryptic phosphopeptides were generated. Finally, staurosporine, a
broadly selective kinase inhibitor, inhibited each of the
stimuli with equal potency, implying a common site of
inhibition for all modes of stimulation (although leaving
unanswered the question of the site of inhibition by this
agent). Thus these three observations strongly point to a
common kinase mechanism causing the NKCC protein
phosphorylation that in turn causes the functional activation of the NKCC.
At this time, workers have been studying regulation
of the NKCC for at least 15 years. What we know for sure
is that NKCC phosphorylation results in the functional
activation of the cotransporter. We do not know the identity of the final, common kinase despite the fact that
consensus sites for three different protein kinases have
been identified in the putative cytoplasmic domain of the
protein. We discuss below the conflicting, or nonexistent
evidence for these three protein kinase candidates. It
seems likely, therefore, that regulation of the functional
activity by protein phosphorylation is going to prove to be
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JOHN M. RUSSELL
effect being measured is due directly to PKC-mediated
phosphorylation of the NKCC protein. The possibility of
PKC exerting its effect on NKCC function distally via a
multistep regulation seems to offer the best ad hoc explanation for the gamut of functional effects caused by PKC
stimulators.
III) Other protein kinases?. There are numerous
consensus protein kinase CK2 phosphorylation sites on
the NKCC protein (53, 79, 281, 358). This suggests a
possible role for protein kinase CK2 as a modulator of
NKCC activity. To date, little functional evidence exists
regarding this potential pathway, although O’Donnell et
al. (254) demonstrated that two inhibitors of protein kinase CK2 (A3 and CKI-7) both reduce the hypertonically
induced stimulation of the NKCC of bovine vascular endothelial cells. Because these agents also inhibit myosin
light chain kinase, the interpretation of this result is uncertain (see Refs. 175, 254).
Two groups have reported that ML-7, an inhibitor of
myosin light chain kinase, will prevent the shrinkageinduced stimulation of the NKCC found in bovine aortic
endothelial cells (175, 254). Klein and O’Neill (175) further
demonstrated that cell shrinkage resulted in increased
phosphorylation of both the myosin light chain as well as
the NKCC (identified using the T4 antibody). As expected,
ML-7 inhibited the shrinkage-induced phosphorylation of
the myosin light chain but, surprisingly, had no effect on
the phosphorylation of the cotransporter. Thus the inhibitory effect of ML-7 on NKCC function does not seem to
be the result of a reduction of the phosphorylation of the
NKCC but may be mediated through a reduction of the
phosphorylation of the myosin light chain or another
as-yet-unidentified protein.
At present, it is safe to say that if a single protein
kinase is responsible for the phosphorylation and subsequent functional activation of the NKCC protein, it either
must be different in different cells or it has not yet been
identified.
C) EVIDENCE FOR PROTEIN PHOSPHATASE INVOLVEMENT. A
protein phosphorylation/dephosphorylation scheme for
NKCC activation/deactivation implies an important role
for protein phosphatases in addition to protein kinases.
Reports from studies on a variety of cells have provided
evidence that protein dephosphorylation reduces NKCC
functional activity (e.g., Refs. 5, 6, 176, 201, 212, 254, 267,
287, 335). If the NKCC is constitutively fully activated
when the phosphatase inhibitor is added, then no effect is
observed (e.g., squid axon, Ref. 7). To demonstrate the
action of protein phosphatase, intracellular ATP was
washed out (using intracellular dialysis). The rate at
which the bumetanide-sensitive unidirectional 36Cl influx
was inactivated in the presence and absence of phosphatase inhibitors was measured (5). In this way, the effects
of intracellular fluoride (5 mM) and vanadate (40 mM)
were studied. Neither agent affected the rate of 36Cl influx
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not mediated by PKA as shown by Tanimura et al. (339).
They immunoprecipitated a 175-kDa protein from rat parotid acini that binds bumetanide and is phosphorylated
after treatment with isoproterenol, dibutyryl cAMP, and
forskolin. However, treatment designed to reduce the
[Cl2]i did not lead to such phosphorylation, leading the
authors to argue the effect of cAMP must be direct (and
not via a change of [Cl2]i).
Although the mammalian NKCC1 presumably does
have PKA phosphorylation sites, the functional evidence
for a direct role of PKA-mediated activation of cotransport function is decidedly mixed. For example, treatments designed to elevate cellular cAMP levels stimulate
NKCC activity in avian erythrocytes (e.g., Refs. 121, 265,
347). However, this stimulation is not via PKA (212).
In contrast to the stimulation reported above, there
are numerous reports of cAMP-induced inhibition of
NKCC function. For example, cAMP has been reported to
inhibit the NKCC function in several RBC types including
human (82) and ferret RBC (263). Other tissues exhibiting
NKCC inhibition after cAMP treatment include PC12 cells
(201), flounder intestine (292), vascular smooth muscle
(328), rat vascular smooth muscle (261), and human fibroblasts (260). In the case of the epithelial preparations,
these inhibitory effects may be mediated, at least partially, by decreasing the membrane conductance to Cl2
(e.g., Ref. 292).
In still other cells, with well-defined NKCC mechanisms, cAMP has no discernible effect whatsoever (squid
giant axon, Ref. 304; MDCK cells, Ref. 311; Ehrlich ascites
tumor cells, Refs. 86, 145, 179; chick heart cells, Ref. 77;
ferret RBC, Ref. 223).
The pattern (or lack thereof) of the forgoing results
argues against a direct role for PKA in the activation of
the NKCC1. However, it must be kept in mind that the
precise NKCC isoform has not been identified in many of
the preparations on which the functional studies outlined
above were conducted. Thus it is possible that there are
some isoforms that are directly phosphorylated by PKA
and others that are not.
II) PKC? As already mentioned, there is structural
evidence for consensus PKC binding sites on the NKCC1
protein in both mammalian and shark isoforms (53, 358).
From a functional point of view, the situation for PKC
playing a direct role in the phosphorylation-induced activation of the NKCC is very similar to that just described
for PKA. There are reports of PKC stimulating NKCC
activity (e.g., rat mesangial cells, Ref. 140; avian salt
gland, Ref. 342), inhibiting NKCC activity (Balb/c 3T3
preadipose cells, Ref. 247; aortic endothelial cells, Ref.
251; HT-29 cells, Ref. 76), and having no effects (chick
heart cells, Ref. 77; shark rectal gland, Ref. 323; avian
RBC, Ref. 287; flounder intestine, Ref. 337; ferret erythrocytes, Ref. 275; eccrine clear cells, Ref. 343). In none of
these studies can it be determined whether the functional
Volume 80
January 2000
Na1-K1-Cl2 COTRANSPORT
times higher concentration of okadaic acid than of calyculin A to cause maximal stimulation of the bumetanidesensitive flux. Given the relative selectivities of these two
agents for protein phosphatase 1 and 2A (I151), this difference in sensitivities of the NKCC has been taken to
mean that protein phosphatase 1 is more important than
protein phosphatase 2A in dephosphorylating the NKCC.
An interesting and, from an operational point of view,
important observation has been that both these inhibitors
exhibit a biphasic dose response (201, 335). Thus, if too
high a concentration of either agent is used, the stimulation of the cotransported ion flux is reduced.
In addition to the functional evidence that protein
phosphatases may be involved in controlling the level of
activity of the NKCC, there has recently been some structural evidence as well. O’Donnell’s group (254) has demonstrated that the NKCC protein (identified with a monoclonal antibody) from endothelial cells has a much higher
level of phosphorylation when otherwise unstimulated
cells are exposed to either okadaic acid or calyculin A.
Using duck RBC, Lytle (212) has recently shown that
treatment with calyculin A stoichiometrically increased
both cotransport flux and phosphorylation of the NKCC.
Recently, Flatman and Creanor (71) have postulated
that the protein phosphatase responsible for dephosphorylating (and thereby inactivating) the NKCC protein itself
must be dephosphorylated to be active. They then suggest
that the effects of a variety of protein kinase inhibitors are
exerted not on the final common kinase but on other
protein kinases responsible for phosphorylating (and inactivating) the protein phosphatase. Thus future work
may well reveal that regulation of NKCC function is accomplished by a variety of cascades of protein kinases
and phosphatases.
B. Role of Intracellular Ions
There are several reasons to consider intracellular
ions as potential modulators of NKCC activity. First, simply from thermodynamic and kinetic considerations,
changes in the intracellular concentration of any of the
cotransported ions would be expected to exert effects on
transport, both net and unidirectional (see sects. VIB and
VII). Because the driving force on the NKCC is believed to
be the result of the combined chemical gradients of the
three cotransported ions (see sect. VIB), any change in
their intracellular concentrations would change the net
driving force on the cotransporter. In addition, the binding and release of the cotransported ions are believed to
be a highly ordered, cooperative process (see sect. VIIA).
Such models predict a variety of effects on unidirectional
fluxes of changing intracellular concentrations of the cotransported ions. Finally, pHi and Ca21 concentration
changes could be expected to secondarily influence the
activity of this cotransport mechanism.
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
in the presence of intracellular ATP (continuously supplied via internal dialysis). Therefore, it was concluded
that neither agent had direct effects on the NKCC itself
nor did they appear to interfere with the process by which
the NKCC is activated by ATP. However, if either agent
was present during the washout of cellular ATP, the inactivation of bumetanide-sensitive 36Cl influx was slowed
by a factor of two- to threefold. Another means of demonstrating protein phosphatase activity in the squid axon
was to reduce the [ATP]i to levels near the apparent
half-saturation constant for the nucleotide (;75 mM; Ref.
5) and then apply an inhibitor of protein phosphatases.
Using a more specific protein phosphatase inhibitor, okadaic acid (151), Altamirano et al. (6) provided further
evidence that the NKCC in the squid axon is inactivated
by protein phosphatase. ATP “washout” studies using
okadaic acid as the protein phosphatase inhibitor confirmed the results originally reported using vanadate and
fluoride (Russell, unpublished observations). If, indeed,
the action of the protein phosphatase inhibitors was to
prevent the dephosphorylation of the “activated” NKCC,
one might reasonably ask why, in the presence of high
concentrations of okadaic acid, was there inactivation at
all? To address this question, we must consider the following. First is the fact that these phosphatase inhibitors
do not block all classes of phosphatases (21, 151). This,
coupled with the fact that phosphoprotein phosphatases
are not particularly substrate specific, means that dephosphorylation of the activated NKCC could continue to occur by other phosphatases not blocked by the particular
agent being used. In addition, because phosphorylated
proteins are relatively “energy rich” (e.g., Ref. 184), spontaneous dephosphorylation will eventually inactivate the
cotransporter. Therefore, it is not surprising that agents
that are believed to be acting to block phosphoprotein
phosphatases might not totally prevent the inactivation of
the NKCC. A similar lack of complete inactivation of an
ATP-dependent Ca21 channel in heart muscle has been
reported after treatment with protein phosphatase inhibitor 2 (133).
Most other cell types that have been studied for
protein phosphatase activity appear to have the NKCC
inactivated under “resting” conditions. Pewitt et al. (287)
studied the effects of okadaic acid on 86Rb influx into
duck RBC. They reported that in otherwise unstimulated
erythrocytes, okadaic acid treatment would increase the
bumetanide-sensitive 86Rb uptake rapidly (within 4 –5
min) with an ED50 of ;5 3 1027 M. Similar effects have
been reported for the NKCC of PC12 cells (201), bovine
aortic endothelial cells (254), and brain microvessel endothelial cells (335) using okadaic acid and/or calyculin A.
Two studies have reported a large difference in the
sensitivity of the NKCC-mediated flux stimulation to these
two protein phosphatase inhibitors. In both PC12 cells
(201) and duck erythrocytes (267), it takes about a 100
247
248
JOHN M. RUSSELL
Volume 80
1. Intracellular Cl2 modifies cotransporter activity
2
FIG. 19. Effect of removing intracellular Cl (replaced isosmotically
with glutamate2) on unidirectional influxes of both Cl2 (●) and K1 (M)
measured simultaneously into an internally dialyzed squid giant axon.
Throughout experiment, intracellular fluid was Na1 free. Although not
shown here, stimulation of both influxes seen upon removal of intracellular Cl2 is not observed in presence of 10 mM bumetanide. SSW, squid
seawater. Ouabain concentration, 1025 M; tetrodotoxin concentration,
1027 M. [From Russell (307).]
Third, and perhaps the strongest argument, is the fact that
raising [Cl2]i not only blocks bumetanide-sensitive influxes in a concentration-dependent manner but also the
bumetanide-sensitive effluxes (29). Figure 20 shows the
effects of increasing [Cl2]i on both NKCC-mediated influx
and efflux in the internally dialyzed squid axon. In the
case of the bumetanide-sensitive Cl2 influx into the squid
axon, the relationship between [Cl2]i and influx is rather
steep, with a Hill coefficient of 4.5. This suggests multiple
sites of action by intracellular Cl2. The relationship between [Cl2]i and bumetanide-sensitive efflux is complex,
since for efflux, intracellular Cl2 is both a substrate and
an inhibitor. The efflux versus [Cl2]i data cannot be fitted
by simply assuming that there are cooperative Cl2 binding sites for transport and another set of cooperative
binding sites for inhibition. Note that the steepest region
of the intracellular Cl2 inhibition is in the [Cl2]i range of
normal intracellular Cl2 levels (normal [Cl2]i for squid
axons is ;125 mM; reviewed in Ref. 307). This suggests
that [Cl2]i plays an important negative-feedback role in
controlling the activity of the NKCC. Because the intracellular ionic conditions for these influx and efflux experiments were different (e.g., efflux experiments required
intracellular Na1, which was omitted from the intracellular fluids for the influx studies), quantitative comparisons
of the two unidirectional fluxes are not useful.
As early as 1982, Ussing (348) reported the results of
studies on the recovery of cell volume after exposure of
the blood side of the frog skin to reduced [K1] or [Cl2]
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In the internally dialyzed squid giant axon, elevation
of [Cl2]i inhibits the unidirectional fluxes of all three
cotransported ions in both the influx and efflux directions
(28, 29, 302, 303, 306, 307). These findings grew out of an
early observation that removal of intracellular Cl2 had the
surprising (at the time) effect of increasing 36Cl influx
(302). This was surprising because it was expected that a
substantial portion of the unidirectional 36Cl influx would
occur via isotopic exchange flux pathways, in which case
reducing [Cl2]i would reduce the unidirectional 36Cl influx. Later, as the pathway for 36Cl influx was being identified as the NKCC, it was shown that in addition to
inhibiting the influx of 36Cl, high levels of [Cl2]i also
inhibited the influxes of 22Na (303) and 42K (306). Figure
19 illustrates the profound and reversible effect of changing the [Cl2]i on simultaneously measured 36Cl and 42K
influxes into an internally dialyzed squid axon. The inhibitory effect of elevated intracellular Cl2 was not observed
when the axons were pretreated with furosemide or bumetanide or when ATP was removed by internal dialysis,
showing it was a specific effect on the NKCC.
Intracellular Cl2 is not the only, or even the most,
effective anion at inhibiting the NKCC. Thiocyanate, I2,
and Br2 are all more potent inhibitors than Cl2, whereas
22
NO2
3 and SO4 exhibit significant but much less inhibitory
22
potentcy (29). Because SCN2, NO2
alone are
3 , and SO4
not suitable substrates for the NKCC (e.g., Refs. 234, 260),
this suggests that the site(s) being affected by the intracellular anions may not be transport sites. Further evidence that the intracellular site(s) may not be the same
sites that Cl2 binds to for cotransport is the fact that
extracellular Cl2 has very little inhibitory effect (29).
Nevertheless, the exact site of inhibition by intracellular
Cl2 is still unknown.
Because the binding and release of substrate ions is
believed to be highly ordered (see sect. VIIA), one possibility is that raising the trans-side concentration of a
substrate could inhibit the reaction by an end-product
inhibition mechanism. Although this mechanism may partially account for the observed inhibition by intracellular
Cl2, there are at least three lines of evidence that argue
against this as the sole explanation. First, as mentioned
above, there is the fact that some nontransported anions
are even more effective at inhibition when presented
intracellularly than is Cl2. However, it is possible that
nontransported ions bind at the transport sites but do not
promote or support cotransport. Second, if the mechanism is simple trans-side inhibition, then removal of extracellular Cl2 ought to stimulate NKCC-mediated ion
efflux. This was not what actually happened when Cl2
(normally 561 mM) was completely replaced by gluconate
in the external fluid bathing the squid axon. In this case,
there was only a small increase of cotransport efflux (29).
January 2000
Na1-K1-Cl2 COTRANSPORT
249
solutions. He reasoned that exposure to KCl-depleted solutions caused the cells to lose K1 and Cl2 and an osmotic
equivalent of water. He showed that the recovery process
was furosemide sensitive and proposed that it was via a
coupled NCC process. Based on his assumption that the
cell shrinkage resulted from a loss of (K1) Cl2, Ussing
(348) suggested that the cotransporter might be activated
by a fall of [Cl2]i, although no definitive proof was offered.
The remarkable effect of intracellular Cl2 on the
NKCC has recently attracted a good deal of attention as
the implications have become better understood. For example, there are a number of cell types which, when
shrunken by direct exposure to hyperosmotic fluids, do
not perform regulatory volume increase (RVI; see Table
6). However, several will perform a pseudo-RVI if they are
first swollen by exposure to hypotonic fluids, then permitted to undergo regulatory volume decrease (RVD), which
they do by losing K1 and Cl2. This has been termed a
“post RVD RVI” (134). Levinson (202–204) pursued this
observation using Ehrlich ascites tumor cells and showed
that the subsequent RVI was the result of a bumetanidesensitive net uptake of K1, Na1, and Cl2 that always
results in an increase of intracellular Cl2 content. Thus,
when [Cl2]i recovered, cell volume recovered, suggesting
that somehow [Cl2]i was playing an important role in
regulating the activity of the NKCC. An insightful observation made in the discussion of the 1990 paper (203) was
that, although the gradient of the chemical potential
(Dm̃net) for the NKCC favored net uptake by the NKCC,
such net uptake did not occur until the [Cl2]i was reduced
by the prior RVI-driven loss of Cl2. This led Levinson
(203) to speculate that the activity of the NKCC might be
“somehow regulated by the intracellular [Cl2] and not by
the gradient of chemical potential.” Similarly, studies on
shark rectal gland (104) and vascular endothelial cells
(257) revealed that there are situations in which when the
net free energy in the chemical gradients of Na1, K1, and
Cl2 favors net uptake by the NKCC, it does not occur.
O’Neill and Klein (257) suggested, based on the report of
trans-inhibition in squid axon (see above), that intracellular Cl2 may play a regulatory role in the operation of the
vascular endothelial cell NKCC.
Homma and Harris (141) showed that soaking cultured rat glomerular mesangial cells in Cl2-free or Na1free media for 60 –90 min resulted in enhanced cotransport flux and [3H]bumetanide binding. Bumetanidesensitive 86Rb uptake was increased between 55 and 70%
by such treatment. [3H]bumetanide binding was increased
50 – 60%. Similarly, in a study using the osteosarcoma cell
line UMR-106 – 01, Whisenant et al. (351) showed that
presoaking the cells in a Cl2-free medium for 30 min
resulted in a 2.5-fold increase in bumetanide-sensitive
86
Rb uptake. These workers had previously shown that
such a pretreatment depleted the cells of Cl2. The preceding results support the view that reduced [Cl2]i stimulates transport by the NKCC. Finally, it has been noted
that to study the functional expression of cloned NKCC, it
is necessary to soak HEK-293 cells in Cl2-free medium,
presumably to reduce [Cl2]i (e.g., Refs. 283, 358).
Recently, Gillen and Forbush (93) restudied the issue
of [Cl2]i and NKCC activity using an epithelial cell line
(HEK-293 cells) transfected with human NKCC1. They
measured both bumetanide-sensitive 86Rb uptake as well
as [Cl2]i. As was the case for squid axon, they found a
very steep relationship between bumetanide-sensitive ion
flux and [Cl2]i. They likewise showed that at the resting
[Cl2]i level, the NKCC-mediated uptake was very low.
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2
FIG. 20. Effect of varying [Cl ]i on bumetanidesensitive unidirectional influx (V) and unidirectional efflux (●) of Cl2 measured using 36Cl in internally dialyzed squid axons. [Cl2]i was adjusted
by dialyzing with fluids identical in all respects
except a varying amount of Cl2 was replaced with
glutamate2. A nonlinear least-squares analysis of
Hill equation was fitted to influx data. Fitting parameters were as follows: Vmax 5 56.6 6 1.4
pmolzcm22zs21; Hill n 5 24.5 6 0.3; K0.5 5 101
6 2.0 mM. Efflux data are fitted by eye. [K1]o was
100 mM (for influx and efflux studies), which promotes a larger influx without affecting efflux. For
influx studies, [K1]i was 400 mM and [Na1]i was 0
mM. For efflux studies, [K1]i was 200 mM and
[Na1]i was 200 mM. All other ionic conditions both
inside and outside were identical for two sets of
data. External fluid contains ouabain (1025 M) and
tetrodotoxin (1027 M). Each point represents data
averaged from no less than 3 axons and in most
cases 6 axons. [Influx data from Breitwieser et al.
(28); efflux data from Breitwieser et al. (29).]
250
JOHN M. RUSSELL
ment that increased the transepithelial Cl2 flux by 10- to
30-fold increased the [Na1]i from 11 to 29 mM while it
decreased the [Cl2]i from 49 to ;40 mM and left [K1]i
unchanged. Using these ion activity measurements and
the net driving force equation (see Table 3), it is clear that
the 10- to 30-fold increase in furosemide-sensitive Cl2 flux
cannot be explained by the change of net driving force on
the NKCC represented by the changes of ion concentrations that these workers measured. In the discussion of
their paper, Greger et al. (104) considered at least six
possible links between the increase of the apical membrane Cl2 conductance and the increased NKCC rate. One
of the possibilities was that reducing the normally high
[Cl2]i itself might act to stimulate the cotransporter, an
effect which had already been clearly demonstrated in the
squid axon (see sect. IXB1).
Forbush’s group (213, 214) has termed this interaction between [Cl2]i and epithelial secretion, the “[Cl2]icoupling hypothesis.” Thus Lytle and Forbush (213; see
also Ref. 72) explicitly postulated that a reduction of
[Cl2]i, resulting from Cl2 loss through the activated apical
Cl2 channels, was the key to the apparent “cross-talk”
between different transport mechanisms located on the
two separate membranes. Furthermore, they have suggested that upregulation of transport by the NKCC results
from an enhanced phosphorylation of the NKCC protein
that in some way is caused by the reduced [Cl2]i (213).
Figure 21 illustrates the relevant anatomical considerations and the Cl2 transport pathways involved in this
hypothesis. Results obtained from several epithelial preparations directly (115, 119) and indirectly (120, 216, 296)
support this [Cl2]i coupling hypothesis.
Haas and co-workers (115, 119, 120) have performed
a series of studies on airway epithelial cells that provide
the best direct evidence in support of the [Cl2]i coupling
hypothesis in epithelia. First, they showed that secretagogue-stimulated NKCC activity (measured 2 ways) could
be reduced by treatments expected to prevent or reduce
the loss of Cl2 across the apical membrane. In one study,
they measured saturable [3H]bumetanide binding to dog
tracheal epithelial cells as an index of NKCC activity (120;
see sect. VIII for a discussion of this approach). As seen in
Figure 22, the apical Cl2 channel inhibitor IAA-94 (94)
reduced or abolished the secretagogue-stimulated saturable [3H]bumetanide binding. When 36Cl efflux was used as
a measure of NKCC activation, IAA-94 blocked 75% of the
cAMP-stimulated 36Cl flux and 69% of the ATP-stimulated
flux. These results support the [Cl2]i coupling hypothesis
by demonstrating that two different measures of NKCC
activity are reduced when the apical Cl2 channels are
inhibited. Presumably, such inhibition of the apical Cl2
channels would prevent a fall in [Cl2]i and thereby prevent the activation of the NKCC.
Increasing [K1] on the basolateral side of the membrane ([K1]b) is an alternative means to reduce or inhibit
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Both these observations nicely confirm in another cell
type earlier reports for the key importance of [Cl2]i to the
functional activity of the NKCC.
2
A) [CL ]i AND REGULATION OF TRANSEPITHELIAL SECRETION:
2
THE [CL ]i-COUPLING HYPOTHESIS. Secretory epithelial organs
(e.g., salivary glands, tracheal epithelium, shark rectal
glands) secrete a Cl2-rich aqueous solution in response to
a variety of stimuli. Secretion requires the transepithelial
movement of Cl2 (plus an attendant cation, usually Na1,
and osmotically obliged water) from the blood to the
lumen of the gland. This means that the Cl2 must enter
the cell across the blood-side membrane (the basolateral
membrane) via the NKCC, diffuse through the epithelial
cytoplasm, then cross the apical membrane, via Cl2 channels, into the lumen of the gland. The attendant cation
follows either via a channel (transcellular) or across the
tight junction (paracellular). Thus secretion requires the
coordination of not less than four ion transport pathways:
at least two Cl2 transport pathways located on two different membranes as well as a basolateral K1 channel and
an apical Na1 (or K1) channel (5 if you count the Na1/K1
pump). In some cases, the direct secretagogue action is
apparently only at the apical membrane. Here the effect is
to cause Cl2 channels to open, thereby permitting Cl2 to
run down its electrochemical gradient, out of the cell and
into the secretory organ lumen (e.g., cAMP-mediated agonists in shark rectal gland, Refs. 100, 104; purinergic agonists in the tracheal epithelium, Refs. 120, 224).
