Am J Physiol Regulatory Integrative Comp Physiol
280: R301–R312, 2001.
invited review
Evolution of the Na-Pi cotransport systems
ANDREAS WERNER AND ROLF K. H. KINNE
Department of Physiological Sciences, University of Newcastle, Newcastle upon Tyne NE2 4HH,
United Kingdom; and Max-Planck-Institut für molekulare Physiologie, 44227 Dortmund, Germany
phosphate; comparative physiology; gene structure
EVERY CELL,
whether of prokaryotic or eukaryotic origin,
depends on Pi for structural and metabolic needs. The
energy inherent in electrical and chemical potentials is
convertible into ATP and vice versa (66, 95, 96). Given
the negative electrochemical potential across the cell
membrane, the anionic Pi cannot be accumulated
within the cytosol by simple diffusion. Because availability of Pi is rate limiting to growth, a transport
system capable of accumulating Pi against the prevailing electrochemical gradient is a vital prerequisite for
evolutionary success. Theoretically, an increase of the
extracellular Pi concentration by one or two orders of
magnitude could drive the anion into the cell. However,
the very limited solubility of Pi in combination with
divalent ions such as Ca2⫹ effectively limits the upper
Pi concentrations available to all organisms and cells.
Therefore, the uptake of Pi is obligatorily coupled to a
downhill movement of H⫹ or Na⫹, depending on the
ionic currency used.
Functional studies using a variety of model organisms have revealed a number of different strategies to
deal with the problem of Pi accumulation. A common
theme is that all organisms do not rely on a single
transport system. In unicellular organisms at low external Pi concentrations, a membrane transporter with
high affinity for the substrate combined with a low
transport capacity would be present (PstA/C in Esche-
Address for reprint requests and other correspondence: A. Werner,
Dept. of Physiological Sciences, The Medical School, Framlington
Place, Univ. of Newcastle upon Tyne, NE2 4HH UK (E-mail:
[email protected]).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
http://www.ajpregu.org
0363-6119/01 $5.00 Copyright © 2001 the American Physiological Society
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Werner, Andreas, and Rolf K. H. Kinne. Evolution of the Na-Pi
cotransport systems. Am J Physiol Regulatory Integrative Comp Physiol
280: R301–R312, 2001.—Membrane transport systems for Pi transport
are key elements in maintaining homeostasis of Pi in organisms as
diverse as bacteria and human. Two Na-Pi cotransporter families with
well-described functional properties in vertebrates, namely NaPi-II and
NaPi-III, show conserved structural features with prokaryotic origin. A
clear vertical relationship can be established among the mammalian
protein family NaPi-III, a homologous system in C. elegans, the yeast
system Pho89, and the bacterial Pi transporter Pit. An alternative
lineage connects the mammalian NaPi-II-related transporters with homologous proteins from Caenorhabditis elegans and Vibrio cholerae. The
present review focuses on the molecular evolution of the NaPi-II protein
family. Preliminary results indicate that the NaPi-II homologue cloned
from V. cholerae is indeed a functional Pi transporter when expressed in
Xenopus oocytes. The closely related NaPi-II isoforms NaPi-IIa and
NaPi-IIb are responsible for regulated epithelial Na-dependent Pi transport in all vertebrates. Most species express two different NaPi-II proteins with the exception of the flounder and Xenopus laevis, which rely on
only a single isoform. Using an RT-PCR-based approach with degenerate
primers, we were able to identify NaPi-II-related mRNAs in a variety of
vertebrates from different families. We hypothesize that the original
NaPi-IIb-related gene was duplicated early in vertebrate development.
The appearance of NaPi-IIa correlates with the development of the
mammalian nephron.
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Fig. 1. Different families of Pi translocating proteins. NaPi-II-related proteins (in light gray) are expressed in vertebrates, C. elegans,
and in Vibrio cholerae, but not in Drosophila and yeast. The nonmammalian homologues of NaPi-II have not been characterized on a
functional level, and their names represent either the identification
in the Wormpep database (clone Zk-563.2, GenBank accession number AAA81148) or the suggested name for the protein in the database
(Pi-permease, GenBank accession number CAA09443). The second
panel (in dark gray) displays the protein family NaPi-III. Homologous proteins are expressed in all model organisms shown and
display a highly conserved structure.
both mediated by NaPi-II-related proteins (45, 76). Our
own work has characterized NaPi-II-related sequences
in various vertebrate species to study more closely the
molecular evolution of NaPi-II. Homolog proteins in
nonvertebrates, namely C. elegans and V. cholerae,
have been identified by sequence comparison. In addition, new evidence for bidirectional transcription of the
NaPi-II encoding gene in C. elegans, winter flounder,
and zebrafish provides a novel distinct molecular
mechanism for regulation of NaPi-II gene expression
(48, 79).
NAPI-III, PHO89, PIT FAMILY
This family of proteins has homolog forms in various
model organisms, namely E. coli, S. cerevisiae, C. elegans, and Homo sapiens. In bacteria, it is called Pit
(27); in yeast, is it Pho89 (84); in human, it includes the
two NaPi-III-related retroviral receptors Glvr and Ram
(Pit-1, Pit-2; Ref. 53); and in C. elegans, six related
isoforms are referred to as phosphate permeases (24).