In 1984, two studies appeared that, in retrospect,
provided the first direct evidence that [Cl2]i might be the
link between the Cl2 channel on the apical membrane and
the NKCC on the basolateral membrane. Both Shorofsky
et al. (322) and Greger et al. (104) used Cl2-sensitive
liquid ion exchanger microelectrodes to monitor [Cl2]i.
(Actually, these microelectrodes measure ion activity aCl
i ,
but no serious error is made if we use the more familiar
term [Cl2]i.) They used two different secretory epithelia,
but both clearly demonstrated that resting [Cl2]i was
much higher than expected if Cl2 were distributed across
the membrane according to electrochemical equilibrium
considerations. Furthermore, they both showed that
when the cells were stimulated to secrete, [Cl2]i declined
significantly. Furthermore, Greger et al. (104), using the
shark rectal gland, showed that the increase in the Cl2
conductance of the apical membrane appeared to precede
the fall of [Cl2]i.
Opening apical Cl2 channels would be expected to
reduce [Cl2]i. Could a reduction of [Cl2]i work by simply
increasing the net chemical driving force on the NKCC?
As discussed in section VIB, the net driving force on the
cotransporter is a function of the transmembrane chemical gradients of Na1, K1, and Cl2. Greger et al. (104)
monitored the intracellular ion activities of Na1, K1, and
Cl2 before and during cAMP stimulation using ion-selective microelectrodes. They showed that the cAMP treat-
Volume 80
January 2000
Na1-K1-Cl2 COTRANSPORT
251
2
FIG. 21. Possible role for [Cl ]i in regulation of secretion by a
secretory epithelium. The “[Cl2]i coupling hypothesis” is as follows: 1)
agonist causes opening of a Cl2 channel on apical membrane of epithelial cell. 2) [Cl2]i, which is normally maintained above its electrochemical equilibrium concentration (see sect. IXA) by NKCC, now falls as a
result of increased apical membrane conductance. 3) Fall of [Cl2]i
reduces [Cl2]i-mediated inhibition of NKCC (see Fig. 20), possibly by
permitting an increase in phosphorylation of NKCC. 4) K1 recycles via
a K1 channel in same membrane in which NKCC is located. 5) Na1
follows Cl2 via a paracellular pathway. See text for details.
Cl2 efflux across the apical membrane. This reduces the
recycling of K1 via basolateral K1 channels, thereby reducing the cell-negative intracellular voltage, which is the
driving force for Cl2 to leave the cell through the apical
channel. The effects of raising [K1]b on [3H]bumetanide
binding were tested in dog and human tracheal epithelial
cells treated with isoproterenol or with UTP. The pattern
of effects was similar to that observed with IAA-94 treatment. Elevated [K1]b prevented the extra [3H]bumetanide
binding caused by UTP, but not that caused by isoproterenol. Thus these results suggested that NKCC activation
caused by ATP/UTP depended on the movement of Cl2
out of the cell across the apical membrane, whereas the
stimulation by isoproterenol/cAMP was only partially accounted for by this mechanism. Because Cl2 loss via the
apical channels might result in a reduction of [Cl2]i and
because the stimulatory effects of reduced [Cl2]i in the
FIG. 22. Effects of IAA-94 (an inhibitor of apical membrane-located
Cl2 channels) on saturable binding of [3H]bumetanide to confluent
cultures of dog tracheal epithelial cells (grown on permeable supports)
caused by various stimulators of transepithelial Cl2 secretion. The
following concentrations of secretagogues were used: isoproterenol, 5
3 1026 M (basolateral medium only); ATP and UTP, 10 mM (apical
membrane only). aSignificantly different from appropriate control (with
or without IAA-94) at P 5 0.05 level by Student’s t-test. bSignificantly
different from identical condition without IAA-94 (P , 0.05). [Modified
from Haas et al. (120).]
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squid axon were known (e.g., Ref. 28), Haas and coworkers suggested that the link between apical Cl2 channel activation and NKCC activation might be the reduced
[Cl2]i.
The [Cl2]i-coupling hypothesis specifies that it is the
reduction of [Cl2]i that leads to the activation or upregulation of the NKCC. Thus a direct test requires experiments in which [Cl2]i is either controlled or measured and
changed independently of other factors known to stimulate Cl2 secretion. Haas and McBrayer (115) achieved
control of [Cl2]i by applying nystatin to the apical membrane of a confluent monolayer of dog tracheal epithelial
cells. Nystatin increases the membrane permeability to
small, univalent ions, especially cations (39, 206, 308),
thereby giving some control over [Cl2]i (but causing
changes in the intracellular concentrations of Na1 and K1
at the same time). The secretagogue effects of isoproterenol and UTP on transepithelial fluxes were measured
under two conditions. In one case, the [Cl2]i was uncontrolled (without nystatin), and in the other case, [Cl2]i
was nystatin-clamped to the level of the [Cl2] in the apical
media (;125–130 mM). Figure 23 compares the effects of
isoproterenol and UTP in the absence or presence of
nystatin treatment on monolayers. When [Cl2]i changes
were prevented by “nystatin clamping,” UTP no longer
stimulated 36Cl transepithelial fluxes. Thus qualitatively, if
not quantitatively, these results agree with those reported
252
JOHN M. RUSSELL
earlier using IAA-94 or high [K1]b and measuring [3H]bumetanide binding and 36Cl fluxes. In the most direct test to
date of the [Cl2]i-coupling hypothesis, Haas and
McBrayer (115) measured the bumetanide sensitivity of
the transepithelial 36Cl flux across confluent epithelia
whose “intracellular” [Cl2] was varied over the range of
124, 66, and 32 mM by applying nystatin to the apical
membrane in media containing the test [Cl2]. Figure 24A
shows that when [Cl2]i was reduced, the total 36Cl flux
was increased, whereas the bumetanide-insensitive flux
was essentially unaffected. When the difference between
these two fluxes, which is the bumetanide-sensitive 36Cl
flux, is plotted against the nominal [Cl2]i in Figure 24B, a
relatively steep relationship is revealed. This relationship
is well-fitted by a Hill function when the Hill coefficient is
set to 2.
Could the UTP effects on transepithelial Cl2 flux
illustrated in Figure 24 be explained entirely on the basis
of a channel-mediated reduction of [Cl2]i? Examination
of Figure 24 shows that the total 36Cl transepithelial flux
(in the absence of nystatin) is increased about 2.3-fold;
this is close to the 2.64-fold stimulation reported by Haas
et al. (119) for the same treatment. Because these latter
measures of UTP stimulation used total 36Cl flux (including the bumetanide-insensitive flux), the stimulation of
the NKCC-mediated flux (the bumetanide-sensitive flux)
is likely to be somewhat larger. According to the relationship illustrated in Figure 24 to achieve a 2.5-fold stimula-
tion of bumetanide-sensitive 36Cl flux by reducing [Cl2]i
from its resting level of [Cl2]i equals 60 mM, [Cl2]i must
fall to ;11 mM. Shorofsky et al. (322), using Cl2-sensitive
microelectrodes, measured the decrease of intracellular
Cl2 ion activity after exposure to epinephrine and found
it decreased from 47.2 to 32.2 mM. Using an activity
coefficient of 0.78, this converts to a [Cl2]i decrease from
60.5 to 41.3 mM. Thus, as we saw earlier in this section for
cAMP-stimulated fluxes in shark rectal gland, the actual
decrease of [Cl2]i is much less than apparently required
according to the relationship in Figure 24. There are at
least two major reasons for this apparent discrepancy
between the data and the prediction of the [Cl2]i-coupling
hypothesis. First, Haas and co-workers have shown that
the stimulatory effects of b-adrenergic agonists such as
isoproterenol and epinephrine do not depend entirely on
a fall of [Cl2]i to stimulate transepithelial fluxes. Second,
it should be pointed out that the conditions of the experiments whose results were used to generate the relationship illustrated in Figure 24 were such that as [Cl2]i was
being varied, there was a concomitant and substantial
increase of [Na1]i as well as a depletion of [K1]i occurring. Because it has been shown that an increase of [Na1]i
will inhibit the NKCC (29, 351; see sect. IXB2), it is likely
that this figure underestimates the actual [Cl2]i sensitivity
of the NKCC of the dog tracheal epithelial cell.
The possibility that [Cl2]i might serve as a proximate
signal for the activation of the NKCC was also addressed
by Foskett and co-workers (74, 75, 296) using another
secretory epithelial preparation, the rat salivary acinar
cell. The results of these studies by Foskett and co-workers taken all together are consistent with a fall of [Cl2]i
being necessary (but not sufficient) for the coordinated
activation of at least three Na1 uptake pathways (74, 75,
296).
In a study that tied together several seemingly disparate lines of evidence regarding muscarinic stimulation
of salivary secretion, Robertson and Foskett (296) measured net Na1 uptake into single salivary cells as a measure of the combined activities of the NKCC, the Na1/H1
exchanger (NHE), and a nonselective cation channel. In
this preparation, carbachol elevates [Ca21]i, which in turn
activates apical Cl2 channels and basolateral K1 channels
(cf. Fig. 21). It was known that secretion by the salivary
glands can be inhibited by bumetanide and by amiloride
analogs. It was further known that the activities of the
NKCC and the NHE were stimulated by carbachol (243).
The authors proposed that a fall of [Cl2]i is critical to the
activation of all three of these Na1 transporting pathways.
They showed that raising [K1]o, which would be expected
to reduce the loss of K1 and Cl2 and hence prevent a fall
of [Cl2]i, prevented the increase of [Na1]i which ordinarily is observed upon application of carbachol. This
result is consistent with their hypothesis, but a complication is that carbachol also causes the salivary cells to
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2
36
FIG. 23. Effect of [Cl ]i on response of apical-to-basolateral
Cl
fluxes across confluent cultures of dog tracheal epithelial cells to isoproterenol and UTP. Control cells had normal [Cl2]i (66 mM, assuming
an activity coefficient of 0.72; see Ref. 212). Exposure of apical membrane to nystatin has effect of “clamping” [Cl2]i to [Cl2] of fluid bathing
apical face of epithelium, in this case, 132 mM. Isoproterenol (5 mM) and
UTP (10 mM) were presented to apical membrane. Each control bar
represents 8 determinations; each isoproterenol bar and each UTP bar
represents 4 determinations each. aSignificantly different from appropriate control (with or without nystatin) by Student’s t-test. bSignificantly
different from same condition without nystatin. [From Haas and
McBrayer (115).]
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January 2000
Na1-K1-Cl2 COTRANSPORT
253
shrink, and cell shrinkage is a well-known stimulant for
both the NKCC and the NHE. Thus it was necessary to
show that the shrinkage was not the proximate signal to
stimulate these Na1 transporters. To address this, the
authors performed a clever study in which they prevented
the carbachol-induced cell shrinkage by simultaneously
exposing cells to carbachol and a gradually reducing extracellular osmolarity such that the tendency to shrink
caused by K1-Cl2 loss was just offset by the extracellular
hypotonicity. Figure 25 shows the results of one of these
experiments. It shows that even when cell volume is
unchanged, the presumed fall of [Cl2]i caused by the loss
of KCl is sufficient to stimulate Na1 uptake. Although
these results support the [Cl2]i coupling hypothesis, they
add two new pieces of information. First, in this cell type,
the fall of [Cl2]i stimulates not only NKCC activity, but
also stimulates NHE-mediated transport. In contrast, others have reported that increases of [Cl2]i stimulate NHEmediated transport (e.g., Refs. 138, 278). It is possible the
different results reflect different isoforms of the NHE. In
fact, the contribution of the NKCC to the measured variable ([Na1]i) is relatively small, being only ;15% of the
total measured Na1 uptake. The second important point
is that Foskett’s group (356) has shown that without a rise
in [Ca21]i, treating the cell in ways that would be expected to reduce [Cl2]i does not stimulate these Na1
transport pathways. This suggests an as yet unknown key
role for Ca21.
2
B) [CL ]i AND PHOSPHORYLATION OF THE NKCC. This brings
us to a consideration of how a reduction of [Cl2]i might
exert a regulatory effect on the NKCC. Lytle and Forbush
(213) showed that activation of the NKCC of the shark
rectal gland by either cAMP-dependent secretagogues
(such as vasoactive intestinal polypeptide, adenosine, or
forskolin) or by cell shrinkage promoted the phosphorylation of the cotransport protein at serine and threonine
residues. They also demonstrated that treatment with
some protein kinase inhibitors could prevent activation of
the NKCC (measured as [3H]benzmetanide binding). They
suggested that [Cl2]i affected the degree of phosphorylation (and hence, activity) of the cotransporter.
Haas et al. (119) directly tested this part of the [Cl2]i
coupling hypothesis using nystatin-treated tracheal epithelial cells. As discussed in section XB1A, the application
of nystatin to the apical membrane permits control over
[Cl2]i in the absence of changes in cell volume. In a series
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2
FIG. 24. Apparent effect of [Cl ]i on bumetanide-sensitive 36Cl flux across confluent monolayers of dog epithelial cells grown on permeable supports. A: primary data
from Reference 115 showing effects of varying [Cl2] of
nystatin-containing media bathing apical membrane on
bumetanide-sensitive 36Cl flux. Circles, 124 mM [Cl2]; triangles, 66 mM [Cl2]; squares, 32 mM [Cl2]; open symbols,
bumetanide treated. B: bumetanide-sensitive transepithelial 36Cl flux, taken from primary data in A plotted against
[Cl2] in apical membrane bathing media. Line is drawn
using Hill equation with Hill coefficient set to 2.0. [A
adapted from Haas and McBrayer (115).]
254
JOHN M. RUSSELL
of parallel studies examining either transepithelial 36Cl
flux or 32P incorporation into the NKCC protein (identified via immunoprecipitation), these workers demonstrated a parallel increase of transport activity and NKCC
protein phosphorylation as the [Cl2]i was reduced.
Lytle and Forbush (216) examined the effects of
reduced [Cl2]i in isolated shark rectal gland tubules on
either [3H]benzmetanide binding (as a measure of
NKCC activity) or 32P incorporation into the NKCC
protein (identified by immunoprecipitation). Two treatments were used to reduce [Cl2]i, which according to
the hypothesis should upregulate [3H]benzmetanide
binding and stimulate phosphorylation of the NKCC
cotransporter. They showed that reducing the Cl2 concentration of the fluid bathing the tubules resulted in
increased [3H]benzmetanide binding and increased the
degree of phosphorylation of the NKCC. Because external Cl2 replacement by an impermeant anion (gluconate) would be expected to result in a net loss of Cl2
plus a cation (to maintain macroscopic electroneutrality), these cells may well have been shrunken. Cell
shrinkage per se is known to stimulate both [3H]benzmetanide binding and NKCC phosphorylation (120, 160,
214). Another means of reducing [Cl2]i was to reduce
[Na1]o (e.g., Ref. 102). Again, they noted that a treat-
ment designed to lower [Cl2]i resulted in a parallel
increase of [3H]benzmetanide binding and NKCC phosphorylation. But again there is the possibility that these
cells might shrink as a result of NaCl loss.
A rise of [Cl2]i has been shown to inhibit fluxes through
the NKCC (e.g., squid axon, Refs. 28, 29; HEK-293 cells, Ref.
93). Lytle and Forbush (216) raised [Cl2]i by raising the K1
concentration of the media bathing tubules from the shark
rectal gland. This treatment would be expected to raise
[Cl2]i as a result of two actions: by stimulating the NKCC on
the basolateral membrane to promote Cl2 uptake and by
reducing Cl2 loss across the apical membrane through a
reduction of the Vm and hence the electrochemical driving
force on Cl2 (i.e., Vm 2 ECl). They observed that this treatment reduced [3H]benzmetanide binding in cells treated
with forskolin. The inhibitory effect required the presence of
extracellular Cl2, consistent with the idea that it is caused
by an increase of [Cl2]i. However, a net increase of [Cl2]i,
along with a presumed increase of [K1]i, might also be
expected to increase cell volume, and the NKCC has been
shown to be inhibited by cell volume increases (see sect.
XC). In fact, the authors provide data which show that the
high [K1]o-treated cells did, in fact, swell. However, it should
be pointed out that exposure to hypotonic media, which also
causes the cells to swell, was shown to stimulate [3H]benzmetanide binding (214). This result might be explained by a
decrease of [Cl2]i that must inevitably occur when a cell
swells (but see sect. XC). However, Pewitt et al. (286) and
Kaji (160) found hypotonicity to reduce [3H]bumetanide
binding. Still others (e.g., Ref. 111) have shown that cell
swelling has no effect on [3H]bumetanide binding. A set of
results such as these raises additional questions about the
quantitative relationship between diuretic binding and
NKCC functional activation state (see sect. VIIIB3).
Despite these uncertainties, there is no question that
raising [Cl2]i promotes the inhibition of the NKCC. Plus,
there is strong evidence to support the view that such
inhibition is the result of a reduction in the degree of
phosphorylation of the cotransporter protein. In principle, decreased phosphorylation could result from either
inhibition of phosphorylation (via a [Cl2]i-dependent inhibition of a protein kinase) or stimulation of dephosphorylation (via a [Cl2]i-dependent stimulation of a protein
phosphatase). At this time, there is insufficient direct
evidence to distinguish between the two possibilities, but
circumstantial evidence favors an inhibitory effect of internal Cl2 on a protein kinase.
A possible role for intracellular Cl2 as an activator of
protein phosphatase was tested by Altamirano et al. (6).
Inhibition of bumetanide-sensitive 36Cl influx caused by
raising [Cl2]i could not be overcome or even offset by the
protein phosphatase inhibitor okadaic acid in the internally dialyzed squid giant axon. The fact that okadaic acid
could stimulate NKCC flux under conditions of limiting
ATP concentration showed the agent to be capable of
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1
FIG. 25. Demonstration that carbachol stimulation of Na uptake by
rat salivary acinar cells (V, bottom line) does not require cell shrinkage
(□, top line). Cell was surperfused with a solution that contained 10 mM
carbachol and whose osmolarity was reduced by reduction of NaCl
concentration to 50 mM. Reduction in tonicity was achieved gradually
by continual perfusion of various gradations of hypotonicity to maintain
a steady-state volume. Thus osmotic water loss (osmotically tied to net
KCl loss) that normally accompanies carbachol treatment is just offset
with hypotonically induced water gain. Ouabain (1 mM) was present to
ensure that change of [Na1]i was reflecting changes of Na1 uptake.
Other experiments showed that this Na1 uptake occurred through three
pathways: NKCC (;30%), Na1/H1 exchanger (;55%), and a nonselective cation channel (;15%). Thus cell volume per se is not a signal to
stimulate Na1 uptake pathways. These data are from a single cell but are
representative of 20 similar cells. V/Vo 5 cell volume (V) normalized to
initial cell volume (Vo). [From Robertson and Foskett (296).]
Volume 80
January 2000
Na1-K1-Cl2 COTRANSPORT
repulsion of positive charges found within the oxygen
binding cavity of hemoglobin. At present, there is no
evidence either for or against this possibility.
2. Effects of other intracellular ions
Given the profound effects of intracellular Cl2 on the
NKCC, it is of interest to consider the possibility that the
two cotransported cations might also have effects on
cotransport activity.
1
1
A) EFFECTS OF INTRACELLULAR NA AND K . In 1993, two
groups reported findings which suggested that NKCC-mediated ion influxes were inversely related to [Na1]i. Whisenant
et al. (351) reported that a 20-min incubation of osteosarcoma cells (UMR-106–01 cells) in a Na1-free media (Nmethyl-D-glucammonium1 replaced Na1) resulted in a tripling of the bumetanide-sensitive 86Rb uptake. Because no
measurements were provided of intracellular ion concentrations or cell volume, one cannot be certain whether the
observed effects were the result of a parallel depletion of
Cl2, a potential result of the inhibition of the NKCC by the
0-[Na1]o condition, and/or by a possible cell shrinkage
caused by a net loss of Na1 plus Cl2.
Ikehara et al. (144) varied the duration of ouabain
treatment to examine the effect of changing [Na1]i on
ouabain-insensitive 86Rb influx into HeLa cells. The fundamental observation was that as [Na1]i increased (and
[K1]i decreased), the ouabain-insensitive 86Rb unidirectional influx decreased. They interpreted this result to
mean that intracellular Na1 inhibits influx via the NKCC,
whereas intracellular K1 is necessary to interact with a
“regulatory cation binding site.” Although this is a very
intriguing hypothesis, the design of the study makes it
difficult to know the basis of the observed changes. In
addition, there are important uncertainties about the effects of their treatment on pHi, Ca21 concentration, and
membrane potential and how these variables might affect
ouabain-insensitive 86Rb influx.
Breitwieser et al. (29) replaced intracellular Na1 with
N-methyl-D-glucammonium1 while maintaining [K1]i,
[Cl2]i, [Ca21]i, pHi, and cell volume constant in the internally dialyzed squid giant axon. They reported that increasing [Na1]i to 200 mM could partially (;65– 80%)
inhibit the bumetanide-sensitive influxes of both Cl2 and
Na1. The apparent affinity of Na1 for the inhibitory “site”
was increased when the [Cl2]i was increased. This is the
expected effect for an increase of intracellular product
concentration (e.g., [Na1]) if the cotransporter follows an
ordered binding and release kinetic model. Importantly,
raising [Na1]i does not inhibit bumetanide-sensitive 22Na
efflux; the relationship between [Na1]i and bumetanidesensitive 22Na efflux is a simple monotonic activation
curve. This latter result is completely different from the
relationship between [Cl2]i and bumetanide-sensitive effluxes of Cl2, Na1, and K1 where a high [Cl2]i can com-
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preventing NKCC dephosphorylation. Taken together,
these results suggest that the inhibitory effect of raising
[Cl2]i was not mediated by an enhancement of the okadaic acid-sensitive protein phosphatases (serine/threonine protein phosphatases 1 and 2A). In addition to the
above experimental evidence against [Cl2]i-triggered regulatory role for protein phosphatases, there is the general
observation that protein phosphorylation is rarely, if ever,
found to be regulated at the protein phosphatase level.
This is presumably because of the relative lack of specificity of such enzymes.
In view of the possibility that the intracellular Cl2
effect is mediated via a protein kinase, a recent report of
an effect by Cl2 on a protein kinase in a nasal epithelium
is quite interesting (344). These workers demonstrated
that protein phosphorylation was a biphasic function of
[Cl2]i. This is highly reminiscent of the effect of [Cl2]i on
NKCC cotransport in the squid giant axon (28,29), tracheal epithelial cells (115), and HEK-293 cells (93). A
particularly interesting part of this observation is that the
protein kinase being affected by the change of [Cl2]i was
one selective for GTP. (Protein kinase CK2 is known to be
able to utilize GTP almost as easily as it uses ATP, e.g.,
Refs. 94, 128.) As discussed previously, there is only one
report of an effect of a CK2 inhibitor on NKCC activity
(254).
Finally, a recent report by Isenring and Forbush (147)
may provide some further insight into the relationship
between [Cl2]i and activation of the NKCC. Xu et al. (358)
had reported that functional activation of expressed
NKCC in HEK-293 cells requires preincubation in Cl2-free
medium. Isenring and Forbush (147) examined the kinetics of activation of bumetanide-sensitive 86Rb uptake in
HEK-293 cells exposed for various times to Cl2-free medium. This treatment would reasonably be expected to
lower [Cl2]i in a time-dependent manner. They observed
that the longer the preincubation time, the greater is the
stimulation of NKCC-mediated flux. The stimulation was
accomplished by an increase of Vmax without a change in
the apparent Km for Cl2. This result seems to indicate that
reducing [Cl2]i activates the NKCC by “turning on” a
population of cotransporters that were previously inoperative. Whether this activation occurs as a result of phosphorylation or translocation from a subcellular site is
presently unknown.
2
C) A DIRECT EFFECT OF INTRACELLULAR CL ? Alternatively, it
2
is possible that intracellular Cl directly affects the NKCC
protein itself, rather than indirectly through an effect on a
protein kinase or phosphatase.
Intracellular Cl2 might exert its inhibitory effect by
an allosteric effect on the NKCC protein directly to hinder
or prevent its phosphorylation. Chloride has been shown
to have an allosteric effect on the binding of oxygen to
hemoglobin (285, 321). An increased [Cl2] leads to a
reduced oxygen affinity by neutralizing the electrostatic
255
256
JOHN M. RUSSELL
86
FIG. 26. Bumetanide-sensitive Rb uptake into ferret red blood cells
as a function of [Mg21]i. [Mg21]i was set by incubating red blood cells in
a solution containing 10 mM A-23187 and variable [Mg21]. Cells were
incubated in variable [Mg21] solutions for 13 min, then 86Rb was added.
Samples were taken at 1 and 3 min to measure uptake. {Replotted from
Flatman (68) by converting [Mg21]o to [Mg21]i by multiplying by 2.2.}
tial bumetanide-sensitive 86Rb uptake (Fig. 26). How is
this possible if NKCC activity depends on the protein
being phosphorylated and such phosphorylation would be
expected to be severely compromised at such low
[Mg21]i? There are at least two possibilities. One is that
not only do protein kinases require Mg21 for their activation, but so do protein phosphatases. Hence, in the nominal absence of ionized magnesium, dephosphorylation
may also be severely compromised, leaving the NKCC
protein in a partially phosphorylated state (but see Ref.
71). Another possibility is that by reducing the [Mg21]i,
KCC was activated. Potasssium-chloride cotransport is
known to be activated by treatments that reduce the
phosphorylation state (e.g., Ref. 154). However, given the
relatively high bumetanide concentration used in this
study (0.1 mM), KCC-mediated fluxes, if present, would be
largely inhibited. The effects reported are generally consistent with [Mg21]i being a cofactor essential in the phosphoryltransferase reactions believed to be involved in the
activation/deactivation of the NKCC.
Hegde and Palfrey (130) reported that Mg21 has a
biphasic effect on [3H]bumetanide binding to duck erythrocyte membranes. Thus, at [Mg21] between 1 and 10 mM,
there was some increase in the specific binding, whereas
at [Mg21] .10 mM, such binding was inhibited. Interestingly, the stimulation of specific binding was unaffected
by the addition of various adenine or guanine nucleotides.
This suggests that the effect of Mg21 on specific [3H]bumetanide binding is not a result of phosphorylation of the
NKCC. Coupled with the results discussed below, it provides another instance in which the same treatment has
different effects on [3H]bumetanide binding and on
NKCC-mediated ion fluxes.
Palfrey and Prewitt (267) also reported effects of
reducing [Mg21]i on NKCC cotransport fluxes. They pretreated duck RBC with 10 mM A-23187 plus 0.5 mM EDTA
for 30 min. Starke (331) showed this treatment to reduce
the [Mg21]i to an undetectable level. Palfrey and Prewitt
(267) demonstrated that activation of the avian NKCC by
cAMP could be reversed by subsequent reduction of
[Mg21]i. This fits with the view that cAMP stimulation
requires a PKA-mediated phosphorylation (although, as
we have seen in sect. IXA2B, the PKA-mediated phosphorylation is unlikely to be the NKCC protein itself) and PKA
activity requires Mg21. Of particular interest was their
finding that NKCC activation by calyculin A (an inhibitor
of type 1 protein phosphatase) or by exposure to hypertonic fluids could not be reversed by subsequent reduction of [Mg21]i. This observation suggests the possibility
that cell shrinkage-induced NKCC activation results from
inhibition of a protein phosphatase rather than by stimulation of a protein kinase.
21
C) EFFECTS OF INTRACELLULAR CA . There are numerous reports linking changes of [Ca21]i with effects on the
NKCC (67, 82, 130, 157, 207, 251, 258, 262, 330, 349). Most
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pletely block the NKCC-mediated cotransport effluxes of
all three ions. Thus intracellular Na1 appears to inhibit by
a kind of “end-product inhibition” mechanism (unlike the
effect of intracellular Cl2, which is more consistent with
action at a non-cotransporting ion binding site).
21
B) EFFECTS OF INTRACELLULAR MG . Given that ATP is
essential for the operation of the NKCC, presumably by
phosphorylating the cotransporter protein, it is not surprising that changes in intracellular Mg21 levels have
significant effects on cotransport function. Although studies on the role of Mg21 in cotransport function have been
relatively few (e.g., Refs. 62, 68), they have yielded clear
results. Flatman (68) measured the bumetanide-sensitive
86
Rb uptake into ferret RBC as a function of the [Mg21]i
using the ionophore A-23187 to adjust the [Mg21]i. Figure
26 shows that cotransporter-mediated 86Rb uptake is increased as the [Mg21]i is increased. Flatman (68) also
showed that the effects of varying the [Mg21]i cannot be
attributed to changes of total ATP content. Two interesting features can be seen in this relationship. First, the
bumetanide-sensitive 86Rb influx rate is dependent on
intracellular Mg21 in a concentration-dependent manner.
The sigmoid shape of the relationship suggests that the
cooperative interaction of more than one Mg21 is involved
in the activating effect. This is consistent with a role for
Mg21 in the phosphorylation of the NKCC. Phosphoryltransferase reactions require Mg21 to bind ATP in a stoichiometry of 2 Mg21:1 ATP (350). Second, even at very
low [Mg21]i (nominally 0.16 mM), there is still a substan-
Volume 80
January 2000
Na1-K1-Cl2 COTRANSPORT
tion of K1 uptake as pHo was lowered. The two studies
differ on whether the observed inhibitory effect on the
NKCC was mediated directly by pHo changes or was
actually a result of pHo-induced changes of pHi.
Paris and Pouysségur (276) performed their studies
on the PS120 mutant of the CCL39 hamster lung fibroblast
cell line. This mutant cell line lacks the amiloride-sensitive Na1/H1 exchanger, so in the nominal absence of
HCO2
3 , it has a very limited ability to regulate its pHi (190).
Their study demonstrated that this mutant possessed a
bumetanide-sensitive cotransporter mediating extracellular Na1- and extracellular Cl2-dependent K1 (86Rb) uptake as well as extracellular K1-dependent 22Na uptake.
As we have already discussed, this is good evidence that
the observed fluxes are mediated by the NKCC. They
further demonstrated that these bumetanide-sensitive
fluxes were greatly stimulated by treatment with the
growth factor a-thrombin. When they varied pHo while
measuring bumetanide-sensitive 86Rb uptake, they found
that reduction of pHo below ;7.2 progressively inhibited
the NKCC-mediated influx (Fig. 27).
Given that these cells are unable to regulate their pHi,
the authors also measured the pHi of these cells (using the
distribution ratio of a weak acid, benzoic acid). When the
bumetanide-sensitive fluxes were plotted as a function of
pHi, the result was that NKCC-mediated uptake was inhibited at pHi values below ;7.0 (Fig. 27). To distinguish
whether pHo or pHi or both was the determining factor in
the acidic pH-mediated inhibition of the cotransporter,
they performed a clever maneuver that permitted them to
measure the bumetanide-sensitive flux at the same pHi
while pHo was held at two widely dissimilar values. Thus,
at pHo 6.5, they added 20 mM of the weak base diethylamine to raise pHi to ;7.0. With cells bathed at pHo 8.0,
they pretreated cells with 1 mM NH1
4 and then removed it
to cause pHi to be ;7.0 (see Ref. 297 for description of the
NH1
4 prepulse technique for intracellular acidification).
The outcome was that when pHi ;7.0, the bumetanidesensitive 86Rb uptake was the same, regardless of whether
pHo was 6.5 or 8.0. This result strongly supports the view
that an intracellular-facing site of the NKCC (or some
important modulator, e.g., a protein kinase) is pH sensitive, whereas the extracellular-facing portion of the NKCC
has little sensitivity to changes of pH.
Hegde and Palfrey (130) performed their studies on
duck erythrocytes and on membranes prepared from
duck erythrocytes. The reader will recall that this preparation was among the first in which the NKCC mechanism
was identified (e.g., Ref. 121). Hegde and Palfrey (130)
measured two properties of the NKCC that had been
maximally stimulated by pretreatment with norepinephrine and/or NaF. First, they examined the effects on
[3H]bumetanide binding to cell membrane fragments prepared from prestimulated cells of varying pH of the bind-
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of the observations fall into three groups: 1) [Ca21]i has
either no effect on NKCC cotransporter activity or inhibits
it; 2) a rise of [Ca21]i stimulates fluxes through the cotransporter, but does so secondary to a direct Ca21 effect
on either a K1 or Cl2 conductance; and 3) a rise of [Ca21]i
stimulates fluxes via the NKCC presumably via a PKC or
calmodulin-mediated mechanism.
Flatman (67) used A-23187 to vary the [Ca21]i in
ferret RBC over the range of 5 3 1025 to 5 3 10210 M and
observed no effect on the bumetanide-sensitive 86Rb uptake. Only when [Ca21]i was increased above 1025 M was
NKCC cotransport inhibited, and this inhibition was only
partially reversible. Similarly, Garay (82) found that increases in [Ca21]i inhibited NKCC-mediated fluxes in human RBC. Hegde and Palfrey (130) demonstrated that
Ca21 as well as a variety of other divalent cations (Mn21,
Zn21, Co21, and Ni21) all inhibited specific [3H]bumetanide binding. They also reported that this inhibition was
noncompetitive in nature.
In a number of epithelial preparations, treatments
that increase [Ca21]i have often been reported to stimulate NKCC-mediated fluxes (e.g., Refs. 207, 258, 349). The
reader will recall that according to the Cl2 coupling hypothesis discussed in section IXB1A, Cl2 and K1 channels
must be activated to lead to a reduction of [Cl2]i, which in
turn stimulates the NKCC. O’Neill and Steinberg (258)
reported that in vascular endothelial cells a rise of [Ca21]i
causes the activation of such a K1 channel, thereby explaining the stimulatory effect of agents that elevate
[Ca21]i. This presumably explains an earlier report by
O’Donnell (251) that NKCC activity of endothelial cells
was stimulated by agents that raise [Ca21]i.
However, there are still reports of activation of
NKCC fluxes by treatments that increase [Ca21]i in a
variety of cells (e.g., vascular smooth muscle cells, Ref.
262; mouse fibroblasts, Ref. 330; and Ehrlich ascites tumor cells, Ref. 157). The mechanism by which a rise in
[Ca21]i stimulates the NKCC is unknown at the present
time. In fact, it may differ from cell type to cell type.
However, from the evidence presently available, it seems
very unlikely that the NKCC protein itself is directly affected by changes of [Ca21]i.
D) EFFECTS OF PHi. Ordinarily both pHi and extracellular pH (pHo) are closely regulated within relatively narrow limits (e.g., Ref. 297). However, a variety of normal
and pathological perturbations are known that have significant effects on both parameters. Thus an important
part of an overall characterization of the NKCC is knowledge about its response to changes in both pHo and pHi.
Two studies have been published that explicitly examine
the effects of pH on the NKCC. Because they arrive at
different conclusions, it will be necessary to consider
their findings in some detail.
Both studies examined the activated cotransporter,
measured K1 (86Rb) uptake, and noted significant inhibi-
257
258
JOHN M. RUSSELL
Volume 80
ing medium. Figure 28A shows that the binding of bumetanide was progressively reduced as the pH of the binding
medium was reduced below ;7.6. Thus this result qualitatively agrees with the observations of Paris and Pouysségur (276) in that an acidic pH inhibits a function or
property of the NKCC. However, this isolated membrane
fragment preparation does not permit one to determine
the sidedness of the pH effect.
To address the sidedness issue, Hegde and Palfrey
(130) examined the effects of changing pH on 86Rb influx
into intact, prestimulated duck RBC. As discussed in section IID, duck RBC possess an anion exchanger (band 3)
that, under ordinary circumstances, mediates near-equilibration of pHi with pHo. To confine their pH changes to
pHo, Hegde and Palfrey (130) pretreated the duck erythrocytes with 1 mM SITS, which irreversibly binds and
inhibits the band 3 anion exchanger (AE1). They then
examined the effects of varying the pHo on 86Rb uptake as
seen in Figure 28B. (It should be noted that the authors
measured total 86Rb uptake, not the bumetanide-sensitive
component.) The results match very well their results of
[3H]bumetanide binding in that the NKCC-mediated uptake of 86Rb uptake is progressively reduced as pHo is
reduced below ;7.6. Thus these results support the view
FIG. 28. Effects of varying pH on NKCC properties of
norepinephrine-stimulated NKCC of duck erythrocytes.
Duck red blood cells were prestimulated by applying 1025
M norepinephrine for 10 –15 min before preparing cells for
experiment. A: effects of varying pH of binding medium on
specific [3H]bumetanide binding to membranes prepared
from stimulated duck red blood cells. B: effects of varying
pHo on 86Rb uptake into duck red blood cells. In addition
to being prestimulated by treatment with norepinephrine,
these cells were pretreated with 1 mM SITS to prevent pHi
equilibration with pHo. [Data replotted from Hedge and
Palfrey (130).]
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
FIG. 27. Effects of varying pH on thrombin-stimulated
bumetanide-sensitive 86Rb uptake by fibroblasts. PS120
mutants of CCL39 hamster lung fibroblast cell line were
preequilibrated in varying external pH solutions for 30 min
before measuring 86Rb uptake in absence or presence of
0.1 mM bumetanide. Intracellular pH was measured in
samples of each treatment by exposing cells to [14C]benzoic acid. A: 86Rb uptake plotted as a function of extracellular pH (pHo). B: 86Rb uptake plotted as function of
intracellular pH (pHi). [Data replotted from Paris and
Pouysségur (276).]
January 2000
Na1-K1-Cl2 COTRANSPORT
C. Role of the Cytoskeleton
An association between ion transporters and the cytoskeleton has been postulated for some time (e.g., Refs.
25, 233). The general idea is that changes of cell volume
will be “sensed” by and affect the cytoskeleton. In turn,
the resultant cytoskeletal changes/responses will be
transmitted to the membrane ion transporters in the plasmalemma through the cortical cytoskeletal network of
actin and associated proteins. Given the prominent role
accorded the NKCC in cell volume regulation, particularly
RVI (see sect. XC), it is not surprising that functional and
structural evidence exists linking NKCC transport activity
with cytoskeletal elements, particularly actin.
Reports using two different preparations (Ehrlich
ascites tumor cells, Ref. 158; and T84 cells, Ref. 226)
suggest that depolymerization of the actin cytoskeleton is
associated with activation of the NKCC fluxes. In both of
these studies, treatment of cells bathed in isosmotic medium with cytochalasin D resulted in actin depolymerization and activation of NKCC fluxes. Some evidence also
exists suggesting that when these two types of cells are
swollen by exposure to hypotonic medium, the NKCC is
also stimulated, and the actin is depolymerized (158, 227).
This result is surprising because the NKCC is generally
associated with activation by cell shrinkage, not swelling
(see sect. XC). These seemingly incongruous results may
be the result of a reduction of [Cl2]i. We have already
reviewed the evidence that reduction of [Cl2]i stimulates
NKCC fluxes (see sect. IXB1). Cell swelling leads to reduced [Cl2]i by simple dilution of cytoplasmic contents,
whereas cytochalasin D treatment appears to activate
Ca21-dependent Cl2 channels (233), thereby depleting
intracellular Cl2. Mathews et al. (227) argue that not all
the effects of cell swelling on NKCC flux activity can be
attributed to a decrease of [Cl2]i. Their argument is based
on results of experiments on T84 cells designed to first
reduce [Cl2]i before their swelling in a hyposmotic fluid.
Cells preincubated in a medium with 72.5 mM Cl2 (control [Cl2]o 5 144 mM) still responded to cell swelling by
a greatly attenuated but still statistically significant increase of bumetanide-sensitive 86Rb uptake. However,
because swelling will further reduce [Cl2]i, this result
does not unequivocally rule out that cell swelling stimulates NKCC activity by reducing [Cl2]i.
When examining the possible role of actin on cell
shrinkage activation of the NKCC, Mathews et al. (227)
reported that cell shrinkage did not further stimulate
NKCC-mediated 86Rb uptake into cells treated with cytochalasin D. However, treatment with phalloidin, an
agent which stabilizes the fibrous form of actin (F-actin),
significantly enhanced the NKCC flux response to cell
shrinkage but nearly abolished the NKCC response to cell
swelling. Interestingly, phalloidin applied to cells bathed
in isosmotic medium had no effect on NKCC-mediated
flux.
There are reasons to favor the view that actin polymerization, not depolymerization, is the physiologically
relevant change in actin state related to regulation of the
NKCC. First, cell shrinkage (known to activate the NKCC;
see sect. XC and Table 6) would tend to increase the
intracellular ionic strength. Actin polymerization is enhanced by increases of ionic strength (e.g., Ref. 10). Second, regulation of cytoskeletal activity includes phosphorylation of the cytoskeleton as well as associated proteins
(e.g., Refs. 30, 333). Grinstein et al. (107) showed that
neutrophils depleted of intracellular Cl2 had an increased
level of protein phosphorylation and actin polymerization.
It is therefore tempting to suggest that an interplay among
actin, [Cl2]i, and ATP is at least partially responsible for
cell shrinkage-induced NKCC activation. According to
this scenario, a reduction of [Cl2]i promotes cytoskeletal
phosphorylation which, either through actin polymerization or some unknown effect, stimulates NKCC flux activity, or cell shrinkage might enhance actin phosphorylation in the face of a [Cl2]i that under normal ionic
strength conditions will not support/permit such actin
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that the pH sensitivity of the NKCC is confined to an
extracellular-facing site.
In light of the different results obtained in these two
studies, one needs to consider what potential problems
might be inherent in each approach. The specific [3H]bumetanide binding results reported by Hegde and Palfrey
(130) are consistent with either an intracellular or extracellular locus of action of protons, since these studies
were conducted on isolated membranes where one would
expect both “sides” of the membranes to be in contact
with the same pH. Therefore, the essential difference
between the two studies comes down to the 86Rb influx
studies. Hegde and Palfrey (130) treated their RBC with
SITS to prevent pHi changes in response to changes of
pHo. In the absence of direct measurements of pHi in this
study, it is possible that the control of pHi in these circumstances was less than perfect. In addition, as noted,
these authors measured total 86Rb influx, not the bumetanide-sensitive fraction. Thus it is likely that some of the
pHo effects would be exerted on other K1 transport processes such as channels. For example, the data in Figure
28 show that at pH 6, the [3H]bumetanide binding is
reduced ;75% relative to maximal level reported (Fig.
28A), whereas the 86Rb influx is reduced ,50% (Fig. 28B).
Thus a combination of uncertainty about pHi and the
possibility of another K1 transport pathway contributing
to the observed effect on 86Rb influx raises important
reservations about the conclusion that it is pHo that inhibits the NKCC. In summary, the available evidence favors the interpretation that acidic pHi inhibits the NKCC,
whereas changes of pHo have little or no effect.
259
260
JOHN M. RUSSELL
phosphorylation. This would explain the apparent shift of
the intracellular Cl2 inhibitory relation caused by cell
shrinkage reported for the squid axon (28).
In summary, there is solid evidence of a cytoskeletal
role in the regulation of NKCC activity. It is presently
unclear whether actin polymerization or depolymerization is important for stimulation of the NKCC. Circumstantial evidence suggests that [Cl2]i interacts with cytoskeleton (perhaps via a [Cl2]i-mediated effect on actin
phosphorylation) to modulate NKCC activity. This is a
rich area for future research.
Of the numerous normal and pathological functions
that have been ascribed to the NKCC, I discuss four in
some detail. I used two main criteria to select these four:
1) there had to be considerable evidence favoring the
function, and 2) the function must be of importance for
many cells or of great importance to the organism as a
whole.
A. Role in Net Cl2 Transport by Epithelial Tissues
The ability of transporting epithelia to move water
and electrolytes between biological compartments is
one of those functions that is key to organismal survival. The NKCC plays a very prominent role in a variety
of epithelial absorptive and secretory processes. This
cotransporter is important not only for normal physiological functions of epithelia but has also been implicated in some diseases (e.g., Bartter’s syndrome, cholera). In epithelia, the NKCC is part of a two-step
mechanism that accomplishes the net transfer of Cl2
across the epithelial cell layer from one biological compartment to another. The NKCC serves to raise [Cl2]i of
the absorptive or secretory epithelial cell above electrochemical equilibrium. A higher than electrochemical
equilibrium [Cl2]i provides the driving force for Cl2 to
exit the cell on the other side, usually via a Cl2 channel
(see Fig. 21). In general, Cl2 absorptive cells have the
NKCC on the apical membrane (99). In the thick ascending limb of the loop of Henle in the kidney, the
apical NKCC is the NKCC2 isoform. It is not yet known
if net Cl2 absorption is always associated with the
NKCC2 isoform. Cells that secrete Cl2 typically have
the NKCC1 isoform located on the basolateral membrane.
B. To Maintain [Cl2]i at Higher Than
Equilibrium Values
As we have seen in the preceding sections, there is
abundant functional and molecular biological evidence
that the NKCC is found in nearly every cell type. Such a
widespread distribution suggests a fundamental cellular
homeostatic role or roles for this cotransporter in addition to its central role in transepithelial solute and water
transport. Because in most cells this ion transport mechanism is thermodynamically poised to perform net influx
(see sect. VIB), its near-universal presence in cells may
derive from its ability to either 1) maintain [Cl2]i at levels
above electrochemical equilibrium and/or 2) to serve as a
route for net Na1 and K1 uptake to achieve either transient increases in the intracellular concentrations of these
two cations or changes in cell volume as a result of their
net uptake (coupled with Cl2). We examine these possibilities in each of the following sections about specific
proposed functions of the NKCC.
There are numerous reports in the literature, particularly before 1985, of [Cl2]i values for a variety of cells
that are higher than expected from passive electrochemical considerations (e.g., cf. sect. VIB). In addition to the
NKCC, there are three Cl2 transport mechanisms described in the literature that, in principle, are capable of
generating and maintaining such high [Cl2]i levels: 1) the
NCC, which seems to be limited to certain epithelial
tissues (but see Ref. 57; cf. sect. IIB); 2) a Cl2-ATPase
pump has been described in Aplysia gut (91) but thus far
nowhere else; and 3) Na1-independent Cl2/HCO2
3 exchanger (AE2) is also widespread, and it is generally more
active at alkaline pHi levels than at normal pHi values
(361).
As mentioned in section VIB, the free energy in the
transmembrane gradients of Na1, K1, and Cl2 favors net
Cl2 uptake by the NKCC for most cells (cf. Table 4).
However, there have been very few studies designed expressly to determine whether the NKCC is the mechanism
by which a cell maintains a high [Cl2]i. This probably
reflects the fact that such studies are technically difficult,
requiring accurate measurement of the [Cl2]i under several test conditions as well as having available accurate
and relevant estimates of the Vm.
The first such study was performed on shark rectal
gland epithelial cells (104). Using ion-selective microelectrodes, they measured an intracellular Cl2 activity (5
[Cl2]i 3 activity coefficient) in “resting” cells of 49 mM.
The calculated Cl2 activity necessary to be in passive
electrochemical equilibrium was 6 mM. Thus intracellular
Cl2 activity was nearly eight times greater than expected
for a passive distribution of the anion. Furthermore, when
these cells were exposed to furosemide (50 –100 mM) in
the external medium, they rapidly lost Cl2. These results
showed that the higher than electrochemical equilibrium
distribution of Cl2 was at least partially dependent on a
furosemide-sensitive process. As the authors showed, the
high intracellular Cl2 activity served as a driving force for
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X. FUNCTIONS OF THE SODIUM-POTASSIUMCHLORIDE COTRANSPORTER
Volume 80
January 2000
Na1-K1-Cl2 COTRANSPORT
C. Cell Volume Regulation and the NKCC
Although it is generally accepted that the NKCC plays
a direct role in cell volume regulation (e.g., Refs. 110, 134,
188, 266), the available evidence is often circumstantial,
and it is usually difficult to determine whether the NKCC
is a physiological volume regulatory mechanism or
merely a volume-sensitive transport mechanism. Before
reviewing these issues, it is useful to first briefly consider
how cells control their volume and the expected properties of a volume regulatory NKCC (as distinct from a
volume-sensitive NKCC).
Animal cells lack rigid cell walls, and their cell membranes are sufficiently water permeable so that water is at
thermodynamic equilibrium across the cell membranes.
This means that intracellular effective osmolality is always equal to extracellular effective osmolality. It therefore follows that cell volume will be determined by the
cell osmolyte content. Thus the maintenance and regulation of cell volume, a fundamental cellular homeostatic
process, is ultimately dependent on the regulation of the
intracellular osmolyte content.
When shrunken by exposure to hyperosmotic solutions, many cells activate solute uptake mechanisms that
result in a recovery of the original volume. This overall
general process has been termed RVI. A variety of solute
transporters have been shown to be capable of accomplishing RVI (e.g., Ref. 124) including ion transporters
such as the NKCC. The particular transport mechanism
utilized apparently varies from cell to cell. Some cells use
a combination of the NKCC plus the pHi-coupled NHE and
the Cl2/HCO2
3 exchanger (e.g., Refs. 272, 298). Such redundancy probably reflects the importance to cell viability of maintaining and regulating cell volume.
In addition to being able to regulate cell volume in
the face of relatively sudden and large cell volume perturbations caused by being exposed to anisosmotic solutions, living cells have another, more subtle, but more
constant cell volume maintenance problem with which
they must cope, namely, as a result of the continuous
leaking of solutes into and out of the cell, there is a
constant potential for cell volume changes. Therefore, to
maintain cell volume constant, even under isosmotic conditions, these solute leaks must be perfectly matched with
solute pumping of the exact same magnitude, but in the
opposite direction.
The NKCC-mediated processes have been associated
with RVI in some cells since before we even knew the ion
movements being measured were via an NKCC (e.g., Refs.