The close homology between these proteins implies
that the transporters share comparable membrane topologies including 10–12 transmembrane spanning
segments (TM). The apparent ambiguous number of
TM helices may reflect the result of different topology
predictions rather than truly divergent structures (51,
68, 104). The hydrophilic loop in rat Pit-2 between TM
6 and 7 (10 TM) or TM 7 and 8 (12 TM), respectively,
has been experimentally assigned to the cytoplasmic
space (17).
The structural parallels within the protein family
are only partly reflected on a functional level. E. coli,
which mostly depends on a bioenergetic H⫹ cycle (66),
couples the uptake of Pi via the Pit system to protons.
The Pit transporter, a low-affinity, high-capacity system, contributes 90% to the total phosphate uptake of
cells grown in high Pi medium (1 mM Pi, Ref. 114). The
Michaelis constants (Kms) for the anion binding were
reported to be in the low micromolar range (9.2 ␮M,
Ref. 70; 11.9 ␮M, Ref. 103; 38.3 ␮M, Ref. 114). Extensive functional characterization of the transporter in
whole cells, membrane vesicles, and proteoliposomes
revealed that to act as a substrate Pi needs to be
complexed with a divalent cation. The requirement of
divalent cations for the Pit system to transport Pi is
underlined by the fact that pitA mutants show resistance to toxic medium concentrations (2.5 mM) of
zinc(II) (5). The metal-HPO42⫺ complex is cotransported
with one proton with a 1:1 stoichiometry as reflected by
the pronounced pH dependence of metal phosphate
uptake (92, 103, 114). Where extracellular pH is increased and/or membrane potential is depolarized, Pi
efflux and exchange occur (92, 103).
In contrast to the bacterial H⫹-MeHPO4 cotransport
system Pit, Pho89 of S. cerevisiae shows the characteristics of a high-affinity Na-Pi cotransport system. The
Kms for Pi binding were reported as 0.5 and 0.6 ␮M.
Roomans et al. (91) proposed two Na-binding sites with
apparent affinities of 0.04 and 29 mM, whereas Martinez and Persson (68) proposed a maximal stimulatory
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richia coli, Pho84 in yeast; Refs. 3, 84, 88). In addition,
a low-affinity, high-capacity system would cover Pi
supply at normal or increased external Pi concentrations (84, 88, 90). In contrast, for multicellular organisms with a balanced internal milieu, the housekeeping
membrane transport system (NaPi-III) is complemented
by one or more highly regulated system(s) at epithelial
surfaces responsible for the control of Pi homeostasis
(NaPi-II; Refs. 76, 109).
The techniques of molecular genetics combined with
the recent explosion of sequence information, including
the entire genomes of E. coli (12), Saccharomyces cerevisiae (18), Caenorhabditis elegans (102), and Drosophila (1), have rendered the structural background of
a number of Pi-transporting genes and added to the
understanding of the phylogenetic relations between
the different systems. However, there is a lack of physiological data to complement the increasing number of
candidate genes identified by database searches. Many
such queries are assigned as “putative” or “similar to”
Pi transporters. Wherever possible, functional and
physiological correlates for the accumulation of Pi to
the related query sequences are included.
This review focuses on comparative aspects of Pi
transport systems, namely on the two protein families,
NaPi-III (Pit, Pho89) and NaPi-II, respectively (Fig. 1).
Sequence comparisons have established homologies between the vertebrate systems NaPi-III and transport
proteins in bacteria and yeast. In vertebrates, these
transport proteins are thought to represent the ubiquitously expressed housekeeping Pi transport system
(52).
From a purely homocentric point of view, the phylogenetic relationship within the epithelial Na-Pi cotransporter family NaPi-II is of major interest. The
members of this protein family are involved in regulated Pi uptake in various epithelia and control Pi
uptake and loss from the organism. Thus, in vertebrates, the slowly adapting intestinal Pi absorption as
well as the acutely regulated renal Pi reabsorption are
INVITED REVIEW
ing, because this protein is highly expressed in a
variety of other tissues, such as lung, prostate, and
pancreas (28). This aspect of NaPi-IIb function is discussed extensively below. The division into the subfamilies NaPi-IIa and IIb allowed the reorganization of the
previously rather heterogeneous protein family comprising NaPi-II-related isoforms from human and rat
(IIa; Ref. 67), mouse (IIa; Ref. 20), opossum (IIa; Ref.
98), rabbit (IIa; Ref. 106), bovine (IIb; Ref. 41), flounder
(IIb; Ref. 111), Xenopus (IIb; Ref. 49), and zebrafish
(IIb; Ref. 79). All these transporters show virtually
identical hydropathy profiles, suggesting a common
three-dimensional structure. Initial topology prediction suggested 11 hydrophobic helices, which are potentially crossing the membrane. However, experimental evidence using rat NaPi-IIa and flounder NaPi-IIb
contradicts a simple in-out arrangement of the predicted helices. Both NH2 and COOH termini have been
confirmed to face the intracellular space, which excludes an uneven number of transmembrane segments
(57, 60). Epitope tags introduced within the first and
the fourth hydrophilic “spacer” were found to be accessible by antibodies without permeabilization of the cell
membrane (60). The fourth intervening sequence, often
referred to as “large, extracellular loop,” is glycosylated
in vivo; however, the modification is not required for
proper sorting and function of the transporter (40).
There is still a controversy about the exact number of
transmembrane helices in NaPi-II: most of the published models comprise eight membrane passages with
one or two hydrophobic helices tilted to form hinge
regions; these are consistent with the experimental
data reported so far (Fig. 2; Refs. 77, 83).