186, 187, 189, 314 –316). Given this long historical association, it is little wonder that the most commonly cited
function of the NKCC in symmetric cells (nonepithelia) is
that of participating in RVI (e.g., Refs. 110, 114, 134, 266).
In most cells, the NKCC is thermodynamically poised to
perform net solute uptake (see sect. VIB). This fact, com-
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the loss of cellular Cl2 across the apical membranes
through Cl2-selective channels activated by cAMP. This
combination of NKCC and Cl2 channels will accomplish
net transepithelial Cl2 movment, in this particular case,
secretion.
Alvarez-Leefmans and co-workers (13, 14) performed
a somewhat more detailed study on nonepithelial cells,
namely, frog dorsal root ganglion cells. These are neurons
of the sympathetic nervous system that reside outside the
central nervous system. By using double-barreled electrodes (one side was Cl2 sensitive, the other measured
Vm), they measured an intracellular Cl2 activity of ;31
mM. Given an [Cl2]o of ;122 mM, the calculated ECl was
233 mV, a value quite different from the measured Vm of
258 mV. For the intracellular Cl2 activity to be in electrochemical equilibrium with a Vm of 258 mV, [Cl2]i
would have to be 12 mM. Therefore, the actual [Cl2]i in
these frog neurons was 2.5 times greater than predicted
by the electrochemical driving force. Such an ionic disequilibrium can only be maintained by an active transport
process. When either extracellular Na1 or K1 was replaced by a cation (N-methyl-D-glucammonium1) that
does not support NKCC cotransport, the neurons underwent a net loss of intracellular Cl2 that could be reversed
by return of the missing cation. The recovery of [Cl2]i
could be blocked by treatment with 10 mM bumetanide.
These results clearly show that the NKCC can raise the
[Cl2]i to levels well above that predicted for the passive
distribution of Cl2 across the membrane.
In these sensory neurons, having [Cl2]i at a relatively
high level serves to provide a depolarizing electrical battery, i.e., ECl is more positive than Vm. In the presynaptic
neuron, this can be used by GABAA receptors to produce
a depolarization of the presynaptic membrane, thereby
inhibiting synaptic transmission, by a process known as
presynaptic inhibition or primary afferent depolarization
(e.g., Ref. 12).
Although the NKCC is poised to raise [Cl2]i above its
predicted electrochemical equilibrium level, its ability to
do so can be limited if there are other, “leak,” pathways to
shunt the cotransported flux. Skeletal muscle appears to
illustrate this principle to the extreme. Thus it has been
shown that when Cl2 electrodiffusive pathways are
blocked, either by a Cl2 channel blocker or by denervation of the muscle, the [Cl2]i increases by a furosemidesensitive mechanism (4, 127). This implies that skeletal
muscle possesses a functional NKCC. However, in the
normal state, Cl2 conductance, or conductive permeability, is very high, keeping [Cl2]i quite low, in apparent
equilibrium with the resting Vm of skeletal muscle. The
purpose of having an NKCC whose net transport effects
are shunted by a high electrical permeability for Cl2 is
unknown.
261
262
JOHN M. RUSSELL
1. Stimulation of bumetanide-sensitive RVI, but not
NKCC-mediated ion fluxes, often depends on the
means of cell shrinkage
Any discussion of the role of the NKCC in RVI has to
address the fact that activation of cotransport fluxes by
cell shrinkage and activation of bumetanide-sensitive RVI
(Table 6, columns A—D) may not always be directly
related. For many cells, whether or not bumetanide-sensitive RVI occurs depends on the means used to reduce
cell volume (see columns A and B in Table 6). At the same
time, for all cells tested (with a single exception), there is
the observation that cell shrinkage stimulates transmembrane ion fluxes mediated by the NKCC (Table 6, column
D). However, in about one-half these cases, RVI is not
observed (Table 6, columns A and B). Thus, although the
available data clearly indicate that the NKCC is sensitive
to cell shrinkage, the interpretation is less clear as to
whether the NKCC has the physiological responsibility to
participate in RVI.
What is the difference in the way the cell-shrinkage
stimulus is brought about in the two approaches summarized in Table 6? The most common means used by workers in this field to reduce cell volume is to expose cells to
an external solution made hyperosmotic (relative to normal media) by the addition of a nonpermeable osmolyte.
To avoid changing the thermodynamic driving force on
the NKCC, this osmolyte is usually sucrose or mannitol.
With this hypertonic experimental system, water leaves
the cell, decreasing cell volume, but increasing the concentrations of intracellular solutes (including Cl2). As
seen in Table 6 (column B), this hyperosmotic method of
cell shrinkage activates a bumetanide-sensitive RVI in
some cells (e.g., duck RBC, trabecular meshwork cells,
and retinal pigment epithelial cells) but not in others (e.g.,
ferret RBC, Ehrlich ascites tumor cells, vascular smooth
muscle cells, and vascular endothelial cells). In many
cases, rather large test osmolalities have been used (e.g.,
2 times normal osmolality). Osmotic challenges of this
magnitude are rarely encountered by the cells of most
multicellular organisms. Thus it is fair to question
whether such a hyperosmotic stimulus is a physiologically relevant means to test whether the NKCC has a
physiological role in cell volume regulation.
An alternative means of shrinking cells, usually
termed “isosmotic cell shrinkage” involves first causing
cells to undergo a net loss of intracellular osmolytes,
mainly, K1 and Cl2 and an osmotic equivalent of water.
This has been accomplished in two ways. One is a twostep process that begins with swelling cells by exposure
to hypotonic fluid. The swollen cells then undergo RVD by
losing K1 and Cl2 and osmotically obliged water. When
such solute-depleted cells are transferred to a solution
with normal osmolality (“isotonic fluid”), they shrink. An
alternative treatment to achieve isosmotic shrinkage is to
bathe the cells in an isosmotic solution from which the
Na1 and K1 have been removed by replacement with
impermeant ions. This causes the net loss of cellular
cations and anions (mostly K1 and Cl2) and an osmotic
equivalent of water. As Table 6 (column A) shows this
mode of cell shrinkage is capable of activating a bumetanide-sensitive RVI by cells that were not stimulated with
hypertonic shrinkage (e.g., Ehrlich ascites tumor cells,
vascular smooth muscle, and vascular endothelial cells;
column B). Cell volume recovery after these kinds of
priming treatments has been termed RVI after RVD, pseudoregulatory volume increase, or secondary RVI (205).
In every case reported (except for HT-29 cells, Ref.
168), cell shrinkage, by whatever means, increased the
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bined with the fact that it moves at least four osmotically
active particles into the cell per turnover, gives the NKCC
the obvious potential to be a powerful determinant of cell
volume. What then is the evidence for a physiological role
of the NKCC in cell volume regulation and maintenance?
In the following discussion, we distinguish between a
physiological role for the NKCC in cell volume regulation/
maintenance and its simply being an ion transport mechanism sensitive to changes of cell volume. By physiological role, I mean that the NKCC is an essential component
in a feedback mechanism designed to maintain cell volume at some constant set-point. The alternative is that by
virtue of changes of intracellular ion concentrations or
transmembrane ion gradients, the NKCC simply responds
to cell volume changes but does not participate in the
maintenance and regulation of cell volume.
If the NKCC fulfills a normal homeostatic function in
the regulation of cell volume as distinct from simply
responding to changes in driving forces secondarily resulting from cell volume changes, there are several testable corollaries. 1) Cell shrinkage should activate a bumetanide-sensitive RVI. 2) In cells with a bumetanidesensitive RVI, cell volume reduction should stimulate
bumetanide-sensitive Na1, K1, and Cl2 fluxes such that
there is net cation-Cl2 uptake and a consequent cell volume increase. Proof of these two corollaries represents
the minimal evidence necessary to show that the NKCC is
involved in RVI. Another closely related question is
whether the NKCC is also involved in the maintenance of
normal cell volume. If so, then the following corollaries
ought to be true as well. 3) Inhibition of the NKCC fluxes
under normally isosmotic conditions would lead to cell
shrinkage (assuming normal cell volume is the dynamic
result of cell shrinkage tendencies and cell swelling tendencies). 4) Stimulation of NKCC fluxes ought to cause
cell swelling. 5) Cell swelling might be expected to reduce
NKCC fluxes. Table 6 is a collation of experimental data
bearing on these corollaries taken from a variety of cells
and produced by a number of different labs often using
different approaches.
Volume 80
Na1-K1-Cl2 COTRANSPORT
January 2000
TABLE
263
6. Regulatory volume increase and the NKCC
Preparation
(A) RVI
Isosmotic
Shrinkage
(B) Hypertonic
Shrinkage
(C) RVI Blocked by
Bumetanide
(E) Effect of
Bumetanide on
Cell Volume
Under Isotonic
Conditions
(F) Effect of
Increased
NKCC Fluxes
on Cell Volume
NT
Stimulates*
(118)
NT
Cell swelling
(118, 187,
215)
NT
Inhibits (118)
NT
NT
Stimulates (158)
Cell shrinkage
(253)
Cell shrinkage
(2, 61)
NT
Cell shrinkage
(57)
Cell shrinkage
(45)
No effect
(259)
Cell swelling
(253)
Cell Swelling
(2)
NT
NT
Inhibits (253)
NT
NT
NT
NT
NT
NT
NT
NT
NT
Stimulates (179,
253, 257) (both
iso- and
hypertonic)
Stimulates (351)
Cell shrinkage
(253)
No effect
(257)
NT
Cell swelling
(253)
Inhibits (253,
257)
NT
NT
Stimulates (170)
(hypertonic); no
effect (340)
(hypertonic)
Stimulates (41)
(both iso- and
hypertonic)
Stimulates (29)
(hypertonic)
Stimulates (201)
(hypertonic)
Stimulates (213)
NT
NT
Stimulates (235)
No effect (41)
NT
Stimulates (41,
236)
NT
NT
NT
NT
Cell swelling
(201)
NT
No effect
(222)
NT
NT
NT
NT
Inhibits (79,
337)
NT
NT
NT
Inhibits (142)
(D) Effect of Cell
Shrinkage on NKCC
Fluxes
NT
Yes‡ (186, 314)
Yes‡ (314)
Stimulates (187,
315)
Ferret RBC
Ehrlich ascites
tumor cells
NT
Yes (89)
Yes‡
(157)
NT
No (223)
NT
Stimulates (223)
No (157, 203)
Yes (89, 157, 284)
Yes (253)
NT
Stimulates (87, 157,
204)
Stimulates (253)
NT
Yes‡ (2, 164)
Yes (2)
Stimulates (164)
NT
NT
NT
No (57)
NT
NT
Stimulates (77)
NT
Nt
NT
NT
NT
Yes
(259)
No (259)
Yes (259)
Rat arterial smooth
muscle
Vascular
endothelial cells
NT
NT
NT
Stimulates (259)
(both iso- and
hypertonic
Stimulates (44)
Yes (253,
257)
No (257)
Yes (253, 257)
Osteosarcoma cells
Yes but slow
(167)
No (170)
Yes (167)
Rat astrocytes
Yes
(167)
NT
C6 glioma
Yes* (41)
No (41)
Yes (41)
Squid giant axon
NT
NT
NT
PC12 cells
NT
Yes (201)
Yes (201)
Trabecular
meshwork cells
Retinal pigment
epithelia
Chick heart cells
Rabbit ventricular
myocytes
Rabbit atrial
myocytes
Vascular smooth
muscle
NT
Shark rectal gland
Guinea pig jejunal
villus cells
Xenopus oocytes
rabbit mTAL
(NKCC2?)
Mouse mTAL
(NKCC1 or -2?)
HT-29 (human
colonic
adenocarcinoma
cells)
Eccrine clear cells
Chinese hamster
ovary cells
NIH 3T3
(fibroblasts)
HEK-293
NT
NT
NT
Stimulates (213,
214)
NT
NT
Yes (222)
Yes (222)
Stimulates (222)
NT
NT
NT
NT
NT
NT
NT
NT
NT
Stimulates (337)
(hypertonic)
Stimulates (64)
(hypertonic)
Stimulates (161)
NT
NT
NT
No effect (169)
(hypertonic)
NT
NT
No effect (169)
Yes
(343)
NT
Yes (343)
Yes (343)
NT
NT
NT
NT
Yes (298)
NO (298)
NT
NT
NT
NT
NT
NT
Stimulated (298)
(isosmotic)
NT
NT
Cell swelling
(231)
NT
NT
NT
NT
NT
Cell shrinkage
(93)
Numbers in parentheses indicate reference for observation. RVI, regulatory volume increase; RBC, red blood cell; mTAL, medullary thick
ascending limb; NT, not tested.
* In absence of norepinephrine, which stimulates NKCC, cells will shrink.
† With 1 mM furosemide.
‡ Requires elevated extracellular [K1].
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Duck RBC
(G) Effect of Cell
Swelling on NKCC
Fluxes
264
JOHN M. RUSSELL
hypertonically shrunken cells to undergo NKCC-mediated
RVI would be that for some cells, shrinkage reduces or
even reverses the thermodynamic gradient that normally
favors net uptake via the NKCC.
Jensen et al. (157), working with Ehrlich ascites tumor cells, have explicitly tested this hypothesis. They
measured changes of cell volume and the intra- and extracellular concentrations of Na1-, K1-, Cl2-, and bumetanide-sensitive 86Rb influx within 0.5 min after the cells
were exposed to the various hypertonic solutions. Their
results appear to support the view that it is the resultant
thermodynamic gradient during the maximal shrinkage
phase that will determine whether the NKCC can mediate
RVI.
In a few cell types, increasing the favorable thermodynamic gradient simply by raising the [K1]o somewhat
will result in a bumetanide-sensitive RVI in hypertonically
shrunken cells (e.g., Ehrlich ascites tumor cells, Ref. 157;
duck RBC, Refs. 185, 186; retinal pigment epithelial cells,
Ref. 2). However, if it is simply a matter of the thermodynamic gradient, it is very surprising that raising [Na1]o
or [Cl2]o does not promote RVI in Ehrlich ascites cells
(e.g., Ref. 204). Thus it appears that a favorable thermodynamic gradient is necessary but not sufficient to cause
the NKCC to promote RVI.
This latter conclusion is supported by results of
O’Neill and Klein (257). These workers showed that vascular endothelial cells responded to isosmotic shrinkage
with both an increase of NKCC flux activity as well as an
RVI. When shrunken by exposure to hyperosmotic fluids,
NKCC fluxes increased, but there was no RVI. [3H]bumetanide binding studies showed that the relative increase in
the number of binding sites in hypertonically shrunken
cells more or less exactly matched the relative increase in
NKCC-mediated transport. They suggested that [Cl2]i may
hold the key as to whether the increased number of NKCC
copies can generate the requisite net influx or not. We
have already discussed the fact that for many cells under
isotonic conditions the net thermodynamic gradient for
cells at rest strongly favors a net uptake by the NKCC, yet
it does not occur. We (e.g., Refs. 8, 306) have presented
evidence that this failure of the NKCC to reach overall
thermodynamic equilibrium may be the result of a kinetic
type of inhibition, an inhibition which is directly proportional to the [Cl2]i perhaps mediated by changes in phosphorylation state of the cotransporter or some critical
cofactor (see sect. IXB1B) Thus O’Neill and Klein (257)
argue that the key difference between shrinking cells
using isosmotic shrinkage and using hyperosmotic shrinkage is the resultant difference in [Cl2]i. It could be that for
many cells (including vascular endothelial cells) the normal [Cl2]i is sufficiently high that when the cell is hypertonically shrunken, [Cl2]i rises into a region that severely
inhibits the NKCC (although the increase in the number of
NKCC copies in the membrane allows an overall increase
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unidirectional fluxes mediated by the NKCC (Table 6,
column D). The question then is, Why is there often no
NKCC-mediated RVI if there is always an increase of
fluxes through the NKCC? For most of these studies,
bumetanide-sensitive 86Rb influx is the flux measured.
Thus it is possible that, for the cells not undergoing RVI,
shrinkage has promoted osmotically neutral exchange
fluxes, i.e., K1/K1 exchange, via the NKCC. Such NKCCmediated exchange fluxes have been reported for several
preparations (e.g., duck RBC, Ref. 121; human RBC, Ref.
58; human platelets, Ref. 55). The NKCC-mediated K1/K1
exchange fluxes are enhanced by a high [K1]i (217), and
hypertonic shrinkage would be expected to increase [K1]i
(along with [Na1]i and [Cl2]i). It has been suggested that
the NKCC turnover rate is faster when performing exchange fluxes compared with performing net fluxes (58).
In this scenario, the increased isotopic flux would simply
reflect an increase in the proportion of cotransporters
performing exchange fluxes (fast) relative to those performing net fluxes (slower).
However, there are at least two types of evidence
that argue against the above sequence of events. First, in
the internally dialyzed squid giant axon, hypertonic
shrinkage increased unidirectional 36Cl influx under carefully controlled conditions that prevented changes of
[K1]i (internal dialysis) and made exchange fluxes impossible (0 mM [Na1]i and [Cl2]i, Ref. 28). Second, in several
studies, workers have demonstrated that coincident with
the shrinkage-induced increase of NKCC-mediated fluxes,
there is a shrinkage-induced increase in [3H]bumetanide
binding (e.g., Refs. 111, 160, 258). In fact, O’Neill and Klein
(257) showed that the increase in specific [3H]bumetanide
binding matches the increase in flux almost perfectly,
resulting in no change in the calculated turnover number
for the cotransporter. A similar set of results has been
reported for the duck RBC (Refs. 111, 118; cf. Fig. 15). The
[3H]bumetanide binding studies support the view that
shrinkage induces an increase in the numbers of functional NKCC copies in the cell membranes. This view is
also supported by the results of Lytle (212) which show
that cell shrinkage increases the degree of phosphorylation of the NKCC protein. If the number of functional
copies of the NKCC is increased as a result of hypertonic
cell shrinkage, why do some cells perform RVI and others
do not?
To perform RVI, the NKCC must mediate a net uptake of ions, and to mediate net uptake, the thermodynamic driving force on the NKCC must favor uptake while
the cells are shrunken (regardless of the number of functional copies). However, hypertonic shrinkage will, of
necessity, result in an increase of the intracellular concentration of all intracellular solutes, including the three
cotransported ions. Thus it is clear that hypertonic shrinkage must reduce the driving force favoring net uptake. A
simple explanation for the ability of some, but not all,
Volume 80
January 2000
Na1-K1-Cl2 COTRANSPORT
2. Role for the NKCC in cell volume maintenance?
If the NKCC plays a key role in cell volume maintenance (e.g., housekeeping role), then inhibition of the cotransporter ought to lead to cell shrinkage. The literature
provides only a few reports of tests of this cell-volume
maintenance role. Table 6, column E, shows that of the nine
studies which examined the effects of applying bumetanide
to cells under isotonic conditions, six reported that the
treated cells shrunk and three observed no change in cell
volume. In another approach to examining the effect of
cotransport inhibition on isotonic cell volume, one group
has compared the effect of phorbol esters on cell volume on
two cell lines: one with NKCC and one deficient in NKCC
(248). In the NKCC-containing cells, treatment with 12-Otetradecanoylphorbol-13-acetate (TPA) inhibited bumetanide-sensitive 86Rb uptake and caused the cells to shrink,
whereas in the NKCC-deficient cells, only slight cell volume
changes were noted. Clemo and Baumgarten (45) have reported that atrial natriuretic factor (ANF) inhibits the NKCC
by raising the intracellular concentration of cGMP in rabbit
atrial myocytes. Treatment with ANF or 8-bromo-cGMP resulted in cell shrinkage. This result suggests a cell volume
maintenance role for the NKCC in atrial myocyte volume
regulation. Finally, Wilcock et al. (354) have reported that
the NKCC-mediated 86Rb influx into proliferating murine
leukemia cells (L1210 leukemic cells) was selectively inhibited by alkylating mustard compounds, and the cells shrunk
after such treatment.
For those cells that do not shrink after inhibition of
the NKCC, the NKCC may not fill a cell volume maintenance regulatory role. Alternatively, the absence of a
decrease in cell volume may simply reflect the fact that,
for these particular cells, the rate of back-leak of ions is
so slow that cell shrinkage resulting from NKCC inhibition might not be detected in these relatively acute experiments. Finally, there is the real possibility that cell volume is subject to redundant mechanisms of regulation
(e.g., NHE coupled with Cl2/HCO2
3 exchange) that are
capable of maintaining cell volume for the periods covered by these studies. Several examples of cells exhibiting
such physiological redundancy in terms of effectors of
cell volume regulation can be cited (e.g., liver cells, Ref.
123; ferret RBC, Ref. 223; C6 glioma cells, Ref. 240; vascular endothelial cells, Ref. 252; vascular smooth muscle
cells, Ref. 259; Ha-Ras expressing NIH-3T3 fibroblasts,
Ref. 193).
Another corollary to the hypothesis that the NKCC
plays a key role in cell volume regulation is that stimulation of NKCC transport activity under isosmotic conditions would be expected to cause cell swelling. Table 6,
column F, shows six studies in which the NKCC was
stimulated in a variety of ways, but always while cells
were bathed in an isosmotic fluid. In every case, cell
swelling occurred.
Further evidence that the NKCC may be importantly
involved in cell volume maintenance is the recent results
by Jensen and Hoffmann (156). They asked the question:
Will prolonged exposure of Ehrlich ascites tumor cells to
a hypertonic medium cause parallel increases in expression of the NKCC protein and bumetanide-sensitive
fluxes? They exposed cells for 5 h to a growth medium
containing 35S-methionine and 35S-cysteine that also contained an additional 60 mosmol/kgH2O of sucrose making
it about 1.2 times normal osmolality. Using polyclonal
antibodies developed to the NKCC protein, they then
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of NKCC-mediated flux), whereas when isosmotically
shrunken the [Cl2]i remains low enough to be permissive
for vigorous NKCC operation in the net influx mode.
Although this is a tempting explanation, it seems the truth
is more complicated. First, there is the observation that
cell shrinkage, however mediated, stimulates unidirectional fluxes mediated by the NKCC. Second is the observation that, in the squid giant axon, shrinkage acts to
increase the NKCC-mediated 36Cl influx even when the
[Cl2]i is held constant and high (28). However, this effect
only occurs in the inhibitory [Cl2]i range; at low [Cl2]i
levels at which intracellular Cl2 does not exert an inhibitory effect, there is no further increase of NKCC-mediated flux. Thus cell shrinkage acts to shift the inhibitory
range for [Cl2]i to a somewhat higher value. This latter
effect argues against an increase in the number of functional NKCC copies in the membrane and returns us to the
issue of whether [3H]bumetanide binding is always an
accurate measure of the actual cotransport function of
the NKCC (cf. sect. VIIIB3).
Jensen et al. (157) found that replacing intracellular
86
Cl2 with NO2
Rb
3 actually reduced bumetanide-sensitive
influx after cell shrinkage of Ehrlich ascites tumor cells.
They argued that this result rules out an intracellular
modulatory role for Cl2. However, the choice of NO2
3 as
the intracellular Cl2 substitute was unfortunate, since
NO2
3 is known to substantially inhibit cotransport fluxes
(130, 345).
Given the apparent relationship between [Cl2]i and
phosphorylation of the NKCC (see sect. IXB2), it is important to point out that several studies have reported that
hypertonic cell shrinkage results in an increase of phosphorylation of the NKCC (e.g., Refs. 119, 212). As yet
however, there are no studies that examine the phosphorylation of the cotransporter in cells that require the RVD/
RVI protocol to effect bumetanide-sensitive RVI. Will the
NKCC of these cells be phosphorylated when exposed to
hypertonic media alone, or will phosphorylation require
the initial cell swelling and RVD (with the assumed decrease of [Cl2]i) before the NKCC is phosphorylated?
Finally, Ferri et al. (66) have presented evidence that
isosmotic and hypertonic activation of the NKCC fluxes
may use different intracellular signaling pathways.
265
266
JOHN M. RUSSELL
immunoprecipitated NKCC protein and found that the
hyperosmotic treatment caused a 3.4-fold increase in the
abundance of the NKCC protein and a 2.6-fold increase of
bumetanide-sensitive 86Rb influx relative to control cells.
Thus the Ehrlich cells exposed for a prolonged period to
a hyperosmotic stress apparently increased the synthesis
and insertion of the NKCC protein into the membrane.
This implies that cell volume maintenance may be a central function of the NKCC in these cells.
D. A Role for the NKCC in the Cell Cycle?
increase in cell K1 (plus associated anion) content that
would in turn cause an increase of cell volume as a result
of osmotic equilibrium. Mitogen-induced increases in cell
volume have been reported that are associated with increased activity of the NKCC (36, 201). Leung et al. (201)
showed that treatment of PC12 cells with nerve growth
factor increased NKCC ccotransport activity and cell volume. Treatment with 10 mM bumetanide inhibited the
NKCC cotransport activity and greatly reduced the cell
volume increase caused by the nerve growth factor.
Working with synchronized NIH 3T3 cells, Bussolati et al.
(36) measured cell K1 content, amino acid content, cell
volume, and numbers of cells. They showed that as the
cells progressed through the cell cycle (stimulated by
return of serum) their K1 and amino acid content nearly
doubled, peaking just before the time of cell division.
When cells were treated with bumetanide, they reported a
60% decrease in cell numbers after 24 h compared with
untreated cells. They interpret their findings to support a
role for the NKCC, together with amino acid uptake mechanisms, in generating the cell volume increase necessary
for cell division. However, the authors used 1 mM bumetanide, which is an extremely high concentration. Presumably this was to maintain an effective bumetanide concentration while allowing for binding of the agent to
serum proteins and degradation of the drug over the 24 h
of the experiment at 37°C. Results from numerous workers (see above) have clearly established that the NKCC is
stimulated as cells progress through the cell cycle. The
results of these latter two studies suggest that stimulation
of the NKCC affects the cell volume increase necessary
for cell division.
If stimulation of the NKCC is a critical component of
the cascade of events culminating in cell proliferation,
then its inhibition with bumetanide might reasonably be
expected to prevent or significantly reduce cell proliferation. However, several groups have reported that bumetanide treatment has only a small effect on DNA synthesis.
In the earliest of these studies, Amsler et al. (16) reported
that treatment of Swiss 3T3 cells with a combination of
insulin, epidermal growth factor, and arginine vasopressin was very effective at stimulating bumetanide-sensitive
86
Rb uptake and [3H]thymidine uptake into cells. However, treatment with bumetanide (100 mM) had no significant effect on [3H]thymidine uptake, while completely
blocking 86Rb uptake. Paris and Pouysségur (276) showed
that treatment of a hamster fibroblast cell line (PS 120)
with a-thrombin or epidermal growth factor greatly increased 86Rb influx, most of which was bumetanide sensitive. However, they reported that bumetanide treatment
only reduced the a-thrombin-induced reinitiation of DNA
synthesis by ;28%. Smyth et al. (329) showed that a cell
line made deficient in NKCC (BALB/c-3T3 cells) could still
be stimulated by mitogens to increase their DNA synthesis. The real question is not whether bumetanide treat-
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It is self-evident that at some stage of cell division
there must be an increase in cell volume. During the
normal cycle of cell proliferation, there may be at least
two check points at which cell size is, in some way,
monitored for suitability for entry into the next phase of
the cell cycle (e.g., Ref. 10). One check point occurs
during the G1 phase before entry into the S or DNA
synthetic phase and the other occurs during the G2 phase
before entry into the period of mitosis and cytokinesis.
The link between cell size and cell division is believed to
be less rigid for mammalian cells than has been described
for yeast (e.g., Refs. 10, 22). Nevertheless, there is evidence that an increase in cell size is an important variable
in determining the timing of cell division (e.g., Ref. 168).
For example, oligodendrocyte precursor cells sometimes
divide asymmetrically such that one daughter cell is significantly larger than the other. The smaller of the two
cells takes almost twice as long to divide as the larger
one, consistent with the view that a certain cell size needs
be reached before cell division will occur (81).
Among the earliest events that occur after mitogenic
treatment of quiescent cells is an increase of K1 influx
(e.g., Refs. 16, 208, 271, 276, 300). From the earliest reports, it was noted that a significant portion of the increased K1 influx was ouabain insensitive (e.g., Ref. 300).
Subsequent studies have clearly shown that virtually all
the ouabain-insensitive K1 influx is via the NKCC (e.g.,
Refs. 16, 260, 270, 276). As a result of the mitogen-induced
increase in K1 uptake, Rozengurt (299) suggested that an
increased [K1]i is necessary to release a block that ordinarily prevents quiescent cells from progressing through
the cell cycle. However, several groups have been unable
to measure such an increase of [K1]i following mitogenic
stimulation (e.g., Refs. 16, 36). This is not so surprising
when one considers that mammalian cells are in osmotic
equilibrium with their surroundings (;285 mosmol/
kgH2O). Such an equilibrium means that the most concentrated [K1] (plus associated anion) solution that could be
taken up by mammalian cells would be one that is ;150
mM. Because normal [K1]i is ;140 mM, it is obvious that
even if K1 uptake is greatly stimulated, a meaningful
increase of [K1]i will not occur; rather, there will be an
Volume 80
January 2000
Na1-K1-Cl2 COTRANSPORT
tured cells, it is necessary to be particularly cautious in
applying the results to native, fully differentiated, tissue.
An example of the importance of this general caveat can
be seen from the results of Raat et al. (290) that compared
the expression levels of the NKCC in freshly isolated
tissue with expression levels in cells that had been cultured for two to five passages. They studied rabbit proximal tubules, rat vascular smooth muscle cells, and pig
aortic endothelial cells. In each case, they found very little
or no bumetanide-sensitive 86Rb uptake in the freshly
isolated cells, but all the cultured cells exhibited a larger
total 86Rb uptake, much of which was bumetanide sensitive. Furthermore, using a Northern probe for NKCC1,
they demonstrated that the freshly isolated tissue had no
NKCC mRNA, whereas the cultured cells exhibited a
strong density band. The authors suggest that NKCC activity may, in general, be suppressed in differentiated
cells, but upregulated in proliferating cells. It is likely that,
like all generalities, this one is too broad. Although the
warning inherent in this report should be well-heeded,
there are several reasons to be cautious about the general
application of this particular finding with respect to the
NKCC. First, it must be remembered that much of the
early work in the NKCC field was performed on differentiated cells (e.g., duck RBC, squid axon, shark rectal
gland, thick ascending limb of the kidney). Second, various probes including bumetanide derivatives (see sect.
VIII), monoclonal antibodies (e.g., Ref. 221), and Northern
probes (e.g., Ref. 358) have identified the NKCC protein or
message in a wide variety of intact tissue and differentiated cells, including vascular endothelial cells (360). Thus
it is clear that the NKCC functions in a variety of differentiated cells. The important message from the work of
Raat et al. (290) is that putting cells into culture conditions is likely to upregulate the cotransporter, and this
potentially makes comparisons with the undifferentiated
cells complicated.
XI. QUESTIONS REMAINING TO BE ANSWERED
Although we have learned a great deal about this
unique triple ion cotransport mechanism in the 20 years
since it was first described, there is obviously much work
to be done to fully understand its mechanism of action,
how it is regulated, and its cellular functions.
As is the case with virtually all active ion transport
processes, we do not understand how the ions are moved
across the membrane. In the case of the NKCC, we do not
yet even know what parts of the molecule are involved,
although some guesses have been made (e.g., Ref. 281)
and promising first steps have been taken to unravel this
key question (148 –150). It can be safely assumed that
molecular structure-function studies will be a hot topic in
this field in the next few years as the powerful tools of
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ment will block mitogenically induced DNA synthesis, but
whether it will prevent cell proliferation. Unfortunately,
none of the forgoing studies examined whether cell division could still take place after bumetanide treatment.
Panet and co-workers (271, 274) have long championed the view that the NKCC plays an important role in
cell proliferation. In 1989 they presented evidence that the
combined activity of the NKCC and the NHE were required for serum-induced activation of BALB/c 3T3 cells
(272). They examined the effects of amiloride and bumetanide on serum-stimulated Na1 pump activity and on
accumulation of cellular Na1. When either amiloride or
bumetanide was applied separately, there was a partial
reduction of the stimulation. In fact, in agreement with
the findings of Amsler et al. (16), the effects of bumetanide alone were quite modest. However, when the two
agents were used in combination, the stimulation of both
the Na1 pump and of cellular Na1 accumulation were
abolished. The same pattern of partial inhibition when
used alone and complete inhibition when used together
was noted for [3H]thymidine incorporation. This suggested that, for these cells, the NKCC and the NHE operate together in a redundant manner to ensure that the
necessary cellular ionic changes can occur.
The experiments described above were conducted
on immortal cell lines rather than primary cell cultures.
To determine whether the NKCC might play a role in
mitogen-induced proliferation of primary cultured cells,
Panet and Atlan (273) studied the effects of bumetanide
treatment on cell exit from G0/G1. They monitored both
DNA synthesis and cell numbers. They showed that bumetanide inhibited both these measures of cell proliferation that it did so reversibly and that inhibition of proliferation occurred when bumetanide was applied 2– 6 h
after the addition of mitogen. This makes a strong case for
a key role for the NKCC in the cell cycle of the human
fibroblast. Similar results were presented for primary cultures of bovine vascular endothelial cells by Panet et al.
(275).
Comparison of the partial effects of bumetanide
treatment on cell proliferation seen when studying immortal cell lines (e.g., Refs. 16, 272, 276) with the much
more dramatic effects of bumetanide alone in the primary
cultures led Panet et al. (275) to suggest there could be
important differences in the way immortal cells and primary cells use ion transporters to effect cell proliferation.
They point out that confluent cultures of immortal cell
lines exit the G0/G1 phase upon addition of serum,
whereas primary cultures do not and suggest that primary
culture cells may be under tighter regulatory control than
immortal cell lines.
It is important to emphasize that all the studies on the
role of the NKCC in cell proliferation have been carried
out in cultured cells, either primary cultures or immortal
cell lines. As always when interpreting results form cul-
267
268
JOHN M. RUSSELL
I take great pleasure in acknowledging the crucial and
important roles that two of my long-time colleagues have played
in my journey to understand the NKCC. Drs. Anı́bal A. Altamirano and Gerda E. Breitwieser have labored long with me, and
I cannot thank either of them enough for the many hours of
pithy discussion (not to mention the years of experimental
work) we have shared that served to focus us on important and
exciting questions relating to this fascinating ion transport
mechanism. For all their help and support I am deeply grateful.
Support for the research by the author cited in this review
came from the National Institute of Neurological Disorders and
Stroke Grant NS-11946; for this I am also most grateful.
Address for reprint requests and other correspondence:
J. M. Russell, Dept. of Biology, Biological Research Laboratories, 130 College Place, Syracuse University, Syracuse, NY 13244
(E-mail: [email protected]).
REFERENCES
1. ADELMAN, W. J., AND R. E. TAYLOR. Leakage current rectification
in the squid giant axon. Nature 190: 883– 885, 1961.
2. ADORANTE, J. S., AND S. S. MILLER. Potassium-dependent volume
regulation in retinal pigment epithelium is mediated by Na,K,Cl
cotransport. J. Gen. Physiol. 96: 1153–1176, 1990.
3. ADRAGNA, N., C. M. PERKINS, AND P. LAUF. Furosemide-sensitive
Na1-K1 cotransport and cellular metabolism in human erythrocytes. Biochim. Biophys. Acta 812: 293–296, 1985.
4. AICKIN, C. C. Chloride transport across the sarcolemma of vertebrate smooth and skeletal muscle. In: Chloride Channels and
Carriers in Nerve, Muscle and Glial Cells, edited by F. J. AlvarezLeefmans and J. M. Russell. New York: Plenum, 1990, p. 209 –249.
5. ALTAMIRANO, A. A., G. E. BREITWIESER, AND J. M. RUSSELL.
Vanadate and fluoride effects on Na1-K1-Cl2 cotransport in squid
giant axon. Am. J. Physiol. 254 (Cell Physiol. 23): C582—C586,
1988.
6. ALTAMIRANO, A. A., G. E. BREITWIESER, AND J. M. RUSSELL.
Effects of okadaic acid and intracellular Cl2 on Na1,K1,Cl2 cotransport. Am. J. Physiol. 269 (Cell Physiol. 38): C878 —C883,
1995.
7. ALTAMIRANO, A. A., G. E. BREITWIESER, AND J. M. RUSSELL.
Activation of Na1,K1,Cl2 cotransport in squid giant axon by extracellular ions: evidence for ordered binding. Biochim. Biophys.
Acta 1416: 195–207, 1999.
8. ALTAMIRANO, A. A., AND J. M. RUSSELL. Coupled Na/K/Cl efflux:
“reverse” unidirectional fluxes in squid giant axons. J. Gen.
Physiol. 89: 669 – 686, 1987.
9. ALTAMIRANO, A. A., B. A. WATTS III, AND J. M. RUSSELL. Binding
of bumetanide to microsomes from optic ganglia of the squid,
Loligo pealei. Am. J. Physiol. 258 (Cell Physiol. 27): C933—C943,
1990.
10. ALBERTS, B., D. BRAY, J. LEWIS, M. RAFF, K. ROBERTS, AND J. D.
WATSON. Molecular Biology of the Cell (3rd ed.). New York:
Garland, 1994, chapt. 17, p. 873–910.
11. ALVAREZ-LEEFMANS, F. J. Intracellular Cl2 regulation and synaptic inhibition in vertebrate and invertebrate neurons. In: Chloride
Channels and Carriers in Nerve, Muscle and Glial Cells, edited by
F. J. Alvarez-Leefmans and J. M. Russell. New York: Plenum, 1990,
p. 109 –158.
12. ALVAREZ-LEEFMANS, F. J. Chloride transport, osmotic balance
and presynaptic inhibition. In: Presynaptic Inhibition and Neural
Control, edited by P. Rudomı́n, R. Romo, and L. Mendell. New York:
Oxford Univ. Press, 1997.
13. ALVAREZ-LEEFMANS, F. J., S. M. GAMIÑO, F. GIRALDEZ, AND I.
NOGUERÓN. Intracellular chloride regulation in amphibian dorsal
root ganglion neurones studied with ion-selective microelectrodes.
J. Physiol. (Lond.) 406: 225–246, 1988.
14. ALVAREZ-LEEFMANS, F. J., F. GIRALDEZ, AND J. M. RUSSELL.
Methods for measuring chloride transport across nerve, muscle
and glial cells. In: Chloride Channels and Carriers in Nerve,
Muscle and Glial Cells, edited by F. J. Alvarez-Leefmans and J. M.
Russell. New York: Plenum, 1990, p. 3– 66.
15. AMLAL, H., M. PAILLARD, AND M. BICHARA. Cl2-dependent NH1
4
transport mechanisms in medullary thick ascending limb cells.
Am. J. Physiol. 267 (Cell Physiol. 36): C1607—C1615, 1994.
16. AMSLER, K., J. J. DONOHUE, C. W. SLAYMAN, AND E. A. ADELBERG. Stimulation of bumetanide-sensitive K1 transport in Swiss
3T3 fibroblasts by serum and mitogenic hormones. J. Cell Physiol.
123: 257–263, 1985.
17. AMSLER, K., AND R. KINNE. Photoinactivation of sodium-potassium-chloride cotransport in LLC-PK1/Cl 4 cells by bumetanide.
Am. J. Physiol. 250 (Cell Physiol. 19): C799 —C806, 1986.
18. ANDRÉ, B., AND B. SCHARENS. The yeast YBR235w gene encodes
a homolog of the mammalian electroneutral Na1-(K1)-Cl2 cotransport family. Biochem. Biophys. Res. Commun. 217: 150 –153, 1995.
19. BAKER, P. F., AND H. G. GLITSCH. Does metabolic energy participate directly in the Na1-dependent extrusion of Ca21 ions from
squid axon? J. Physiol. (Lond.) 233: 44 – 46, 1973.
20. BAKER, P. F., AND S. J. POTASHNER. Glutamate transport in
invertebrate nerve: the relative importance of ions and metabolic
energy. J. Physiol. (Lond.) 232: 26 –27, 1973.
21. BALLOU, L. M., AND E. H. FISCHER. Phosphoprotein phosphatases.
In: The Enzymes. Control by Phosphorylation (3rd ed.), edited by
P. D. Boyer and E. G. Krebs. Orlando, FL: Academic, 1986, vol. XVII,
pt. A, p. 312–365.
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
molecular biology are used to tease out the important
parts of the molecular architecture.
Although the evidence is substantial and strong that
the NKCC is phosphorylated before engaging in ion transport, the identity of the kinase or kinases responsible is
still unknown (see sect. IXA). Lytle (212) has suggested
that a single kinase is responsible for the final phosphorylation of the NKCC protein. Thus the fact that so many
workers have identified a variety of protein kinases as
being capable of either stimulating or inhibiting the NKCC
may mean that overall regulation of NKCC activity involves a cascade of protein kinases and/or phosphatases,
depending on the cell type and the stimulus. In addition,
the mechanism by which the phosphorylation renders the
cotransporter capable of ion transport is completely unknown. As this mechanism is apparently a secondary
active transporter, it is unlikely that the phosphorylation
induces a transient “high-energy” intermediate as in the
case of the ATPase ion pumps. It is assumed the effect of
phosphorylation is to increase the ion affinity and/or the
maximal velocity of the cotransporter, but there is at
present little direct evidence in this regard.
The nature of the inhibition by intracellular Cl2 of
cotransporter activity is currently ascribed to a reduction
of phosphorylation of the NKCC protein as was discussed
in section IXB1B. However, this just brings us back to the
issues addressed above. How is the cotransporter phosphorylated? What is the target of the intracellular Cl2?
How does cell volume change this interrelationship?
In addition to the obvious questions mentioned
above, there are many others associated with cell- and
function-specific regulation of the NKCC. The effects of
various diseases and pathogens, not touched on in this
review, will become increasingly obvious and important,
perhaps serving as starting points in disease treatment.
Volume 80
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46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
uretic factor regulate cell volume of rabbit atrial myocytes. Circ.
Res. 77: 741–749, 1995.
CRANE, R. K. Hypothesis of the mechanism of intestinal active
transport of sugars. Federation Proc. 21: 891– 895, 1962.
CRANE, R. K. The gradient hypothesis and other models of carriermediated active transport. Rev. Physiol. Biochem. Pharmacol. 78:
99 –159, 1977.
DAGHER, G., C. BRUGNARA, AND M. CANESSA. Effects of metabolic depletion on the furosemide-sensitive Na and K fluxes in
human red blood cells. J. Membr. Biol. 86: 145–155, 1985.
DAWSON, W. D., AND T. C. SMITH. Intracellular Na1, K1 and Cl2
activities in Ehrlich ascites tumor cells. Biochim. Biophys. Acta
860: 293–300, 1986.
DELPIRE, E., M. R. KAPLAN, M. D. PLOTKIN, AND S. C. HEBERT.
The Na-(K)-Cl cotransporter family in the mammalian kidney. Molecular identification and functions. Nephrol. Dial. Transplant. 11:
1967–1973, 1996.
DELPIRE, E., AND P. K. LAUF. Kinetics of Cl-dependent K fluxes in
hypotonically swollen low K sheep erythrocytes. J. Gen. Physiol.
97: 173–193, 1991.
DELPIRE, E., AND P. K. LAUF. Kinetics of DIDS inhibition of
swelling-activated K-Cl cotransport in low K sheep erythrocytes. J.
Membr. Biol. 126: 89 –96, 1992.
DELPIRE, E., M. I. RAUCHMAN, D. R. BEIR, S. C. HEBERT, AND
S. R. GULLANS. Molecular cloning and chromosome localization of
a putative basolateral Na-K-2Cl cotransporter from mouse inner
medullary collecting duct (mIMCD-3) cells. J. Biol. Chem. 269:
25677–25683, 1994.
DESILETS, M., AND C. M. BAUMGARTEN. K1, Na1, and Cl2 activities in ventricular myocytes isolated from rabbit heart. Am. J.
Physiol. 251 (Cell Physiol. 20): C197—C208, 1986.
DESILVA, H. A., J. G. CARVER, AND J. K. ARONSON. Effects of high
external concentration of K1 on 86Rb efflux in human platelets:
evidence for Na1/K1/2Cl2 co-transport. Clin. Sci. 91: 725–736,
1996.
DIPOLO, R. Effect of ATP on the calcium efflux in dialyzed squid
giant axons. J. Gen. Physiol. 64: 503–517, 1974.
DREWNOWSKA, K., AND C. M. BAUMGARTEN. Regulation of cellular volume in rabbit ventricular myocytes: bumetanide, chlorothiazide and ouabain. Am. J. Physiol 260 (Cell Physiol. 29): C122—
C131, 1991.
DUHM, J. Furosemide-sensitive K1(Rb1) transport in human erythrocytes: modes of operation, dependence on extracellular and intracellular Na1, kinetics, pH dependency and the effect of cell
volume on N-ethylmaleimide. J. Membr. Biol. 98: 15–32, 1987.
DUNHAM, P. B., G. W. STEWART, AND J. C. ELLORY. Chlorideactivated passive potassium transport in human erythrocytes. Proc.
Natl. Acad. Sci. USA 77: 1711–1715, 1980.
DUNN, M. J. Ouabain-uninhibited sodium transport in human
erythrocytes. J. Clin. Invest. 52: 658 – 670, 1970.
EDELMAN, J. L., G. SACHS, AND J. S. ADORANTE. Ion transport
asymmetry and functional coupling in bovine pigmented and nonpigmented ciliary epithelial cells. Am. J. Physiol. 266 (Cell Physiol.
35): C1210 —C1221, 1994.
ELLORY, J. C., P. W. FLATMAN, AND G. W. STEWART. Inhibition of
human red cell sodium and potassium transport by divalent cations. J. Physiol. (Lond.) 340: 1–17, 1983.
EVANS, M. G., A. MARTY, Y. P. TAN, AND A. TRAUTMANN. Blockage of Ca-activated Cl conductance by furosemide in rat lacrimal
glands. Pflügers Arch. 406: 65– 68, 1986.
EVELOFF, J. L., AND J. CALAMIA. Effect of osmolarity on cation
fluxes in medullary thick ascending limb cells. Am. J. Physiol. 250
(Renal Fluid Electrolyte Physiol. 19): F176 —F180, 1986.
EVELOFF, J., AND D. G. WARNOCK. K-Cl transport systems in
rabbit renal basolateral membrane vesicles. Am. J. Physiol. 252
(Renal Fluid Electrolyte Physiol. 21): F883—F889, 1986.
FERRI, C., R. L. EVANS, M. PAULAIS, A. TANIMURA, AND R. J.
TURNER. Evidence that up-regulation of Na1-K1-2Cl2 cotransporter by hypertonic shrinkage is not duplicated by isotonic shrinkage. Mol. Cell. Biochem. 169: 21–25, 1997.
FLATMAN, P. W. The effects of calcium on potassium transport in
ferret red cells. J. Physiol. (Lond.) 386: 407– 423, 1987.
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
22. BASERGA, R. Growth in size and cell DNA replication. Exp. Cell
Res. 151: 1–5, 1984.
23. BEAR, R. S., AND F. O. SCHMITT. Electrolytes in the axoplasm of
the giant nerve fibers of the squid. J. Cell. Comp. Phyiol. 14:
205–215, 1939.
24. BENJAMIN, E. A., AND E. A. JOHNSON. A quantitative description
of the Na-K-2Cl cotransporter and its conformity to experimental
data. Am. J. Physiol. 273 (Renal Fluid Electrolyte Physiol. 42):
F473—F482, 1997.
25. BENNETT, V., AND D. M. GILLIGAN. The spectrin-based membrane
structure and micron-scale organization of the plasma membrane.
Annu. Rev. Cell Biol. 9: 27– 66, 1993.
26. BLAUSTEIN, M. P. Effects of internal and external cations and of
ATP on sodium-calcium and calcium-calcium exchange in squid
axons. Biophys. J. 20: 79 –111, 1977.
27. BORON, W. F., AND J. M. RUSSELL. Stoichiometry and ion dependencies of the intracellular-pH-regulating mechanism in squid giant
axon. J. Gen. Physiol. 81: 373–399, 1983.
28. BREITWIESER, G. E., A. A. ALTAMIRANO, AND J. M. RUSSELL.
Osmotic stimulation of Na1-K1-Cl2 cotransport in squid giant axons is [Cl2]i dependent. Am. J. Physiol. 258 (Cell Physiol. 27):
C749 —C753, 1990.
29. BREITWIESER, G. E., A. A. ALTAMIRANO, AND J. M. RUSSELL.
Elevated [Cl2]i and [Na1]i inhibit Na1,K1,Cl2 cotransport by different mechanisms in squid giant axons. J. Gen. Physiol. 107:
261–270, 1996.
30. BRETSCHER, A. Microfilaments and membranes. Curr. Opin. Cell
Biol. 5: 653– 660, 1993.
31. BRINLEY, F. J., AND L. J. MULLINS. Sodium extrusion by internally
dialyzed squid axons. J. Gen. Physiol. 50: 2303–2332, 1967.
32. BROCK, T. A., C. BRUGNARA, M. CANESSA, AND M. A. GIMBONE,
JR. Bradykinin and vasopressin stimulate Na1-K1-Cl2 cotransport
in cultured endothelial cells. Am. J. Physiol. 250 (Cell Physiol. 19):
C888 —C895, 1986.
33. BROWN, C. D. A., AND H. MURER. Characterization of a Na1:K1:
2Cl2 cotransport system in the apical membrane of a renal epithelial cell line (LLC-PK1). J. Membr. Biol. 87: 131–139, 1985.
34. BRUGNARA, C., T. VAN HA, AND D. C. TOSTESON. Role of chloride
in K transport through a cotransport system in human red blood
cells. Am. J. Physiol. 256 (Cell Physiol. 25): C994 —C1003, 1989.
35. BURG, M., L. STONER, J. CARDINAL, AND N. GREEN. Furosemide
effect on isolated perfused tubules. Am. J. Physiol. 225: 119 –124,
1973.