Despite obvious parallels with regard to topology,
the two isoforms NaPi-IIa and NaPi-IIb show distinct
differences in three hydrophilic regions (Fig. 3). These
include the two termini and a segment within the
glycosylated extracellular loop (Fig. 2). The NH2 terminus in either isoform lacks structural hallmarks and
may be deleted without affecting expression in Xenopus oocytes (A. Werner, unpublished). The COOH terminus of both isoforms includes a PDZ binding domain
NAPI-II FAMILY: PROTEIN STRUCTURE
Two Na-dependent Pi cotransport systems can be
functionally distinguished and are expressed in intestinal and renal epithelia. These transport proteins
prominently influence mammalian Pi homeostasis. The
cloning of the relevant proteins from human (28, 67,
73) and mouse (20, 45) revealed a close structural
similarity between the systems. Both are members of
the NaPi-II protein family, with the renal isoforms
belonging to the subfamily IIa and the intestinal transporters assigned to the subfamily NaPi-IIb, respectively. The attribute “intestinal,” however, is mislead-
Fig. 2. Model of NaPi-II based on secondary structure predictions
and experimental findings as suggested by Murer et al. (Ref. 77). The
predicted topology displays 8 transmembrane helices and 2 hydrophobic hinge regions. Both NH2 and COOH termini have been
confirmed experimentally to be cytoplasmic, whereas the first loop
and the glycosylated loop (G) are extracellular (shown in black). The
number of putative NH2-glycosylation sites varies between NaPi-II
proteins from different species. The conserved PDZ binding domain
is shown striped.
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effect on transport rate at a concentration of 25 mM
sodium. Interestingly, a stimulatory effect of divalent
cations on Pi transport rate has been reported for
Pho89. However, this finding was attributed to a decrease in surface potential, and MeHPO4 transport was
not considered (91). Pho89 shows a pronounced pH
dependence with virtually no activity at pHo of 4.5 with
an optimum at 9.5 (68). Therefore, Pho89 maintains Pi
supply in alkaline environments where the protondriven system Pho84 is inactive (84).
The six membrane proteins homologous to NaPi-IIIPit-Pho89 in C. elegans have not been characterized on
a functional base.
The mammalian homologues to the Pit-Pho89 systems comprise the two retroviral receptors Glvr and
Ram (80, 104). The functional properties of both human isoforms and rat Ram have been addressed using
different expression systems, i.e., Xenopus oocytes and
fibroblasts (53, 81). The proteins show comparable
functional characteristics. A two-electrode voltage
clamp using Xenopus oocytes showed a strict Na-dependence of Pi-mediated current by Glvr and Ram,
with 1–2 Na⫹ ions translocated with one Pi per cycle.
Half-maximal transport rate was at concentrations of
40–50 mM Na⫹. The apparent affinity for Pi was reported to be 24 and 25 ␮M for human Glvr and rat
Ram, respectively (53). With transfected fibroblasts
assayed by flux measurements, a Km of 53 ␮M Pi was
reported for human Glvr (81). However, this difference
is likely to reflect experimental rather than functional
discrepancies. Both isoforms show increased transport
rates at low pH, which may reflect a preference for
H2PO43⫺ over HPO43⫺ (52).
Interestingly, both the expression level and thus the
transport activity of the NaPi-III protein family are
regulated by the level of extracellular Pi (17, 53, 68,
114). However, this phenomenon is poorly studied.
This is largely due to the presence of alternative Pitransporting systems that seemed of higher scientific
interest. In E. coli, this is the high-affinity Pi-uptake
system composed of the proteins PstA, PstC, PstB, and
PstS (88, 108). In S. cerevisiae, the regulated pathway
via Pho84, which mediates Pi uptake at low external Pi
concentrations, has been thoroughly investigated (16,
61, 84). In vertebrates, research on Pi transport has
concentrated mostly on the highly regulated epithelial
Na-Pi cotransport system NaPi-II (76–78, 109).
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at the very end of the protein (A/X-T-X-L/F). In addition, the IIb-related proteins contain a stretch of six to
ten cysteine residues that are absent in NaPi-IIa. The
divergent structure in the extracellular loop consists of
45 amino acids (NaPi-IIb) and 27 or 28 amino acids
(NaPi-IIa), respectively (Fig. 3).
To obtain insights into the stage in vertebrate evolution at which gene duplication and the subsequent
development of the two distinct isoforms occurred, we
characterized a number of species for NaPi-II expression. The divergent island flanked by two highly conserved regions allowed us to perform a simple RT-PCR.
Three different degenerate primers in both sense- and
antisense direction were designed on the basis of a
sequence alignment comprising the NaPi-IIa isoforms
from human (67), mouse (20), opossum (98), rabbit
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Fig. 3. A: sequence alignment of NaPi-IIa and NaPi-IIb isoforms from human (GenBank accession numbers
AAA36354 and AAC98695), mouse (AAC42026 and AAC80007), flounder (AAB16821), and the related sequences
from C. elegans (AAA81148) and V. cholerae (CAA09443). The divergent regions between NaPi-IIa and NaPi-IIb
are boxed. Two structural features are highlighted: the COOH-terminal PDZ binding motif is shown in bold, and
the cysteine cluster is represented in the window near the COOH terminus. B: prediction of transmembrane helices
in the NaPi-II homologues from V. cholerae, C. elegans, and human (NaPi-IIa). A web-accessible program based on
a hidden Markov model was used (97, www.cbs.dtu.dk/services/TMHMM-1.0/). Note that the helices do not
necessarily have to cross the membrane. Thin lines connect structures with high sequence similarities. The
number of hydrophobic domains is not constant during evolution; whether this affects the topology of the related
proteins remains to be established (top). Schematic picture of the human NaPi-IIa cDNA with the exon/intron
organization indicated. The given length of exons and introns are from Hartmann et al. (Ref. 37; bottom).
INVITED REVIEW
1
The sequences of the degenerate primers used in screening for
NaPi-II-related sequences in different species are: TMI-1, TCCAAT T/C
A/G T T/C/G GTCAGCATGGT; TMI-2, GGCACCTCT A/G T C/T/G AC
C/A AACAC; TMI-3, CGGTGCATGACT T/G CTT T/C AACTG; TMII-1,
TAGAG G/C A/C C A/G/C GC A/G AACCAGCGGT; TMII-2, GAA G/A
AAGAA G/A TG G/A CA G/C/A AG G/T GC G/A ATCTG; TMII-3,
GCTGCTCTGGACAA C/T GAA G/A GTCAT. Wobbles are shown in
bold. After oligo(dT) primed reverse transcription, a touch-down PCR
protocol was used. If no distinct product was identifiable, a nested PCR
was performed under similar cycling conditions.
sion in oocytes, although at a very low level. Such a
rudimentary form of NaPi-II would not only provide a
useful tool to study aspects of function and topology but
also open up possibilities for overexpressing the protein.
NAPI-II FAMILY: STRUCTURE-FUNCTION
RELATIONSHIP
The functional properties of a variety NaPi-II-related transporters have been reported (14, 15, 30, 32).
Only studies based on cloned transporters will be included at this point, although previous experiments
using isolated brush-border vesicles and cell culture
models show comparable results (35, 78). This reflects
the primary influence of NaPi-II in these preparations
(72, 101). The oocytes of the clawed frog Xenopus laevis
are the expression system of choice for the characterization of transport proteins. Consequently, the overwhelming majority of functional data on NaPi-II-mediated Na-Pi cotransport is based on oocyte expression.
Alternative expression systems have been investigated, but with the intentions to study other aspects
rather than functional characterization (e.g., reconstitution of physiological/regulatory features; protein
overexpression; Refs. 9, 33, 87). The initial experiments in Xenopus oocytes relied on radiotracer flux
measurements, because electroneutrality of the overall
transport was assumed. Busch et al. (15), however,
showed that rat NaPi-IIa-mediated transport elicited a
Pi-inducible inward current into oocytes in the presence of Na⫹ ions. Electrophysiological methods have
subsequently been used to characterize a variety of
NaPi-II members, including human NaPi-IIa (15), rat
NaPi-IIa (14, 30), mouse NaPi-IIa (38) and NaPi-IIb
(45), flounder NaPi-IIb (32), and zebrafish NaPi-IIb
(79). To summarize, the systems show comparable apparent substrate affinities, 30–250 ␮M for Pi and 35–
60 mM for Na, respectively, independent of their origin
and subtype. The coupling ratio is 3 Na⫹ to 1 Pi (31).
The maximal transport rate as well as the affinity for
Na⫹ was found to be dependent on the membrane
potential. For a detailed description of the functional
characteristics of rat NaPi-IIa and flounder NaPi-IIb,
including pre-steady-state analysis of single transport
steps and kinetic modeling refer to the publications by
Forster et al. (30–32). Interestingly, there are no functional correlates of the structural differences noted
between the two isoforms. The characteristic difference
in susceptibility to protons of renal and intestinal
transport as observed in brush-border membrane vesicles (8, 47) could be reconstituted in Xenopus oocytes
expressing murine NaP-IIa and NaPi-IIb, respectively
(14, 45). With the use of chimeras of the latter two
isoforms, the domain responsible for pH dependence of
the transport could be narrowed to just three amino
acids. The sequence 478REK in mouse NaPi-IIa was
shown to confer increased activity with rising pH, i.e.,
to mediate IIa or “renal” characteristics. Orthologous
replacement of the amino acids 478REK by 500GNT
from murine NaPi-IIb resulted in the loss of proton
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(106), and rat (67), as well as the NaPi-IIb sequences
from bovine (41), mouse (46), flounder (111), and zebrafish (79).1 We used the primers to amplify NaPi-IIrelated fragments of renal and/or intestinal RNA from
chicken, carp, flounder, shark, skate, trout, Xenopus,
and Drosophila. The amplified DNA fragments were
cloned and sequenced. The results, including all vertebrate NaPi-II-related sequence information available
to date, are summarized in Fig. 4. The occurrence of
two NaPi-IIb isoforms in most of the fish species
(shark, skate, trout, and zebrafish) establishes IIb as
the ancient isoform. Because C. elegans expresses a
single NaPi-II homologue, we hypothesize duplication
of the original NaPi-IIb-related gene early in the development of vertebrates. Such a scenario is in agreement with the so-called 1-2-4 rule in early vertebrate
evolution (71). The model assumes, on the basis of
symmetry in gene clusters, that the entire genome
underwent two rounds of duplications, with most of the
duplicated copies eventually developing to pseudogenes or even junk DNA. However, a number of genes
have apparently avoided such fate due to, for example,
a specialized expression pattern of the isoforms (4).
Whether the presence of a single transporter in Xenopus and flounder represents the consequence of a gene
loss or is due to a lack of the proposed duplication(s) is
purely speculative.