36. BUSSOLATI, O., J. UGGERI, S. BELLETTI, V. DALL’ASTA, AND
G. C. GAZZOLA. The stimulation of Na,K,Cl cotransport and of
system A for neutral amino acid transport is a mechanism for cell
volume increase during the cell cycle. FASEB J. 10: 920 –926, 1996.
37. CALDER, J. A., M. SCHACHTER, AND P. S. SEVER. Direct vascular
actions of hydrochlorothiazide and indapamide in isolated small
vessels. Eur. J. Pharmacol. 220: 19 –26, 1992.
38. CANESSA, M., C. BRUGNARA, D. CUSI, AND D. C. TOSTESON.
Modes of operation and variable stoichiometry of the furosemidesensitive Na and K fluxes in human red blood cells. J. Gen. Physiol.
87: 113–142, 1986.
39. CASS, A., AND M. DALMARK. Equilibrium dialysis of ions in nystatin-treated red cells. Nature 244: 47– 49, 1973.
40. CHANG, H., K. TASHIRO, M. HIRAI, K. IKEDA, K. KUROKAWA,
AND T. FUJITA. Identification of a cDNA encoding a thiazide-sensitive sodium-chloride cotransporter from the human and its
mRNA expression in various tissues. Biochem. Biophys. Res. Commun. 223: 324 –328, 1996.
41. CHASSANDE, O., C. FRELIN, D. FARAHIFAR, T. JEAN, AND M.
LAZDUNSKI. The Na1/K1/Cl2 cotransport in C6 glioma cells. Properties and role in volume regulation. Eur. J. Biochem. 171: 425–
433, 1988.
42. CHIPPERFIELD, A. R. An effect of chloride on (Na - K) co-transport in human red blood cells. Nature 286: 281–282, 1980.
43. CHIPPERFIELD, A. R. The (Na1-K1-Cl2) co-transport system.
Clin. Sci. 71: 465– 476, 1986.
44. CHIPPERFIELD, A. R., J. P. L DAVIS, AND A. A. HARPER. Evidence
for volume regulatory increase by Na-K-Cl co-transport in rat arterial smooth muscle. Exp. Physiol. 76: 971–974, 1991.
45. CLEMO, H. F., AND C. M. BAUMGARTEN. cGMP and atrial natri-
269
270
JOHN M. RUSSELL
90. GECK, P., C. PIETRZYK, B.-C. BURCKHARDT, B. PFEIFFER, AND
E. HEINZ. Electrically silent cotransport of Na1, K1 and Cl2 in
Ehrlich cells. Biochim. Biophys. Acta 600: 432– 447, 1980.
91. GERENCSER, G. A. The chloride pump: a Cl2-translocating P-type
ATPase. Crit. Rev. Biochem. Mol. Biol. 31: 303–337, 1996.
92. GILLEN, C. M., S. BRILL, J. A. PAYNE, AND B. FORBUSH III.
Molecular cloning and functional expression of the K-Cl cotransporter from rabbit, rat and human. A new member of the cationchloride cotransporter family. J. Biol. Chem. 271: 16237–16244,
1996.
93. GILLEN, C. M., AND B. FORBUSH III. Functional interaction of the
K-Cl cotransporter (KCC1) with the Na-K-Cl cotransporter in HEK293 cells. Am. J. Physiol. 276 (Cell Physiol. 45): C328 —C336, 1999.
94. GIRAULT, J.-A., H. C. HEMMINGS, JR., S. H. ZORN, E. L.
GUSTAFSON, AND P. GREENGARD. Characterization in mammalian brain of a DARPP-32 serine kinase identical to casein kinase II.
J. Neurochem. 55: 1772–1783, 1990.
95. GLYNN, I. M, V. L. LEW, AND U. LÜTHI. Reversal of the potassium
entry mechanism in red cells, with and without reversal of the
entire pump cycle. J. Physiol. (Lond.) 207: 371–391, 1970.
96. GOOD, D. W., M. A. KNEPPER, AND M. B. BURG. Ammonia and
bicarbonate transport by thick ascending limb of rat kidney. Am. J.
Physiol. 247 (Renal Fluid Electrolyte Physiol. 16): F35—F44, 1984.
97. GRANT, B. F., T. B. BREITHAUPT, AND E. B. CUNNINGHAM. An
adenosine 39:59 monophosphate-dependent protein kinase from the
human erythrocyte: purification and characterization. J. Biol.
Chem. 254: 5726 –5733, 1979.
98. GREEN, J., D. T. YAMAGUCHI, C. R. KLEEMAN, AND S. MUALLEM.
Cytosolic pH regulation in osteoblasts. Regulation of anion exchange by intracellular pH and Ca21 ions. J. Gen. Physiol. 95:
121–145, 1990.
99. GREGER, R. The membrane transporters regulating epithelial NaCl
secretion. Pflügers Arch. 432: 579 –588, 1996.
100. GREGER, R., H. GOGELEIN, AND E. SCHLATTER. Stimulation of
NaCl secretion in the rectal gland of the dogfish, Squalus acanthias. Comp. Biochem. Physiol. A Physiol. 90: 733–737, 1988.
101. GREGER, R., AND E. SCHLATTER. Presence of luminal K1, a
prerequisite for active NaCl transport in the cortical thick ascending limb of Henle’s loop of rabbit kidney. Pflügers Arch. 392: 92–94,
1981.
102. GREGER, R., AND E. SCHLATTER. Mechanism of NaCl secretion in
rectal gland tubules of spiny dogfish (Squalus acanthias). II. Effects of inhibitors. Pflügers Arch. 402: 364 –375, 1985.
103. GREGER, R., E. SCHLATTER, AND F. LANG. Evidence for electroneutral sodium chloride cotransport in the cortical thick ascending
limb of Henle’s loop of rabbit kidney. Pflügers Arch. 396: 308 –314,
1983.
104. GREGER, R., E. SCHLATTER, F. WANG, AND J. N. FORREST, JR.
Mechanism of NaCl secretion in rectal gland tubules of spiny
dogfish (Squalus acanthias). III. Effects of stimulation of secretion
by cyclic AMP. Pflügers Arch. 402: 376 –384, 1984.
105. GREVEN, J., B. KÖLLING, B. V. BRONEWSKI-SCHWARZER, M.
JUNKER, B. NEFFGEN, AND R. M. NILIUS. Evidence for a role of
the Tamm-Horsfall protein in the tubular action of furosemide-like
loop diuretics. In: Diuretics, edited by J. B. Puschett. Amsterdam:
Elsevier Science, 1984, p. 203–214.
106. GRIFFITHS, N. M., AND N. L. SIMMONS. Attribution of [3H]bumetanide binding to the Na-K-Cl “co-transporter” in rabbit renal cortical plasma membrane: a caveat. Q. J. Exp. Physiol. 72: 313–329,
1987.
107. GRINSTEIN, S., W. FURUYA, AND G. P. DOWNEY. Activation of
permeabilized neutrophils: role of anions. Am. J. Physiol. 263 (Cell
Physiol. 32): C78 —C75, 1992.
108. GUNN, R. B. Bumetanide inhibition of anion exchange in human
red blood cells (Abstract). Biophys. J. 47: 326a, 1985.
109. HAAS, M. Properties and diversity of (Na-K-Cl) cotransporters.
Annu. Rev. Physiol. 51: 443– 457, 1989.
110. HAAS, M. The Na-K-Cl cotransporters. Am. J. Physiol. 267 (Cell
Physiol. 36): C869 —C885, 1994.
111. HAAS, M., AND B. FORBUSH III. [3H]bumetanide binding to duck
red cells. Correlation with inhibition of (Na-K-Cl) co-transport.
J. Biol. Chem. 261: 8434 – 8441, 1986.
112. HAAS, M., AND B. FORBUSH III. Photolabeling of a 150 kDa (Na-
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
68. FLATMAN, P. W. The effects of magnesium on potassium transport
in ferret red cells. J. Physiol. (Lond.) 397: 471– 487, 1988.
69. FLATMAN, P. W. Stoichiometry of net sodium and potassium
fluxes mediated by the Na-K-Cl co-transport system in ferret red
cells. Q. J. Exp. Physiol. 74: 939 –941, 1989.
70. FLATMAN, P. W. The effects of metabolism on Na-K-Cl cotransport
in ferret red cells. J. Physiol. (Lond.) 437: 495–510, 1991.
71. FLATMAN, P. W., AND J. CREANOR. Regulation of Na-K-2Cl cotransport by protein phosphorylation in ferret erythrocytes.
J. Physiol. (Lond.) 517: 699 –708, 1999.
72. FORBUSH, B., III., M. HAAS, AND C. LYTLE. Na-K-Cl cotransport in
the shark rectal gland. I. Regulation in the intact perfused gland.
Am. J. Physiol. 262 (Cell Physiol. 31): C1000 —C1008, 1992.
73. FORBUSH, B., III, AND H. C. PALFREY. [3H]bumetanide binding to
membranes isolated from dog kidney outer medulla: relationship to
the Na,K,Cl co-transport system. J. Biol. Chem. 258: 11787–11792,
1983.
74. FOSKETT, J. K. [Ca21]i modulation of Cl2 content controls cell
volume in single salivary acinar cells during fluid secretion. Am. J.
Physiol. 259 (Cell Physiol. 28): C998 —C1004, 1990.
75. FOSKETT, J. K., M. M. WONG, G. SUE-A-QUAN, AND M. A. ROBERTSON. Isosmotic modulation of cell volume and intracellular ion
activities during stimulation of single exocrine cells. J. Exp. Zool.
268: 104 –110, 1994.
76. FRANKLIN, C. C., J. T. TURNER, AND H. D. KIM. Regulation of
Na1/K1/Cl2 cotranport and [3H]bumetanide binding site density by
phorbol esters in HT29 cells. J. Biol. Chem. 264: 6667– 6673, 1989.
77. FRELIN, C., O. CHASSANDE, AND M. LAZDUNSKI. Biochemical
characterization of the Na1/K1/Cl2 co-transport in chick cardiac
cells. Biochem. Biophys. Res. Commun. 134: 326 –331, 1986.
78. FUNDER, J., AND J. O. WIETH. Effects of some monovalent anions
on fluxes of Na and K and on glucose metabolism of ouabaintreated human red blood cells. Acta Physiol. Scand. 71: 168 –185,
1967.
79. GAMBA, G., A. MIYANOSHITA, M. LOMBARDI, J. LYTTON, W.-S.
LEE, M. A. HEDIGER, AND S. C. HEBERT. Molecular cloning,
primary structure, and characterization of two members of the
mammalian electroneutral sodium-(potassium)-chloride cotransporter family expressed in kidney. J. Biol. Chem. 269: 17713–17722,
1994.
80. GAMBA, G., S. N. SALZBERG, M. LOMBARDI, A. MIYANOSHITA, J.
LYTTON, M. A. HEDIGER, B. M. BRENNER, AND S. C. HEBERT.
Primary structural and functional expression of a cDNA encoding
the thiazide-sensitive, electroneutral sodium-chloride cotransporter. Proc. Natl. Acad. Sci. USA 90: 2749 –2753, 1993.
81. GAO, F.-B., AND M. RAFF. Cell size control and a cell-intrinsic
maturation program in proliferating oligodendrocyte precursor
cells. J. Cell Biol. 138: 1367–1377, 1997.
82. GARAY, R. P. Inhibition of the Na1/K1 cotransport system by
cyclic AMP and intracellular Ca21 in human red cells. Biochim.
Biophys. Acta 688: 786 –792, 1982.
83. GARAY, R. P., P. A. HANNAERT, C. NAZERET, AND E. J. CRAGOE,
JR. The significance of the relative effects of loop diuretics and
anti-brain edema agents on the Na1,K1,Cl2 cotransport system and
the Cl2/NaCO3 anion exchanger. Naunyn-Schmiedebergs Arch.
Pharmacol. 334: 202–209, 1986.
84. GARRAHAN, P. J., AND I. M. GLYNN. The sensitivity of the sodium
pump to external sodium. J. Physiol. (Lond.) 192: 159 –174, 1967.
85. GECK, P., AND E. HEINZ. Coupling of ion flows in cell suspension
systems. Ann. NY Acad. Sci. 341: 57– 66, 1980.
86. GECK, P., AND E. HEINZ. The Na-K-2Cl cotransport system. J.
Membr. Biol. 91: 97–105, 1986.
87. GECK, P., E. HEINZ, AND B. PFEIFFER. Influence of high ceiling
diuretics on ion fluxes and cell volume of Ehrlich ascites tumor
cells. Scand. Audiol. Suppl. 14: 25–36, 1981.
88. GECK, P., E. HEINZ, C. PIETRYZYK, AND B. PFEIFFER. The effect
of furosemide on the ouabain-insensitive K1 and Cl2 movements in
Ehrlich cells. In: Cell Membrane Receptors for Drugs and Hormones: A Multidisciplinary Approach, edited by R. W. Straub and
L. Bolis. New York: Raven, 1978.
89. GECK, P., AND B. PFEIFFER. Na1-K1-2Cl2 cotransport in animal
cells–its role in volume regulation. Ann. NY Acad. Sci. 456: 166 –
182, 1985.
Volume 80
January 2000
113.
114.
115.
116.
117.
119.
120.
121.
122.
123.
124.
125.
126.
127.
128.
129.
130.
131.
132.
133.
134.
K-Cl) cotransport protein from dog kidney with a bumetanide
analogue. Am. J. Physiol. 253 (Cell Physiol. 22): C243—C250, 1987.
HAAS, M., AND B. FORBUSH III. Photoaffinity labeling of a 150-kDa
(Na-K-Cl) cotransport protein from duck red cells with an analog of
bumetanide. Biochim. Biophys. Acta 939: 131–144, 1988.
HAAS, M., AND B. FORBUSH III. The Na-K-Cl cotransporters.
J. Bioenerg. Bioeng. 30: 161–172, 1998.
HAAS, M., AND D. MCBRAYER. Na-K-Cl cotransport in nystatintreated tracheal cells: regulation by isoproterenol, apical UTP and
[Cl2]i. Am. J. Physiol. 266 (Cell Physiol. 35): C1440 —C1452, 1994.
HAAS, M, AND T. J. MCMANUS. Bumetanide inhibition of (Na - K 2Cl) co-transport in K/Rb exchange at a chloride site in duck red
cells mediated by external cations (Abstract). Biophys. J. 37: 214a,
1982.
HAAS, M., AND T. J. MCMANUS. Bumetanide inhibits (Na-K-Cl)
co-transport at a chloride site. Am. J. Physiol. 245 (Cell Physiol.
14): C235—C240, 1983.
HAAS, M., AND T. J. MCMANUS. Effect of norepinephrine on swelling-induced potassium transport in duck red cells. Evidence
against a volume regulatory decrease under physiological conditions. J. Gen. Physiol. 85: 649 – 667, 1985.
HAAS, M., D. MCBRAYER, AND C. LYTLE. [Cl2]i-dependent phosphorylation of the Na-K-Cl cotransport protein of dog tracheal
epithelial cells. J. Biol. Chem. 270: 28955–28961, 1995.
HAAS, M., D. MCBRAYER, AND J. R. YANKASKAS. Dual mechanisms for Na-K-Cl cotransport regulation in airway epithelial cells.
Am. J. Physiol. 264 (Cell Physiol. 33): C189 —C200, 1993.
HAAS, M., W. F. SCHMIDT III, AND T. J. MCMANUS. Catecholaminestimulated ion transport in duck red cells. Gradient effects in
electrically neutral (Na-K-2Cl) co-transport. J. Gen. Physiol. 80:
125–147, 1982.
HALL, A. C., AND J. C. ELLORY. Measurement and stoichiometry of
bumetanide-sensitive (2Na:1K:3Cl) cotransport in ferret red cells.
J. Membr. Biol. 85: 205–213, 1985.
HALLBRUCKER, C., S. VON DAHL, F. LANG, W. GEROK, AND D.
HÄUSSINGER. Modification of liver cell volume by insulin and
glucagon. Pflügers Arch. 418: 519 –521, 1991.
HALLOWS, K. R., AND P. A. KNAUF. Fundamental principles and
physiological roles of cell volume regulation. In: Cellular and Molecular Physiology of Cell Volume Regulation, edited by K. Strange.
Boca Raton, FL: CRC, 1994, p. 3–30.
HANNAFIN, J. A., AND R. KINNE. Active chloride transport in rabbit
thick ascending limb of Henle’s loop and elasmobranch rectal
gland: chloride fluxes in isolated plasma membranes. J. Comp.
Physiol. B Biochem. Syst. Environ. Physiol. 155: 415– 421, 1985.
HANNAFIN, J., E. KINNE-SAFFRAN, D. FRIEDMAN, AND R.
KINNE. Presence of a sodium-potassium-chloride cotransport system in the rectal gland of Squalus acanthias. J. Membr. Biol. 75:
73– 83, 1983.
HARRIS, G. L., AND W. J. BETZ. Evidence for active chloride
accumulation in normal and denervated rat lumbrical muscle.
J. Gen. Physiol. 90: 127–144, 1987.
HATHAWAY, G. M., AND J. A. TRAUGH. Casein kinases, multipotential protein kinases. Curr. Top. Cell Regul. 21: 101–127, 1982.
HEBERT, S. C., AND S. R. GULLANS. Editorial comment on “The
molecular basis of inherited hypokalemic alkalosis: Bartter’s and
Gitelmans syndromes.” Am. J. Physiol. 271 (Renal Fluid Electrolyte Physiol. 40): F957—F959, 1996.
HEGDE, R. S., AND H. C. PALFREY. Ionic effects on bumetanide
binding to the activated Na/K/2Cl cotransporter: selectivity and
kinetic properties of ion binding sites. J. Membr. Biol. 126: 27–37,
1992.
HEINZ, E., P. GECK, C. PIETRZYK, G. BURCKHARDT, AND B.
PFEIFFER. Energy sources for amino acid transport in animal
cells. J. Supramol. Struct. 6: 125–133, 1977.
HERBERT, D. N., AND A. CARRUTHERS. Direct evidence for ATP
modulation of sugar transport in human erythrocyte ghosts. J. Biol.
Chem. 261: 10093–10099, 1986.
HESCHELER, J., M. KAMEYAMA, W. TRAUTWEIN, G. MIESKES,
AND H.-D. SOLING. Regulation of the cardiac calcium channel by
protein phosphatases. Eur. J. Biochem. 165: 261–265, 1985.
HOFFMANN, E. K., AND P. B. DUNHAM. Membrane mechanisms
135.
136.
137.
138.
139.
140.
141.
142.
143.
144.
145.
146.
147.
148.
149.
150.
151.
152.
153.
154.
271
and intracellular signalling in cell volume regulation. Int. Rev.
Cytol. 161: 173–262, 1995.
HOFFMANN, E. K., M. SCHIØDT, AND P. DUNHAM. The number of
chloride-cation cotransport sites on Ehrlich ascites cells measured
with [3H]bumetanide. Am. J. Physiol. 250 (Cell Physiol. 19):
C688 —C693, 1986.
HOFFMANN, E. K., C. SJØHOLM, AND C. O. SIMONSEN. Na1,Cl2
cotransport in Ehrlich ascites tumor cells activated during volume
regulation (regulatory volume increase). J. Membr. Biol. 76: 269 –
280, 1983.
HOFFMAN, J. F., AND F. W. KREGENOW. The characterization of
new energy dependent cation transport processes in red blood
cells. Ann. NY Acad. Sci. 137: 566 –576, 1966.
HOGAN, E. M., B. A. DAVIS, AND W. F. BORON. Intracellular Cl2
dependence on Na-H exchange in barnacle muscle fibers under
normotonic and hypertonic conditions. J. Gen. Physiol. 110: 62– 69,
1997.
HOLTZMAN, E. J., S. KUMAR, C. A. FAALAND, F. WARNER, P. J.
LOGUE, S. J. ERICKSON, G. RICKEN, J. WALDMAN, S. KUMAR,
AND P. B. DUNHAM. Cloning, characterization and gene organization of the K-Cl cotransporter from pig and human kidney and C.
elegans. Am. J. Physiol. 275 (Renal Physiol. 44): F550 —F564, 1998.
HOMMA, T., K. D. BURNS, AND R. C. HARRIS. Agonist stimulation
of Na1/K1/Cl2 cotransport in rat glomerulas mesangial cells.
J. Biol. Chem. 265: 17613–17620, 1990.
HOMMA, T., AND R. C. HARRIS. Time-dependent biphasic regulation of Na1/K1/Cl2 cotransport in rat glomerular mesangial cells.
J. Biol. Chem. 266: 13553–13559, 1991.
HUNTER, M. J. Human erythrocyte permeabilities measured under
conditions of net charge transfer. J. Physiol. (Lond.) 268: 35– 49,
1977.
IGARASHI, P., G. B. VENDEN HEUVEL, J. A. PAYNE, AND B.
FORBUSH III. Cloning, embryonic expression and alternative splicing of a murine kidney-specific Na-K-Cl cotransporter. Am. J.
Physiol. 269 (Renal Fluid Electrolyte Physiol. 38): F405—F418,
1995.
IKEHARA, T., H. YAMAGUCHI, K. HOSOKAWA, AND H. MIYAMOTO. Kinetic studies on the effects of intracellular K1 and Na1
and Na1,K1,Cl2 cotransport of HeLa cells by Rb1 influx determination. J. Membr. Biol. 132: 115–123, 1993.
IKEHARA, T., H. YAMAGUCHI, K. HOSOKAWA, A. TAKAHASHI,
1
AND H. MIYAMOTO. Kinetic mechanism of ATP action in the Na K1-Cl2 cotransport of HeLa cells determined by Rb1 influx studies.
Am. J. Physiol. 258 (Cell Physiol. 27): C599 —C609, 1990.
INOUE, I. Voltage-dependent chloride conductance of the squid
axon membrane and its blockade by some disulfonic stilbene derivatives. J. Gen. Physiol. 85: 519 –538, 1985.
ISENRING, P., AND B. FORBUSH III. Ion and bumetanide binding by
the Na-K-Cl cotransporter. Importance of transmembrane domains.
J. Biol. Chem. 272: 24556 –24562, 1997.
ISENRING, P., S. C. JACOBY, J. CHANG, AND B. FORBUSH III.
Mutagenic mapping of the Na-K-Cl cotransporter for domains involved in ion transport and bumetanide binding. J. Gen. Physiol.
112: 549 –558, 1998.
ISENRING, P., S. C. JACOBY, AND B. FORBUSH III. The role of
transmembrane domain 2 in cation transport by the Na-K-Cl cotransporter. Proc. Natl. Acad. Sci. USA 95: 7179 –7184, 1998.
ISENRING, P., S. C. JACOBY, J. A. PAYNE, AND B. FORBUSH III.
Comparision of the Na,K,Cl cotransporters. NKCC1, NKCC2, and
the HEK-203 cell Na-K-Cl cotransporter. J. Biol. Chem. 273: 11295–
11301, 1998.
ISHIHARA, H., B. L. MARTIN, D. L. BRAAUTIGAN, H. KARAKI, H.
OZAKI, Y. KATO, N. FUSETANI, S. WATABE, K. HASHIMOTO, D.
VEMURA, AND D. J. HARTSHORNE. Calyculin A and okadaic acid:
inhibitors of protein phosphatase activity. Biochem. Biophys. Res.
Commun. 159: 871– 877, 1989.
ITO, S., AND O. CARRETERO. An in vitro approach to the study of
macula densa-mediated glomerular hemodynamics. Kidney Int. 38:
1206 –1210, 1990.
JENNINGS, M. L. Kinetics and mechanisms of anion transport in
red blood cells. Annu. Rev. Physiol. 47: 519 –533, 1985.
JENNINGS, M. L., AND R. K. SCHULTZ. Okadaic acid inhibition of
KCl cotransport. Evidence that protein dephosphorylation is nec-
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
118.
Na1-K1-Cl2 COTRANSPORT
272
155.
156.
157.
158.
159.
161.
162.
163.
164.
165.
166.
167.
168.
169.
170.
171.
172.
173.
174.
175.
essary for activation of transport by either cell swelling or Nethylmaleimide. J. Gen. Physiol. 97: 799 – 818, 1991.
JENSEN, B. S., AND E. K. HOFFMANN. On the K1-dependence of
the cation-anion cotransport system in Ehrlich ascites tumor cells
(Abstract). Acta Physiol. Scand. 140: 34A, 1990.
JENSEN, B. S., AND E. K. HOFFMANN. Hypertonicity enhances
expression of functional Na1/K1/2Cl2 cotransporters in Ehrlich
ascites tumor cells. Biochim. Biophys. Acta 1329: 1– 6, 1997.
JENSEN, B. S., F. JESSEN, AND E. K. HOFFMANN. Na1,K1,Cl2
cotransport and its regulation in Ehrlich ascites tumor cells. Ca21/
calmodulin and protein kinase C dependent pathways. J. Membr.
Biol. 131: 161–178, 1993.
JESSEN, F., AND E. K. HOFFMANN. Activation of Na1/K1/Cl2
cotransport system by reorganization of the actin filaments in
Ehrlich ascites tumor cells. Biochim. Biophys. Acta 1110: 199 –201,
1992.
JØRGENSEN, P. L., J. PETERSEN, AND W. D. REES. Identification
of a Na1,K1,Cl2 cotransport protein of Mr 34000 from kidney by
photolabelling with [3H]bumetanide. Biochim. Biophys. Acta 775:
105–110, 1984.
KAJI, D. Na1/K1/2Cl2 cotransport in medullary thick ascending
limb cells: kinetics and bumetanide binding. Biochim. Biophys.
Acta 1152: 289 –299, 1993.
KAJI, D. Effects of membrane potential on K:Cl cotransport in
human erythrocytes. Am. J. Physiol. 264 (Cell Physiol. 33): C376 —
C382, 1993.
KAJI, D. M., J. DIAZ, AND J. C. PARKER. Urea inhibits Na-K-2Cl
cotransport in medullary thick ascending cells. Am. J. Physiol. 272
(Cell Physiol. 41): C615—C621, 1997.
KAPLAN, M. R., D. B. MOUNT, E. DELPIRE, G. GAMBA, AND S. C.
HEBERT. Molecular mechanisms of NaCl cotransport. Annu. Rev.
Physiol. 58: 649 – 668, 1996.
KENNEDY, B. G. Volume regulation in cultured cells derived from
human retinal pigment epithelium. Am. J. Physiol. 266 (Cell
Physiol. 35): C676 —C683, 1994.
KERKUT, G. A., AND R. C. THOMAS. The effect of anion injection
and changes in the external potassium and chloride concentrations
on the reversal potential of the IPSP and acetylcholine. Comp.
Biochem. Physiol. 11: 199 –209, 1964.
KEYNES, R. D. Chloride in the squid giant axon. J. Physiol. (Lond.)
169: 690 –705, 1963.
KHADEMAZAD, M., B.-X. ZHANG, P. LOESSBERG, AND S. MUALLEM. Regulation of cell volume by the osteosarcoma cell line
UMR-106 – 01. Am. J. Physiol. 261 (Cell Physiol. 30): C441—C447,
1991.
KILLANDER, D., AND A. ZETTERBERG. A quantitative cytochemical investigation of the relationship between cell mass and initiation of DNA synthesis in mouse fibroblasts in vitro. Exp. Cell Res.
40: 12–20, 1965.
KIM, H. D., Y.-S. TSAI, C. C. FRANKLIN, AND J. T. TURNER.
Characterization of Na1/K1/Cl2 cotransport in cultured HT29 human colonic adenocarcinoma cells. Biochim. Biophys. Acta 946:
397– 404, 1988.
KIMMELBERG, H. K., AND M. V. FRANGAKIS. Furosemide- and
bumetanide-sensitive ion transport and volume control in primary
astrocyte cultures from rat brain. Brain Res. 361: 125–134, 1985.
KINNE, R. Role of the NaCl-KCl cotransport system in active
chloride absorption and secretion. Ann. NY Acad. Sci. 456: 198 –
206, 1985.
KINNE, R. Molecular properties of the sodium-potassium-chloride
cotransport system in the kidney. In: Diuretics II: Chemistry,
Pharmacology, and Clinical Applications, edited by J. B. Puschett
and A. Greenberg. Amsterdam: Elsevier Science, 1987.
KINNE, R., B. KOENIG, J. HANNAFIN, E. KINNE-SAFFRAN, D. M.
SCOTT, AND K. ZIEROLD. The use of membrane vesicles to study
the NaCl/KCl cotransporter involved in active transepithelial chloride transport. Pflügers Arch. 405, Suppl. 1: S101—S105, 1985.
KINNE, R., E. KINNE-SAFFRAN, B. SCHÖLERMANN, AND H.
SCHÜTZ. The anion specificity of the sodium-potassium-chloride
cotransporter in rabbit kidney outer medulla: studies on medullary
plasma membranes. Pflügers Arch. 407: S168 —S173, 1986.
KLEIN, J. D., AND W. C. O’NEILL. Volume-sensitive myosin phosphorylation in vascular endothelial cells: correlation with Na-K-2Cl
176.
177.
178.
179.
180.
181.
182.
183.
184.
185.
186.
187.
188.
189.
190.
191.
192.
193.
194.
195.
196.
197.
Volume 80
cotransport. Am. J. Physiol. 269 (Cell Physiol. 38): C1524 —C1531,
1995.
KLEIN, J. D., P. B. PERRY, AND W. C. O’NEILL. Regulation by cell
volume of Na1-K1-2Cl2 cotransport in vascular endothelial cells:
role of protein phosphorylation. J. Membr. Biol. 132: 243–252, 1993.
KNEPPER, M., AND M. BURG. Organization of nephron function.
Am. J. Physiol. 244 (Renal Fluid Electrolyte Physiol. 13): F579 —
F589, 1983.
KOENIG, B., S. RICAPITO, AND R. KINNE. Chloride transport in the
thick ascending limb of Henle’s loop: K dependence and stoichiometry of NaCl cotransport in plasma membrane vesicles. Pflügers
Arch. 399: 173–179, 1983.
KORT, J. J., AND G. KOCH. The Na1,K1,2Cl2-cotransport system in
HeLa cells: aspects of its physiological regulation. J. Cell. Physiol.
145: 253–261, 1990.
KRACKE, G. R., M. A. ANATRA, AND P. B. DUNHAM. Asymmetry of
Na-K-Cl cotransport in human erythrocytes. Am. J. Physiol. 254
(Cell Physiol. 23): C243—C250, 1983.
KRACKE, G. R., AND P. B. DUNHAM. Effect of membrane potential
on furosemide-inhibitable sodium influxes in human red blood
cells. J. Membr. Biol. 98: 117–124, 1987.
KRAMHOFT, B., I. H. LAMBERT, E. K. HOFFMANN, AND F. J.
JØRGENSON. Activation of Cl-dependent K transport in Ehrlich
ascites tumor cells. Am. J. Physiol. 251 (Cell Physiol. 20): C369 —
C379, 1986.
KRARUP, T., B. S. JENSEN, AND E. K. HOFFMAN. Occlusion of K1
in the Na1/K1/2Cl2 cotransporter of Ehrlich ascites tumor cells.
Biochim. Biophys. Acta 1284: 97–108, 1996.
KREBS, E. G. The enzymology of control by phosphorylation. In:
The Enzymes. Control by Phosphorylation (3rd ed.), edited by P. D
Boyer and E. G. Krebs, Orlando, FL: Academic, 1986, vol. XVII, pt.
A, p. 3–20.
KREGENOW, F. W. The response of duck erythrocytes to nonhemolytic media. Evidence for a volume controlling mechanism.
J. Gen. Physiol. 58: 372–395, 1971.
KREGENOW, F. W. The response of duck erythrocytes to hypertonic media: further evidence for a volume-controlling mechanism.
J. Gen. Physiol. 58: 396 – 412, 1971.
KREGENOW, F. W. The response of duck erythrocytes to norepinephrine and an elevated extracellular potassium: volume regulation in isotonic media. J. Gen. Physiol. 61: 509 –527, 1973.
KREGENOW, F. W. Osmoregulatory salt transporting mechanisms:
control of cell volume in anisosmotic media. Annu. Rev. Physiol.
43: 493–505, 1981.
KREGENOW, F. W., AND T. CARYK. Cotransport of cations and Cl
during the volume regulatory responses of duck erythrocytes (Abstract). Physiologist 22: 73, 1979.
L’ALLEMAIN, G., S. PARIS, AND J. POUYSSÉGUR. Growth factor
action and intracellular pH regulation in fibroblasts. Evidence for a
major role of the Na1/H1 antiport. J. Biol. Chem. 259: 5809 –5815,
1984.
LAMB, J. F. The chloride content of rat auricle. J. Physiol. (Lond.)
157: 415– 425, 1961.
LANDRY, D. W., M. REITMAN, E. J. CRAGOE, JR., AND Q. ALAWQATI. Epithelial chloride channel: development of inhibitory
ligands. J. Gen. Physiol. 90: 779 –798, 1987.
LANG, F., M. RITTER, E. WOLL, H. WEISS, D. HÄUSSINGER, J.
HOFLACHER, K. MALY, AND H. GRUNICKE. Altered cell volume
regulation in ras oncogene expressing NIH fibroblasts. Pflügers
Arch. 420: 424 – 427, 1992.
LAPOINTE, J.-Y., P. D. BELL, AND J. D. CARDINAL. Direct evidence
for apical Na1:2Cl2:K1 co-transport in macula densa cells. Am. J.
Physiol. 258 (Renal Fluid Electrolyte Physiol. 27): F1466 —F1469,
1990.
LAPOINTE, J.-Y., A. LAAMARTI, A. M. HURST, B. C. FOWLER, AND
P. D. BELL. Activation of Na:2Cl:K cotransport by luminal chloride
in macula densa cells. Kidney Int. 47: 752–757, 1995.
LARSON, M., AND K. R. SPRING. Bumetanide inhibition of NaCl
transport by Necturus gallbladder. J. Membr. Biol. 74: 123–129,
1989.
LAUF, P. K., AND N. C. ADRAGNA. Proton (H) modulation of K-Cl
cotransport through both internal and external sites in DIDS-pH-
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
160.
JOHN M. RUSSELL
January 2000
198.
199.
200.
201.
203.
204.
205.
206.
207.
208.
209.
210.
211.
212.
213.
214.
215.
216.
217.
218.
219.
clamped low magnesium LK sheep erythrocytes (Abstract). Biophys. J. 72: A408, 1997.
LAUF, P. K., AND N. C. ADRAGNA. Functional evidence for a pH
sensor of erythrocyte K-Cl cotransport through inhibition by internal protons and diethylpyrocarbonate. Cell. Physiol. Biochem. 8:
46 – 60, 1998.
LAUF, P. K., J. BAUER, N. C. ADRAGNA, H. FUJISE, A. M. M.
ZADE-OPPEN, K. H. RYU, AND E. DELPIRE. Erythrocyte K-Cl cotransport: properties and regulation. Am. J. Physiol. 263 (Cell
Physiol. 32): C917—C932, 1992.
LAUF, P. K., T. J. MCMANUS, M. HAAS, B. FORBUSH III, J. DUHM,
P. W. FLATMAN, M. H. SAIER, JR., AND J. M. RUSSELL. Physiology
and biophysics of chloride and cation cotransport across cell membranes. Federation Proc. 46: 2377–2394, 1987.
LEUNG, S., M. E. O’DONNELL, A. MARTINEZ, AND H. C. PALFREY.
Regulation by nerve growth factor and phosphorylation of Na/K/
2Cl cotransport and cell volume in PC12 cells. J. Biol. Chem. 269:
10581–10589, 1994.
LEVINSON, C. Volume regulatory activity of Ehrlich ascites tumor
cells and its relationship to ion transport. J. Membr. Biol. 100:
182–191, 1987.
LEVINSON, C. Regulatory volume increase in Ehrlich acsites tumor
cells. Biochim. Biophys. Acta 1021: 1– 8, 1990.
LEVINSON, C. Inability of Ehrlich ascites tumor cells to volume
regulate following a hyperosmotic challenge. J. Membr. Biol. 121:
279 –288, 1991.
LEWIS, S. A., AND P. DONALDSON. Ion channels and cell volume
regulation: chaos in an organized system. News Physiol. Sci. 5:
112–119, 1990.
LEWIS, S. A., D. C. EATON, C. CLAUSEN, AND J. M. DIAMOND.
Nystatin as a probe for investigating the electrical properties of a
tight epithelium. J. Gen. Physiol. 70: 427– 440, 1977.
LIEDTKE, C. M. Bumetanide-sensitive Na-Cl cotransport in bovine
tracheal epithelial cells. Am. J. Physiol. 262 (Lung Cell. Mol.
Physiol. 6): L621—L627, 1992.
LOPEZ-RIVAS, A., A. ADELBERGIE, AND E. ROZENGURT. Intracellular K1 and the mitogenic response of 3T3 cells to peptide
factors in serum-free medium. Proc. Natl. Acad. Sci. USA 79:
6275– 6279, 1982.
LORENZ, J. N., H. WEIHPRECHT, J. SCHNERMANN, O. SKOTT,
AND J. P. BRIGGS. Renin release from isolated juxtaglomerular
apparatus depends on macula densa chloride transport. Am. J.
Physiol. 266 (Renal Fluid Electrolyte Physiol. 35): F486 —F493,
1991.
LUBOWITZ, H., AND R. WHITTAM. Ion movements in human red
cells independent of the sodium pump. J. Physiol. (Lond.) 202:
111–131, 1969.
LUDENS, J. H. Nature of the inhibition of Cl2 transport by furosemide: evidence for competitive inhibition of active transport of
toad cornea. J. Pharmacol. Exp. Ther. 223: 25–29, 1982.
LYTLE, C. Activation of the avian erythrocyte Na-K-Cl cotransport
protein by cell shrinkage, cAMP, fluoride, and calyculin-A involves
phosphorylation at common sites. J. Biol. Chem. 272: 15069 –15077,
1997.
LYTLE, C., AND B. FORBUSH III. The Na-K-Cl cotransport protein of
shark rectal gland. II. Regulation by direct phosphorylation. J. Biol.
Chem. 267: 25438 –25443, 1992.
LYTLE, C., AND B. FORBUSH III. Na-K-Cl cotransport in shark
rectal gland. II. Regulation in isolated tubules. Am. J. Physiol. 262
(Cell Physiol. 31): C1009 —C1017, 1992.
LYTLE, C., AND B. FORBUSH III. Is [Cl2]i the switch controlling
Na-K-Cl cotransport in shark rectal gland? (Abstract). Biophys. J.
61: A384, 1992.
LYTLE, C., AND B. FORBUSH III. Regulatory phosphorylation of the
secretory Na-K-Cl cotransporter: modulation by cytoplasmic Cl.
Am. J. Physiol. 270 (Cell Physiol. 39): C437—C448, 1996.
LYTLE, C., M. HAAS, AND T. J. MCMANUS. Chloride-dependent
obligate cation exchange: a partial reaction of [Na1-K1-2Cl2] cotransport (Abstract). Federation Proc. 45: 548, 1986.
LYTLE, C., AND T. J. MCMANUS. A minimal kinetic model of [NaK-2Cl] cotransport with ordered binding and glide symmetry (Abstract). J. Gen. Physiol. 88: 36a, 1986.
LYTLE, C., T. J. MCMANUS, AND M. HAAS. A model of Na-K-2Cl
220.
221.
222.
223.
224.
225.
226.
227.
228.
229.
230.
231.
232.
233.
234.
235.
236.
237.
238.
273
cotransport based on ordered ion binding and glide symmetry.
Am. J. Physiol. 274 (Cell Physiol. 43): C299 —C309, 1998.
LYTLE, C., J.-C. XU, D. BIEMSDERFER, M. HAAS, AND B. FORBUSH III. The Na-K-Cl cotransport of shark rectal gland. I. Development of monoclonal antibodies, immunoaffinity purification, and
partial biochemical characterization. J. Biol. Chem. 267: 25428 –
25437, 1992.
LYTLE, C., J.-C. XU, D. BIEMSDERFER, AND B. FORBUSH III.
Distribution and diversity of Na-K-Cl cotransport proteins: a study
with monoclonal antibodies. Am. J. Physiol. 269 (Cell Physiol. 38):
C1496 —C1505, 1995.
MACLEOD, R. J., AND J. R. HAMILTON. Regulatory volume increase
in mammalian jejunal villus cells is due to bumetanide-sensitive
NaKCl2 cotransport. Am. J. Physiol. 258 (Gastrointest. Liver
Physiol. 21): G665—G674, 1990.
MAIRBÄURL, H., AND C. HERTH. Na1-K1-2Cl2 cotransport,
Na1/H1 exchange, and cell volume in ferret erythrocytes. Am. J.
Physiol. 271 (Cell Physiol. 40): C1603—C1611, 1996.
MASON, S. J., A. M. PARADISO, AND R. C. BOUCHER. Regulation of
transepithelial ion transport and intracellular calcium by extracellular adenosine triphosphate in human normal and cystic fibrosis
airway epithelium. Br. J. Pharmacol. 103: 1649 –1656, 1991.
MASTROIANNI, N., H. DE FUSCO, M. ZOLLO, G. ARRIGO, O.
ZUFFARDI, A. BETTINELLI, A. BALLABIA, AND G. CASARI. Molecular cloning, expression pattern, and chromosomal location of
the human Na-Cl thiazide-sensitive cotransporter (SLC12A3).
Genomics 35: 486 – 493, 1996.
MATHEWS, J., J. SMITH, AND B. HRNJEZ. Effects of F-actin stabilization or disassembly on epithelial Cl secretion and Na-K-2Cl
cotransport. Am. J. Physiol. 272 (Cell Physiol. 41): C254 —C262,
1997.
MATHEWS, J. B., J. A. SMITH, E. C. MUN, AND J. K. SICKLICK.
Osmotic regulation of intestinal epithelial Na1-K1-Cl2 cotransport:
role of Cl2 and actin. Am. J. Physiol. 274 (Cell Physiol. 43):
C697—C706, 1998.
MATHEWS, J. B., J. SMITH, K. TALLY, C. AWTREY, H. NGUYEN, J.
RICH, AND J. MADARA. Na-K-2Cl cotransport in intestinal cells:
influence of Cl efflux and F-actin on regulation of cotransporter
activity and bumetanide binding. J. Biol. Chem. 269: 15703–15709,
1994.
MCROBERTS, J. A., S. ERLINGER, M. J. RINDLER, AND M. H.
SAIER, JR. Furosemide-sensitive salt transport in the Madin-Darby
canine kidney cell line: evidence for the cotransport of Na1, K1 and
Cl2. J. Biol. Chem. 257: 2260 –2266, 1982.
MERCER, R. W., AND P. B. DUNHAM. Membrane-bound ATP fuels
the Na/K pump: studies on membrane-bound glycolytic enzymes on
inside-out vesicles from human red cell membranes. J. Gen.
Physiol. 78: 547–568, 1981.
MEYER, M., K. MALY, F. UBERALL, J. HOFLACHER, AND H. GRUNICKE. Stimulation of K1 transport by Ha-ras. J. Biol. Chem. 266:
8230 – 8235, 1991.
MILANICK, M. Ferret red cells: Na/Ca and Na-K-Cl cotransport.
Comp. Biochem. Physiol. A Physiol. 102: 619 – 624, 1992.
MILLS, J. W., AND L. J. MANDEL. Cytoskeletal regulation of membrane transport events. FASEB J. 8: 1161–1165, 1994.
MIYAMOTO, H., T. IKEHARA, H. YAMAGUCHI, K. HOSOKAWA, T.
YONEZU, AND T. MASURA. Kinetic mechanism of the Na1,K1,Cl2
cotransport as studied by Rb1 influx into HeLa cells: effects of
extracellular monovalent ions. J. Membr. Biol. 92: 135–150, 1986.
MONGIN, A. A., S. L. AKSENTSEV, S. N. ORLOV, N. G. SLEPKO,
M. V. KOZLOVA, G. V. MAXIMOV, AND S. V. KONEV. Swellinginduced K1 influx in cultured primary astrocytes. Brain Res. 655:
110 –114, 1994.
MONGIN, A. A., S. L. AKSENTSEV, S. N. ORLOV, Z. B. KVACHEVA,
N. I. MEZEN, A. S. FEDULOV, AND S. V. KONEV. Swelling-induced
activation of Na1,K1,2Cl2 cotransport in C6 glioma cells: kinetic
properties and intracellular signalling mechanisms. Biochim. Biophys. Acta 1285: 229 –236, 1996.
MOORE, M. L., J. N. GEORGE, AND R. J. TURNER. Anion dependencies of bumetanide binding and ion transport by the rabbit
parotid Na1-K1-2Cl2 co-transporter: evidence for an intracellular
modifier site. Biochem. J. 309: 637– 642, 1995.
MOTOKIZAWA, F., J. F. REUBEN, AND H. GRUNDFEST. Anion
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
202.
Na1-K1-Cl2 COTRANSPORT
274
239.
240.
241.
242.
243.
245.
246.
247.
248.
249.
250.
251.
252.
253.
254.
255.
256.
257.
258.
permeability of the inhibitory post-synaptic membrane of lobster
muscle. J. Gen. Physiol. 50: 2491–2504, 1967.
MOUNT, D. B., E. DELPIRE, G. GAMBA, A. E. HALL, E. POCH, R. S.
HOOVER, AND S. C. HEBERT. The electroneutral cation-chloride
cotransporters. J. Exp. Biol. 201: 2091–2102, 1998.
MOUNTIAN, I., K. Y. CHOU, AND W. VAN DRIESSCHE. Electrolyte
transport mechanisms involved in regulatory volume increase in C6
glioma cells. Am. J. Physiol. 271 (Cell Physiol. 40): C1041—C1048,
1996.
MUSCH, M. W., AND M. FIELD. K-independent Na-Cl cotransport in
bovine tracheal epithelial cells. Am. J. Physiol. 256 (Cell Physiol.
25): C658 —C665, 1989.
MUSCH, M. W., S. A. ORELLANA, L. S. KIMBURG, M. FIELD, D. R.
HALM, E. J. KRASNY, JR., AND R. A. FRIZZELL. Na1-K1-Cl2 cotransport in the intestine of a marine teleost. Nature 300: 351–353,
1982.
NAUNTOFTE, B. Regulation of electrolyte and fluid secretion in
salivary acinar cells. Am. J. Physiol. 263 (Gastrointest. Liver
Physiol. 26): G823—G837, 1992.
NITSCHKE, R., E. SCHLATTER, O. EIDELMAN, H. J. LANG, H. C.
ENGLERT, Z. I. CABANTCHIK, AND R. GREGER. Piretanide-dextran and piretanide-polyethylene glycol interact with high affinity
with the Na1 2 Cl2 K1 cotransporter in the thick ascending limb of
the loop of Henle. Pflügers Arch. 413: 559 –561, 1989.
OBERLEITNER, H., W. GUGGINO, AND G. GIEBISCH. The effect of
furosemide on luminal sodium, chloride and potassium transport in
the early distal tubule of Amphiuma kidney: effects of potassium
adaptation. Pflügers Arch 396: 27–33, 1983.
OBERLEITNER, H., F. LANG, R. GREGER, W. WANG, AND G.
GIEBISCH. Effect of luminal potassium on cellular sodium activity
in the early distal tubules of the Amphiuma kidney. Pflügers Arch.
396: 34 – 40, 1983.
O’BRIEN, T. G., AND K. KRZEMINSKI. Phorbol ester inhibits furosemide-sensitive potassium transport in Balb/3T3 preadipose cells.
Proc. Natl. Acad. Sci USA 80: 4334 – 4338, 1983.
O’BRIEN, T. G., R. PRETTYMAN, K. S. GEORGE, AND H. R.
HIRSCHMAN. A phorbol ester-non proliferative variant of Swiss
3T3 cells is deficient in Na1,K1,Cl2 cotransport activity. J. Cell
Physiol. 134: 302–306, 1988.
O’DONNELL, M. E. [3H]bumetanide binding in vascular endothelial
cells: quantitation of Na-K-Cl cotransporters. J. Biol. Chem. 264:
20326 –20330, 1989.
O’DONNELL, M. E. Regulation of Na-K-Cl cotransport in endothelial cells by atrial natriuretic factor. Am. J. Physiol. 257 (Cell
Physiol. 26): C36 —C44, 1989.
O’DONNELL, M. E. Endothelial cell sodium-potassium-chloride cotransport. Evidence of regulation by Ca21 and protein kinase C.
J. Biol. Chem. 266: 11559 –11566, 1991.
O’DONNELL, M. E. Role of Na-K-Cl cotransport in vascular endothelial volume regulation. Am. J. Physiol. 264 (Cell Physiol. 33):
C1316 —C1326, 1993.
O’DONNELL, M. E., J. D. BRANDT, AND F. R. E. CURRY. Na-K-Cl
cotransport regulates intracellular volume and monolayer permeability of trabecular meshwork cells. Am. J. Physiol. 268 (Cell
Physiol. 37): C1067—C1074, 1995.
O’DONNELL, M. E., A. MARTINEZ, AND D. SUN. Endothelial NaK-Cl cotransport regulation by tonicity and hormones: phosphorylation of cotransport protein. Am. J. Physiol. 269 (Cell Physiol. 38):
C1513—C1523, 1995.
O’GRADY, S. M., H. C. PALFREY, AND M. FIELD. Characteristics
and functions of the Na-K-Cl cotransport in epithelial tissues.
Am. J. Physiol. 253 (Cell Physiol. 22): C177—C192, 1987.
O’GRADY, S. M., H. C. PALFREY, AND M. FIELD. Na-K-2Cl cotransport in winter flounder intestine and bovine outer medulla: [3H]bumetanide binding and effects of furosemide analogues. J. Membr.
Biol. 96: 11–18, 1987.
O’NEILL, W. C., AND J. D. KLEIN. Regulation of vascular endothelial
cell volume by Na-K-2Cl cotransport. Am. J. Physiol. 262 (Cell
Physiol. 31): C436 —C444, 1992.
O’NEILL, W. C., AND J. F. STEINBERG. Functional coupling of
Na1-K1-2Cl2 cotransport and Ca21-dependent K1 channels in vascular endothelial cells. Am. J. Physiol. 267 (Cell Physiol. 36):
C267—C274, 1995.