The expression of the NaPi-IIa isoform is restricted
to mammals and chicken. Interestingly, the avian kidney contains two types of nephrons. The first one is
similar to reptilian nephrons, the other type has a loop
of Henle that enables birds to produce hyperosmotic
urine (13, 23). A putative expression of NaPi-IIa in the
latter nephron type would corroborate the hypothesis
that the evolution of NaPi-IIa paralleled the development of the mammalian-type nephron.
We could not detect an NaPi-II homologue in Drosophila, a fact that has now been corroborated by the
full sequence of the fly genome (1). A blast search using
the highly conserved putative substrate-binding region
as a query sequence failed to identify an NaPi-II homologue in the Drosophila genome. However, a comparable strategy identified an NaPi-II homologue in both
C. elegans and V. cholerae. It seems that NaPi-II was
not required in Drosophila Pi homeostasis, resulting in
a degeneration of the gene. This raises the question
whether NaPi-II candidate genes in C. elegans and V.
cholerae represent Pi carriers or precursor structures
with undefined function (Fig. 3). We have cloned the
relevant bacterial gene, and preliminary results indicate that the protein enhances Pi uptake after expres-
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sensitivity, i.e., in “intestinal” pH dependence (25).
These experiments, as well as the different voltage
dependence of Pi transport between flounder and zebrafish NaPi-IIb (79), corroborate the hypothesis that
functional hallmarks of the two NaPi-II isoforms are
not related to the divergent structures at the termini
and the extracellular loop. Therefore, these structural
differences between NaPi-IIa and NaPi-IIb must carry
alternative functional characteristics, perhaps on a
regulatory level.
NAPI-II FAMILY: GENE STRUCTURE
The genomic structure and chromosome localization
of a variety of different NaPi-II genes from human (37,
100), mouse (37), rat (93), winter flounder (48), and C.
elegans (24) have now been determined. These data are
summarized in Table 1. The size of the NaPi-II genes
parallels the apparent complexity of the organism’s
genome, according to the general relationship for intron-genome suggested by Vinogradov (107). The differences in NaPi-II between C. elegans and vertebrates
are too pronounced to provide insights into the relationship between protein structure and gene development. However, if attention is restricted to the verte-
brate NaPi-II genes, possible relationships may indeed
be dissected.
Complete gene structures are available for human
(Fig. 3B; Refs. 37, 100), mouse (37), and flounder (48).
Table 1. NaPi-II gene structure and
chromosomal localization
Human
NaPi-IIa
NaPi-IIb
Mouse
NaPi-IIa
NaPi-IIb
Flounder
NaPi-IIb
C. elegans
Length
Exons
Introns
15.5 kb
13
12
Chromosome
5q35
Refs.
4p15-p16
37, 58, 59,
69, 74, 100
113
15.3 kb
13
12
13B
116
6.44 kb
4.1 kb
13
10
12
9
X
48
24
Genomic information of NaPi-II-related genes from human,
mouse, winter flounder, and C. elegans. The approximate length of
the gene, the number of exons/introns, the chromosomal assignment,
and the related references are indicated. Information from additional
organisms, rat (93), opossum (43) and zebrafish (Werner, unpublished) are not included because only fragments of the gene are
sequenced.
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Fig. 4. Summary of the NaPi-II-related protein sequences from vertebrates. The multiple sequence alignment and
the related evolutionary tree was performed using the GCG software package. Top, NaPi-IIa subfamily; bottom,
NaPi-IIb subfamily. For each identified transporter, the adjacent table shows the source of the RNA used for cDNA
cloning or RT-PCR, the length of the translated protein (translated cDNA fragments are in italic), the percent
identity with the related human isoform and finally the GenBank accession number.
INVITED REVIEW
transcription rate in cells already expressing NaPi-IIa
(62, 63). Northern blot experiments using mRNA from
superficial and medullary tissue, respectively, corroborate the expression of NaPi-IIa in medullary nephrons
in response to long-term stimulation of Pi reabsorption
(99).
Initial attempts to identify 5⬘-sequences involved in
the regulation of the NaPi-IIa gene (NPT2/Npt2 according to Hartmann et al., Ref. 37) were inconclusive,
suggesting that a constitutively active promoter drives
the expression of NaPi-II (37, 43). In more recent
studies, successful combinations of promoter length
and inducing stimuli [parathyroid hormone (PTH), Pi,
vitamin D3] and cell lines (OK cells, COS-7 cells, fibroblasts) have been reported. Hilfiker et al. (44) identified a C/EBP-like region in the promoter of the human
NaPi-IIa gene that drives cell-specific expression of the
luciferase reporter gene. On the basis of similar experiments, Shachaf et al. (93) established an effect of
tandemly repeated AP2 sites on promoter activity of
the rat NaPi-IIa gene. Furthermore, the identification
of two specific elements responsive to either 1,25-dihydroxyvitamin D3 or dietary Pi, respectively, has added
significant complexity to the transcriptional regulation
of human NaPi-IIa gene (55, 99). Using a COS-7 cell
line expressing the human vitamin D receptor, a fragment of 15 nucleotides in the NaPi-IIa promoter
(⫺1977 to ⫺1963) was shown to bind to the vitamin D
receptor resulting in the stimulation of the expression
of a reporter gene (99). In the same lab, a phosphate
response element (PRE) (at ⫺1010 to ⫺985) was identified. In addition, it was shown that the expression of
the murine transcription factor TFE3, which binds to
PRE, was increased in response to Pi restriction (55).
An unusual but highly interesting feature of some
NaPi-II genes is the recent evidence of the transcription of the complement of the NaPi-II DNA strand
giving rise to a fully processed antisense mRNA. This
phenomenon has been clearly documented in the
NaPi-II homologues of both C. elegans and flounder
and has recently been reported in zebrafish (24, 48, 79).
The C. elegans transcript encodes a transposase without a characterized function. The flounder antisense
mRNA exhibits a short open reading frame, and the
translation product is a 68-amino acid peptide (48).
The presence of an antisense transcript, which shows
partial complementarity to the transporter encoding
mRNA, raises the question of biological relevance. Is
the translation product relevant for Na-Pi cotransporter function, or is the antisense mRNA itself regulatory? The recent cloning of the homologous antisense
product from zebrafish, however, revealed that the
open reading frame as detected in the flounder was not
conserved. This suggests a biological function not at a
protein level but via RNA-RNA interaction. The newly
identified antisense transcript related to one of the two
zebrafish NaPi-IIb genes shows a reciprocal expression
pattern compared with the Na-Pi cotransporter mRNA
(79). Our interpretation of these data is that the formation of sense/antisense duplexes effectively eliminates NaPi-II protein expression. The biology and
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The two NaPi-IIa-related genes show only minor differences with fluctuations in the size of the longer
introns. The location of the introns with respect to the
protein sequence is identical (37, 100). The flounder
gene shows similar features. Interestingly, the divergent protein structure between human/mouse NaPi-IIa
and flounder NaPi-IIb is associated with a difference in
the size of exons 1 and 2 and, consequently, a difference
in the localization of introns I and II. This suggests
that splice site mutations may account for the divergent NH2 terminus of the NaPi-IIa and NaPi-IIb isoforms. A similar mechanism has been proposed for the
smaller NH2 terminus, which increases the hydrophilicity of transthyretin isoforms (2). The fact that a
deletion of the complete NH2 terminus of flounder
NaPi-II did not interfere with the expression of the
truncated protein in Xenopus oocytes questions
whether the NaPi-II NH2 terminus bears any functional significance (Werner, unpublished). The next
five exons and the relevant splice sites in the homologous genes of human, mouse, and flounder are conserved. The picture changes at exon 9 (Fig. 3), where
unrelated protein sequences are expressed in NaPi-IIa
and NaPi-IIb, respectively. The fact that divergence is
restricted to exon 9 again suggests that splice-site
mutations account for the observed changes in protein
structure. Note that exon 9 encodes mostly hydrophilic
amino acids, which are integrated in the large extracellular loop. The remaining gene structure is well
conserved between NaPi-IIa and NaPi-IIb, including
the large exon 13, which encodes ⬃150 amino acids for
the terminal. Because 50–60 amino acids at the COOH
terminus of the two transporter families are divergent,
this may have resulted from a frame shift mutation.
Interestingly two prominent motifs are found within
this region: a string of cysteine residues in the NaPi-IIb
protein family and a PDZ domain binding motive at the
very COOH terminus of both isoforms. The changes are
likely to contribute to differences in cellular expression
of the two NaPi-II isoforms (Figs. 2 and 3).
The promoter region of the NaPi-II gene is of considerable interest because of the likely control of gene
transcription to maintain Pi homeostasis. It is plausible that an increase in NaPi-II mRNA expression accounts for the long-term stimulation of renal Pi reabsorption via brush-border NaPi-IIa function (54, 63,
110); NaPi-IIb transcription in contrast seems to be
unaffected (39). Apparently contradictory results from
adaptation experiments with mice and opossum kidney
(OK) cells show no alteration in NaPi-II mRNA in
response to Pi restriction (85, 101). However, experimental differences (Northern blot vs. ribonuclease protection) or cell-specific ambiguities related to the phenotype of OK cells may account for these differences.
Interestingly, an increase in NaPi-IIa mRNA in OK
cells was observed in response to high bicarbonatecarbon dioxide tension (50). Immunocytochemical demonstration of cell-specific NaPi-IIa protein expression
indicates that a recruitment of new cells expressing
NaPi-IIa mRNA accounts for the observed overall increase of mRNA rather than an upregulation of the
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mechanism of RNA interference (Refs. 29, 115) is of
increasing interest, and the occurrence of the NaPi-II
antisense transcript raises the prospect of a related
mechanism to regulate NaPi-II gene expression.