Volume 80
259. ORLOV, S. N., J. TREMBLAY, AND P. HAMET. Cell volume in
vascular smooth muscle is regulated by bumetanide-sensitive ion
transport. Am. J. Physiol. 270 (Cell Physiol. 39): C1388 —C1397,
1996.
260. OWEN, N., AND M. L. PRASTEIN. Na/K/Cl cotransport in cultured
human fibroblasts. J. Biol. Chem. 260: 1445–1451, 1985.
261. OWEN, N. E. Regulation of Na/K/Cl cotransport in vascular smooth
muscle cells. Biochem. Biophys. Res. Commun. 125: 500 –508,
1984.
262. OWEN, N. E., AND K. M. RIDGE. Mechanism of angiotensin II
stimulation of Na-K-Cl cotransport of vascular smooth muscle
cells. Am. J. Physiol. 257 (Cell Physiol. 26): C629 —C636, 1989.
263. PALFREY, H. C. Na/K/Cl transport in avian red cells: reversible
inhibition by ATP depletion (Abstract). J. Gen. Physiol. 82: 10a,
1983.
264. PALFREY, H. C., P. W. FEIT, AND P. GREENGARD. cAMP-stimulated cation cotransport in avian erythrocytes: inhibition by “loop”
diuretics. Am. J. Physiol. 238 (Cell Physiol. 7): C139 —C148, 1980.
265. PALFREY, H. C., AND P. GREENGARD. Hormone-sensitive ion
transport system in erythrocytes as models for epithelial ion transport pathways. Ann. NY Acad. Sci. 372: 291–308, 1981.
266. PALFREY, H. C., AND M. E. O’DONNELL. Characteristics and regulation of the Na/K/2Cl cotransporter. Cell Physiol. Biochem. 2:
293–307, 1992.
267. PALFREY, H. C., AND E. B. PREWITT. The ATP and Mg21 dependence of Na1-K1-2Cl2 cotransport reflects a requirement for protein phosphorylation: studies using calyculin A. Pflügers Arch. 425:
321–328, 1993.
268. PALFREY, H. C., AND M. C. RAO. Na/K/Cl co-transport and its
regulation. J. Exp. Biol. 106: 43–54, 1983.
269. PALFREY, H. C., P. SILVA, AND F. H. EPSTEIN. Sensitivity of
cAMP-stimulated salt secretion in shark rectal gland to “loop”
diruetics. Am. J. Physiol. 246 (Cell Physiol. 15): C242—C246, 1984.
270. PANET, R. Serum-induced net K1 influx performed by the diureticsensitive transport system in quiescent NIH 3T3 mouse fibroblasts.
Biochim. Biophys. Acta 813: 141–144, 1985.
271. PANET, R., I. AMIR, AND H. ATLAN. Fibroblast growth factor
induces a transient net K1 influx carried by the bumetanide-sensitive cotransporter in quiescent BALB/c 3T3 fibroblasts. Biochim.
Biophys. Acta 859: 117–121, 1986.
272. PANET, R., I. AMIR, D. SNYDER, L. ZONENSHEIN, H. ATLAN, R.
LASKOV, AND A. PANET. Effect of Na1 influx inhibitors on induction of c-fos, c-myc, and ODC genes during cell cycle. J. Cell.
Physiol. 140: 161–168, 1989.
273. PANET, R., AND H. ATLAN. Stimulation of bumetanide-sensitive
Na1/K1/Cl2 by different mitogens in synchronized human fibroblasts is essential for cell proliferation. J. Cell Biol. 114: 337–342,
1991.
274. PANET, R., I. FROMER, AND H. ATLAN. Differentiation between
serum stimulation and of ouabain-resistant and sensitive Rb influx
in quiescent NIH 3T3 cells. J. Membr. Biol. 70: 165–169, 1992.
275. PANET, R., M. MARKUS, AND H. ATLAN. Bumetanide and furosemide inhibited vascular endothelial cell proliferation. J. Cell.
Physiol. 158: 121–127, 1994.
276. PARIS, S., AND J. POUYSSÉGUR. Growth factors activate the bumetanide-sensitive Na1/K1/Cl2 cotransport in hamster fibroblasts.
J. Biol. Chem. 261: 6177– 6183, 1986.
277. PARK, J. H., AND M. H. SAIER, JR. Phylogenetic, structural and
functional characteristics of the Na-K-Cl cotransporter family. J.
Membr. Biol. 149: 161–168, 1996.
278. PARKER, J. C. Volume-responsive sodium movements in dog red
blood cells. Am. J. Physiol. 244 (Cell Physiol. 13): C324 —C330,
1983.
279. PAYNE, J. A. Functional characterization of the neuronal-specific
K-Cl cotransporter: implications for [K1]o regulation. Am. J.
Physiol. 273 (Cell Physiol. 42): C1516 —C1525, 1997.
280. PAYNE, J. A., AND B. FORBUSH III. Alternatively spliced isoforms
of the putative Na-K-Cl cotransporter are diffferentially distributed
within the rabbit kidney. Proc. Natl. Acad. Sci. USA 91: 4544 – 4548,
1994.
281. PAYNE, J. A., AND B. FORBUSH III. Molecular characterization of
the epithelial Na-K-Cl cotransporter isoforms. Curr. Opin. Cell
Biol. 7: 493–503, 1995.
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
244.
JOHN M. RUSSELL
January 2000
Na1-K1-Cl2 COTRANSPORT
304. RUSSELL, J. M. Anion transport mechanisms in neurons. Ann. NY
Acad. Sci. 341: 510 –523, 1980.
305. RUSSELL, J. M. K1-dependent Na-Cl uptake by squid giant axon
(Abstract). Biophys. J. 33: 40a, 1981.
306. RUSSELL, J. M. Cation-coupled chloride influx in squid axon: role
of potassium and stoichiometry of the transport process. J. Gen.
Physiol. 81: 909 –925, 1983.
307. RUSSELL, J. M. Chloride in the squid giant axon. Curr. Top.
Membr. Transp. 22: 177–193, 1984.
308. RUSSELL, J. M., D. C. EATON, AND M. S. BRODWICK. Effects of
nystatin on membrane conductance and internal ion activities in
Aplysia neurons. J. Membr. Biol. 37: 137–156, 1977.
309. SACHS, J. Ouabain-insensitive sodium movements in the human
red blood cell. J. Gen. Physiol. 57: 259 –282, 1971.
310. SACHS, J. R., AND L. G. WELT. The concentration dependence of
active potassium transport in the human red blood cell. J. Clin.
Invest. 46: 65–76, 1967.
311. SAIER, M. H., JR., AND D. A. BOYDEN. Mechanism, regulation and
physiological significance of the loop diuretic-sensitive NaCl/KCl
symport in animal cells. Mol. Cell. Biochem. 59: 11–32, 1984.
312. SANTOSO, A. W. B., D. M. SCOTT, AND R. KINNE. Localization of
Tamm-Horsfall protein in chloride transporting epithelia: lack of
correlation with the Na,K,Cl cotransporter. Eur. J. Cell Biol. 43:
104 –109, 1987.
313. SCHLATTER, E., R. GREGER, AND C. WEIDTKE. Effect of “high
ceiling” diuretics on active salt transport in the cortical thick
ascending limb of Henle’s loop of rabbit kidney. Pflügers Arch. 396:
210 –217, 1983.
314. SCHMIDT, W. F., AND T. J. MCMANUS. Ouabain-insensitive salt and
water movements in duck red cells. I. Kinetics of cation transport
under hypertonic conditions. J. Gen. Physiol. 70: 59 –79, 1977.
315. SCHMIDT, W. F., AND T. J. MCMANUS. Ouabain-insensitive salt and
water movements in duck red cells. II. Norepinephrine stimulation
of sodium plus potassium cotransport. J. Gen. Physiol. 70: 81–97,
1977.
316. SCHMIDT, W. F., AND T. J. MCMANUS. Ouabain-insensitive salt and
water movements in duck red cells. III. The role of chloride in the
volume response. J. Gen. Physiol. 70: 99 –121, 1977.
317. SEGAL, I. H. Enzyme Kinetics. New York: Wiley, 1975.
318. SHANES, A. M. Electrochemical aspects of physiological and pharmacological action in excitable cells. Part 1. The resting cell and its
alteration by extrinsic factors. Pharmacol. Rev. 10: 59 –164, 1958.
319. SHENNAN, D. B., AND A. R. CHIPPERFIELD. The dependence on
chloride ions of the loop diuretic sensitive component of passive
sodium efflux from human red cells. Pflügers Arch. 406: 333–339,
1986.
320. SHETLAR, R. E., B. SCHÖLERMANN, A. I. MORRISON, AND R. K. H.
KINNE. Characteization of a Na1-K1-2Cl2 cotransport system in
oocytes from Xenopus laevis. Biochim. Biophys. Acta 1023: 184 –
190, 1990.
321. SHIH, D. T.-B., AND M. F. PERUTZ. Influence of anions and protons
on the Adair coefficients of haemoglobins A and Cowton [His
HC3(146) b3 Leu]. J. Mol. Biol. 195: 419 – 422, 1987.
322. SHOROFSKY, S. R., M. FIELD, AND H. A. FOZZARD. Mechansim of
Cl secretion in canine trachea: changes in intracellular chloride
activity with secretion. J. Membr. Biol. 81: 1– 8, 1984.
323. SILVA, P., R. SOLOMON, H. BRIGNULL, S. HORNUNG, J. LANDSBERG, H. SOLOMON, D. WOLF, AND F. EPSTEIN. Effect of protein
kinase C activation of chloride secretion by the rectal gland of
Squalus acanthias. Bull. Mt. Desert Isl. Biol. Lab. 31: 65– 67, 1992.
324. SILVA, P., J. STOFF, M. FIELD, L. FINE, J. N. FORREST, AND F. H.
EPSTEIN. Mechanism of active chloride secretion by shark rectal
gland: role of Na-K-ATPase in chloride transport. Am. J. Physiol.
233 (Renal Fluid Electrolyte Physiol. 2): F298 —F306, 1977.
325. SIMON, D. B., F. E. KARET, J. M. HAMDAN, A. DIPIETRO, S.
SANJAD, AND R. P. LIFTON. Bartter’s syndrome, hypokalemic alkalosis with hypercalcuria, is caused by mutations in the Na-K-2Cl
cotransporter NKCC2. Nature Genet. 13: 183–188, 1996.
326. SIMON, D. B., C. NELSON-WILLIAMS, M. J. BIA, D. ELLISON, F. E.
KARET, A. M. MOLINA, I. VAARA, F. IWATA, H. M. CUSHNER, M.
KOOLEN, F. J. GAINZA, H. J. GITELMAN, AND R. P LIPTON.
Gitelman’s variant of Barter’s syndrome, inherited hypokalemic
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
282. PAYNE, J. A., T. J. STEVENSON, AND L. F. DONALDSON. Molecular
characterization of a putative K-Cl cotransporter in rat brain. A
neuronal-specific isoform. J. Biol. Chem. 271: 16245–16252, 1996.
283. PAYNE, J. A., J.-C. XU, M. HAAS, C. Y. LYTLE, D. WARD, AND B.
FORBUSH III. Primary structure, functional expression and chromosomal localization of the bumetanide-sensitive Na-K-Cl cotransporter in human colon. J. Biol. Chem. 270: 17977–17985, 1995.
284. PEDERSEN, S. F., B. KRAMHØFT, N. K. JØRGENSEN, AND E. K.
HOFFMANN. Shrinkage-induced activation of the Na1/H1 exchanger in Ehrlich ascites tumor cells: mechanisms involved in the
activation and a role for the exchanger in cell volume regulation. J.
Membr. Biol. 149: 141–159, 1996.
285. PERUTZ, M. F., D. T.-B. SHIH, AND D. WILLIAMSON. The chloride
effect in human hemoglobin. A new kind of allosteric mechanism.
J. Mol. Biol. 239: 555–560, 1994.
286. PEWITT, E. B., R. S. HEGDE, M. HAAS, AND H. C. PALFREY. The
regulation of Na/K/2Cl cotransport and bumetanide binding in
avian erythrocytes by protein phosphorylation and dephosphorylation. Effects of kinase inhibitors and okadaic acid. J. Biol. Chem.
265: 20747–20756, 1990.
287. PEWITT, E. B., R. S. HEGDE, AND H. C. PALFREY. [3H]bumetanide
binding to avian erythrocyte membranes. Correlation with activation and deactivation of Na/K/2Cl cotransport. J. Biol. Chem. 265:
14364 –14370, 1990.
288. PLOTKIN, M. D., M. R. KAPLAN, L. N. PETERSON, S. R. GULLANS,
S. C. HEBERT, AND E. DELPIRE. Expression of the Na1-K1-Cl2
cotransporter BSC2 in the nervous system. Am. J. Physiol. 272
(Cell Physiol. 41): C173—C183, 1997.
289. QUAGGIN, S. E., J. A. PAYNE, B. FORBUSH III, AND P. IGARASHI.
Localization of the renal Na-K-Cl cotransporter gene (SLC12a1) on
mouse chromosome 2. Mamm. Genomics 6: 557–561, 1995.
290. RAAT, N. J. H., E. DELPIRE, C. H. VAN OS, AND R. J. M. BINDELS.
Culturing induced expression of basolateral Na1-K1-2Cl2 cotransporter BSC2 in proximal tubule, aortic endothelium and vascular
smooth muscle. Pflügers Arch. 431: 458 – 460, 1996.
291. RANDALL, J., T. THORNE, AND E. DELPIRE. Partial cloning and
characteriation of Slc12a2: the gene encoding the secretory Na1K1-2Cl2 cotransporter. Am. J. Physiol. 273 (Cell Physiol. 42):
C1266 —C1277, 1997.
292. RAO, M. C., N. T. NASH, AND M. FIELD. Differing effects of cGMP
and cAMP on ion transport across flounder intestine. Am. J.
Physiol. 246 (Cell Physiol. 15): C167—C171, 1984.
293. RESHKIN, S. J., S. LEE, J. N. GEORGE, AND R. J. TURNER. Identification, characterization and purification of a 160 kD bumetanide-binding glycoprotein from rabbit parotid. J. Membr. Biol.
136: 243–251, 1993.
294. REUSS, L. R. Basolateral KCl cotransport in a NaCl-absorbing
epithelium. Nature 305: 723–726, 1983.
295. RINDLER, R. J., J. A. MCROBERTS, AND M. H. SAIER, JR. (Na1,K1)
cotransport in the Madin-Darby canine kidney cell line. J. Biol.
Chem. 257: 2254 –2259, 1982.
296. ROBERTSON, M. A., AND J. K. FOSKETT. Na1 transport pathways
in secretory acinar cells: membrane cross talk mediated by [Cl2]i.
Am. J. Physiol. 267 (Cell Physiol. 36): C146 —C156, 1994.
297. ROOS, A., AND W. F. BORON. Intracellular pH. Physiol. Rev. 61:
296 – 434, 1981.
298. ROTIN, D., AND S. GRINSTEIN. Impaired cell volume regulation in
Na1-H1 exchange-deficient mutants. Am. J. Physiol. 257 (Cell
Physiol. 26): C1158 —C1165, 1989.
299. ROZENGURT, E. Early signals in the mitogenic response. Science
234: 161–166, 1986.
300. ROZENGURT, E., AND L. A. HEPPEL. Serum rapidly stimulates
ouabain sensitive 86Rb influx in quiescent 3T3 cells. Proc. Natl.
Acad. Sci. USA 72: 4492– 4495, 1975.
301. RUBIN, C. S., J. EHRLICHMAN, AND O. M. ROSEN. Cyclic adenosine 39,59-monophosphate-dependent protein kinase of human
erythrocyte membrane. J. Biol. Chem. 247: 6135– 6139, 1972.
302. RUSSELL, J. M. ATP-dependent chloride influx into internally dialyzed squid giant axons. J. Membr. Biol. 28: 335–349, 1976.
303. RUSSELL, J. M. Chloride and sodium influx: a coupled uptake
mechanism in the squid giant axon. J. Gen. Physiol. 73: 801– 818,
1979.
275
276
327.
328.
329.
330.
331.
333.
334.
335.
336.
337.
338.
339.
340.
341.
342.
343.
alkalosis, is caused by mutations in the thiazide-sensitive Na-Cl
cotransporter. Nature Genet. 12: 24 –30, 1996.
SLOTKI, I., W. V. BREUR, R. GREGER, AND Z. I. CABANTCHIK.
Long-term cAMP activation of the Na1,K1,Cl2 cotransporter activity in HT-29 human adenocarcinoma cells. Am. J. Physiol. 264 (Cell
Physiol. 33): C857—C865, 1993.
SMITH, J. B., AND L. SMITH. Na1/K1/Cl2 cotransport in cultured
vascular smooth muscle cells: stimulation by angiotensin and calcium ionophore, inhibition by cAMP and calmodulin antagonists. J.
Membr. Biol. 99: 51– 63, 1987.
SMYTH, M. J., T. G. O’BRIEN, AND W. WHARTON. Complex mitogenic requirements for Na1-K1-Cl2-cotransport-deficient BALB/c
3T3 fibroblasts. J. Cell. Physiol. 145: 531–535, 1990.
SNYDER, D., H. ATLAN, M. MARKUS, AND R. PANET. Na1/K1/Cl2
cotransport is stimulated by a Ca21-calmoldulin-mediated pathway
in BALB/c 3T3 fibroblasts. J. Cell. Physiol. 149: 497–502, 1991.
STARKE, L. C. Regulation of [Na-K-2Cl] and [K-Cl] Cotransport
in Duck Red Cells. (PhD thesis). Durham, NC: Duke University,
1989.
STOKES, J. B., I. LEE, AND M. D’AMICO. Sodium chloride absorption by the urinary bladder of the winter flounder. A thiazidesensitive, electrically neutral transport system. J. Clin. Invest. 74:
7–16, 1984.
STOSSEL, T. P. From signal to pseudopod: how cells control
cytoplasmic actin assembly. J. Biol. Chem. 264: 18261–18264, 1989.
SUN, A., E. B. GROSSMAN, M. LOMBARDI, AND S. C. HEBERT.
Vasopressin alters the mechanism of apical Cl2 entry from Na1:Cl2
to Na1K1:2Cl2 cotransport in mouse medullary thick ascending
limb. J. Membr. Biol. 120: 83–94, 1991.
SUN, D., AND M. E. O’DONNELL. Astroglial-mediated phosphorylation of the Na-K-Cl cotransporter in brain microvessel endothelial
cells. Am. J. Physiol. 271 (Cell Physiol. 40): C620 —C627, 1996.
SUVITAYAVAT, W., P. B. DUNHAM, M. HAAS, AND M. C. RAO.
Characterization of the proteins of the intestinal Na1-K1-2Cl2 cotransporter. Am. J. Physiol. 267 (Cell Physiol. 36): C375—C384,
1994.
SUVITAYAVAT, W., H. C. PALFREY, M. HAAS, P. B. DUNHAM, F.
KALMAR, AND M. C. RAO. Characterization of the endogenous
Na1-K1-2Cl2 cotransporter in Xenopus oocytes. Am. J. Physiol.
266 (Cell Physiol. 35): C284 —C292, 1994.
TAMM, I., AND F. L. HORSFALL, JR. Characterization and separation
of an inhibitor of viral hemagglutination present in urine. Proc. Soc.
Exp. Biol. Med. 74: 108 –114, 1950.
TANIMURA, A., K. KURIHARA, S. J. RESHKIN, AND R. J. TURNER.
Involvement of direct phosphorylation in the regulation of the rat
parotid Na1-K1-2Cl2 cotransporter. J. Biol. Chem. 270: 25252–
25258, 1995.
TAS, P. W. L., P. T. MASSA, H. G. KRESS, AND K. KOESCHEL.
Characterization of an Na1/K1/Cl2 co-transport in primary cultures
of rat astrocytes. Biochim. Biophys. Acta 903: 411– 416, 1987.
TORCHIA, J., C. LYTLE, D. J. PON, B. FORBUSH III, AND A. K. SEN.
The Na-K-Cl cotransporter of avian salt gland. Phosphorylation in
response to cAMP-dependent and calcium-dependent secretogogues. J. Biol. Chem. 267: 25444 –25450, 1992.
TORCHIA, J., Q. YI, AND A. K. SEN. Carbachol-stimulated phosphorylation of the Na-K-Cl cotransporter of avian salt gland. Requirement for Ca21 and PKC activation. J. Biol. Chem. 269: 29778 –
29784, 1994.
TOYOMOTO, T., D. KNUDSEN, G. SOOS, AND K. SATO. Na-K-2Cl
cotransporters are present and regulated in simian eccrine clear
cells. Am. J. Physiol. 273 (Regulatory Integrative Comp. Physiol.
42): R270 —R277, 1997.
Volume 80
344. TREHARNE, K. J., L. J. MARSHALL, AND A. MEHTA. A novel
chloride-dependent GTP-utilizing protein kinase in plasma membranes from human respiratory epithelium. Am. J. Physiol. 267
(Lung Cell. Mol. Physiol. 11): L592—L601, 1994.
345. TURNER, R. J., AND J. N. GEORGE. Ionic dependence of bumetanide binding to the rabbit parotid Na/K/Cl cotransporter. J.
Membr. Biol. 102: 71–77, 1988.
346. TURNER, R. J., J. N. GEORGE, AND B. J. BAUM. Evidence for a
Na1/K1/Cl2 cotransport system in basolateral membrane vesicles
from the rabbit parotid. J. Membr. Biol. 94: 143–152, 1986.
347. UEBERSCHAR, S., AND T. BAKKER-GRUNWALD. Effects of ATP
and cyclic AMP on the (Na1-K1-Cl2) cotransport system in turkey
erythrocytes. Biochim. Biophys. Acta 818: 260 –266, 1985.
348. USSING, H. H. Volume regulation of frog skin epithelium. Acta
Physiol. Scand. 114: 363–369, 1982.
349. VALDEZ, I. H., M. PAULAIS, P. C. FOX, AND R. J. TURNER. Microfluormetric studies of intracellular Ca21 and Na1 concentrations in normal human labial gland acini. Am. J. Physiol. 267
(Gastrointest. Liver Physiol. 30): G601—G607, 1994.
350. WACKER, W. E. C. Man and Magnesium. Cambridge, MA: Harvard
University, 1980.
351. WHISENANT, N., M. KHADEMAZAD, AND S. MUALLEM. Regulatory interaction of ATP, Na1 and Cl2 in the turnover cycle of the
NaK2Cl cotransporter. J. Gen. Physiol. 101: 889 –908, 1993.
352. WHISENANT, N., B.-X. ZHANG, M. KHADEMAZAD, P. LOESSBERG, AND S. MUALLEM. Regulation of Na-K-2Cl cotransport in
osteoblasts. Am. J. Physiol. 261 (Cell Physiol. 30): C433—C440,
1991.
353. WIENER, H., AND C. VAN OS. Rabbit distal colon epithelium. II.
Characterization of (Na1,K1,Cl2)-cotransport and [3H]bumetanide
binding. J. Membr. Biol. 110: 163–174, 1989.
354. WILCOCK, C., S. B. CHAHWALA, AND J. A. HICKMAN. Selective
inhibition by bis(2-chloroethyl)methyamine (nitrogen mustard) of
the Na1/K1/Cl2 cotransporter of murine L 1210 leukemia cells.
Biochim. Biophys. Acta 946: 368 –378, 1988.
355. WILEY, J. S., AND R. A. COOPER. A furosemide-sensitive cotransport of sodium plus potassium in the human red cell. J. Clin.
Invest. 53: 745–755, 1974.
356. WONG, M. M. Y., AND J. K. FOSKETT. Oscillations of cytosolic
sodium during calcium oscillations in exocrine acinar cells. Science
254: 1014 –1016, 1991.
357. WRIGHT, E. M., AND J. M. DIAMOND. Anion selectivity in biological
systems. Physiol. Rev. 57: 109 –156, 1977.
358. XU, J. C., C. LYTLE, T. ZHU, J. A. PAYNE, E. BENZ, AND B.
FORBUSH III. Molecular cloning and functional expression of the
bumetanide-sensitive Na-K-Cl cotransporter. Proc. Natl. Acad. Sci.
USA 91: 2201–2205, 1994.
359. YANG, T., Y. G. HUANG, I. SINGH, J. SCHNERMAN, AND J. P.
BRIGGS. Localization of bumetanide- and thiazide-sensitive NaK-Cl cotransporters along the rat nephron. Am. J. Physiol. 271
(Renal Fluid Electrolyte Physiol. 40): F931—F939, 1996.
360. YERBY, T. R., C. R. VIBAT, D. SUN, J. A. PAYNE, AND M. E.
O’DONNELL. Molecular characterization of the Na-K-Cl cotransporter of bovine aortic endothelial cells. Am. J. Physiol. 273 (Cell
Physiol. 42): C188 —C197, 1997.
361. ZHANG, Y., M. N. CHERNOVA, A. K. STUART-RILEY, L. JIANG, AND
S. L. ALPER. the cytoplasmic and transmembrane domains of AE2
both contribute to regulation of anion exchange by pH. J. Biol.
Chem. 272: 5741–5749, 1996.
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332.
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