A COMPARATIVE APPROACH TO EXPLAIN RENAL
EXPRESSION OF NAPI-IIa AND NAPI-IIb
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The principle of whole body homeostasis of Pi requires a balanced intestinal absorption and renal excretion of Pi (7, 22), the latter closely being related to
water availability (13, 23). The need either to retain
water (in terrestrial and marine environments) or to
excrete water (in a freshwater environment) is reflected by specific adaptations in renal structure and
function (13, 23). Whether the molecular physiology of
NaPi-II parallels such broad adaptations will be discussed by comparing just three of our model organisms,
namely winter flounder (seawater), zebrafish (freshwater), and rat (terrestrial). Bijouvet and Reitsma (11)
suggested both fish show net tubular Pi secretion by
simply comparing the daily intake of Pi, plasma Pi
level, and the glomerular filtration rate (GFR). Using
this analysis, the rat, and mammals in general, shows
tubular Pi reabsorption (11). The prediction was confirmed by clearance studies using whole animals (13,
23) and primary cell culture models (36, 89). The molecular identification of the relevant NaPi-II homologues and subsequent immunocytochemical studies
have now resulted in a detailed picture of how NaPi-II
participates in reabsorption/secretion of Pi in the different systems. The flounder (which is a euryhaline
teleost) shows a small number of fully functional glomeruli, which are poorly vasculated. Consequently, the
volume of the filtered primary urine is low (26). Flounder renal tubules consist of a short proximal tubular
segment, denoted PI, which shows morphological features characteristic for a reabsorbing epithelium. The
much longer proximal tubular portion PII is presumed
to be involved in ion secretion (10). The adjacent collecting tubule and collecting duct lead to the bladder,
whose epithelium has morphological and functional
similarities with the mammalian distal tubule. (This is
exemplified by the expression of the thiazide-sensitive
Na-Cl cotransporter in flounder bladder epithelial
cells; Ref. 34). A series of immunocytochemical studies
using an anti-NaPi-IIb antiserum as well as RT-PCR
experiments with isolated tubular segments showed
that the transporter was confined to tubular segment
PII and the collecting tubule. Interestingly, the Na-Pi
cotransporter was found in the basolateral membrane
of the secreting tubule PII and in the apical membrane
in the collecting tubule (26, 56). The membrane localization of the protein is consistent with a mechanism of
secondary active secretion in PII followed by reabsorptive fine tuning of the urine in the adjacent collecting
segment.
It must be emphasized that such an hypothesis is
based on immunocytochemical evidence. Flounder renal tubular primary cultures in a reabsorptive/secretive state express NaPi-II-related mRNA, although im-
munohistochemical detection of the transporter failed
(H. Hentschel, L. Renfro, and A. Werner, unpublished
observation). Efficient secretion of Pi may interfere
with another physiological task of the proximal tubular
segment PII, namely the excretion of Mg2⫹ (10). Efficient secretion of both ions in the same segment would
result in the formation of a precipitate. However, the
nephrons of flounder and other fish do not represent a
uniform population (26, 82). Such heterogeneity in
length may reflect functional differences, i.e., that not
all the nephrons secrete divalent cations and Pi with
the same efficiency.
The renal tubule of zebrafish shows a similar tubular
morphology compared with flounder, although zebrafish nephrons contain functional glomeruli. The recently reported NaPi-IIb-related transporter from zebrafish could be detected only in the apical membrane
of the collecting tubule (Nalbant and Werner, unpublished observation). Both the reabsorptive segment PI
and the secreting segment PII were negative. This
suggests that zebrafish excrete Pi by glomerular filtration followed by NaPi-IIb-mediated reabsorption in the
collecting tubule.
Because renal Pi handling in rats has been extensively studied and reviewed (7, 62, 76–78), only a brief
summary is given here. Rat NaPi-IIa is expressed
apically in the convoluted proximal tubule (21, 63).
Regulation of tubular Pi reabsorption by PTH or Pi
availability is achieved by either incorporation or retrieval of functional NaPi-IIa transporter from the
brush-border membrane (19, 64, 65, 85, 86). With the
use of immunocytochemical methods, NaPi-IIa has not
been detected in other tubular segments, i.e., the distal
tubule, which still accounts for ⬃15% of the absorption
of the filtered Pi load. Interestingly, NaPi-II mRNA
was detected in rat collecting duct by RT-PCR; however, the cognate protein could not be detected (21).
Gene knockout experiments corroborated the central
role of NaPi-IIa in renal Pi handling. Npt2⫺/⫺ mice
(NaPi-IIa knockouts) were viable but showed severe
phosphaturia and skeletal abnormalities (6). Interestingly, the remaining renal Pi reabsorption in these
animals was no longer sensitive to PTH (117).
The expression pattern of NaPi-II in the kidney of
the three model organisms reflects a fundamental difference between fish and mammals with respect to
renal Pi handling: the site of Pi reabsorption shifted
from the collecting tubule in teleosts to the proximal
convoluted tubule in mammals. The proximal Pi reabsorption in mammals may be required due to the background of the high GFR and the reabsorptive capacity
of the proximal tubule. Because terrestrial animals
conserve water by efficient tubular reabsorption, drastic reduction in urinary volume without a parallel
reduction of Pi by reabsorption would result in elevated
concentrations of the anion in later tubular segments,
and, concomitantly, this would increase the risk of
precipitate formation. The divergent expression pattern obviously parallels the structural evolution of the
NaPi-IIa and NaPi-IIb isoforms as suggested in Fig. 4.
INVITED REVIEW
NAPI-IIa AND NAPI-IIb, WHAT DOES THE ONE DO
AND THE OTHER CANNOT?
regulatory sites and are thus not required to be very
efficiently controlled. In contrast, mammals tend to
keep the GFR constant and adapt the rate of reabsorption. Because the filtered load in a mammalian
nephron is high, such regulation has to be both rapid
and efficient to minimize the loss to urine. The evolution of NaPi-IIa may reflect such a tendency: NaPi-IIa,
the phylogenetically younger isoform, harbors the molecular information to participate in rapid regulatory
steps such as endo- and exocytosis. The protein is
expressed in only a limited number of cells and is
committed to the control of whole body Pi homeostasis.
NaPi-IIb, the phylogenetically older isoform, shows a
relatively broad expression pattern and exhibits plasticity with regard to intracellular sorting with the task
of accumulating intracellular Pi in a variety of epithelial tissues.
We thank H. Rimpel for skillful technical assistance and Dr. N. L.
Simmons for fruitful discussions and critical reading of the manuscript.
The work was supported by the Max-Planck Society.
REFERENCES
1. Adams MD, Celniker SE, Holt RA, Evans CA, Gocayne JD,
et al. The genome sequence of Drosophila melanogaster. Science 287: 2185–2195, 2000.
2. Aldred AR, Prapunpoj P, and Schreiber G. Evolution of
shorter and more hydrophilic transthyretin N-termini by stepwise conversion of exon 2 into intron 1 sequences. Eur J Biochem 246: 401–409, 1997.
3. Andre B. An overview of membrane transport proteins in
Saccharomyces cerevisiae. Yeast 11: 1575–1611, 1995.
4. Aparicio S. Vertebrate evolution: recent perspectives from
fish. Trends Genet 16: 54–56, 2000.
5. Beard SJ, Hashim R, Wu G, Binet MR, Hughes MN, and
Poole RK. Evidence for the transport of zinc(II) ions via the pit
inorganic phosphate transport system in Escherichia coli.
FEMS Microbiol Lett 184: 231–235, 2000.
6. Beck L, Karaplis AC, Amizuka N, Hweson AS, Ozawa H,
and Tenenhouse HS. Targeted inactivation of Npt2 in mice
leads to severe renal phosphate wasting, hypercalciuria and
skeletal abnormalities. Proc Natl Acad Sci USA 95: 5372–5377,
1998.
7. Berndt TJ and Knox FG. Renal regulation of phosphate
excretion. In: The Kidney, Physiology and Pathophysiology,
edited by Seldin DW and Giebisch G. New York: Raven, 1992, p.
2511–2532.
8. Berner WR, Kinne R, and Murer H. Phosphate transport
into brush-border membrane vesicles isolated from rat small
intestine. Biochem J 160: 467–474, 1976.
9. Bernhardt F, Schoner W, Schroeder B, Breves G, and
Scheiner-Bobis G. Functional expression and characterization of the wild-type mammalian renal cortex sodium/phosphate cotransporter and an 215R mutant in Saccharomyces
cerevisiae. Biochemistry 38: 13551–13559, 1999.
10. Beyenbach KW and Liu PL. Mechanism of fluid secretion
common to aglomerular and glomerular kidneys. Kidney Int 49:
1543–1548, 1996.
11. Bijouvet OL and Reitsma PH. Phylogeny of renal phosphate
transport in the vertebrates. Adv Exp Med Biol 81: 41–53,
1977.
12. Blattner FR, Plunkett G III, Bloch CA, Perna NT, Burland V, Riley M, Collado-Vides JD, Glasner JD, Rode CK,
Mayhew GF, Gregor J, Davis NW, Kirkpatrick HA, Goeden MA, Rose DJ, Mau B, and Shao Y. The complete genome
sequence of Escherichia coli K-12. Science 277: 1453–1474,
1997.
Downloaded from http://ajpregu.physiology.org/ by 10.220.32.246 on June 14, 2017
At the present time, NaPi-IIa has been detected in
the kidney and the brain (46). As already discussed,
NaPi-IIa is responsible for the controlled renal reabsorption of Pi. What is the function of NaPi-IIa in the
brain? It was argued that NaPi-IIa would cover the
increased demands of Pi in metabolically active cells.
Recent evidence, however, suggests that even in the
brain NaPi-IIa function is related to Pi homeostasis.
Mulroney et al. (75) established a link between the Pi
concentration in the cerebrospinal fluid (CSF) and renal Pi clearance, i.e., experimentally elevated Pi concentration in the CSF resulted in decreased renal Pi
reabsorption despite low or normal levels of Pi in
plasma. Immunocytochemistry has revealed NaPi-IIa
expression in cells lining the third ventricle and amygdala (46, 75). It is hypothesized that NaPi-IIa in the
brain is involved in Pi sensing and consequently that
renal expression of NaPi-IIa can be regulated centrally
(75).
In contrast to the isoform NaPi-IIa, NaPi-IIb is expressed in a variety of tissues, including lung, small
intestine, kidney, prostate, and salivary gland in mammals (28). In zebrafish, a relatively broad expression
pattern of NaPi-IIb was also found (79). In addition,
NaPi-IIb is thought to be instrumental in mediating Pi
secretion in the flounder nephron (26, 56). A reversal of
the Pi flux is achieved by the basolateral sorting of the
transporter. Interestingly, in a number of secreting
human tissues, such as human mammary gland or
prostate, NaPi-IIb-related mRNA was detected (28,
112). Firm evidence has demonstrated Na-dependent
Pi transport in basolateral membrane vesicles from
sheep parotid gland (105). In human milk, sperm fluid,
or in the saliva of ruminants, Pi is an essential component for buffering and nutritional and/or metabolic
needs (94). It is likely that NaPi-IIb may mediate
secondary active Pi accumulation at the basolateral
membrane and consequently reverse Pi flux in mammalian secretory tissue.
An alternative approach to address the issue of protein sorting includes the exogenous expression of NaPiIIa and NaPi-IIb in established cell lines. Such strategy does not necessarily unravel physiologically
relevant sorting mechanisms but may eventually lead
to the identification of sorting motifs within the transport proteins or the cognate interacting factors. Hernando et al. (42) showed that rat NaPi-IIa is sorted
correctly to the brush-border membrane in OK cells
but not in an established pig kidney cell line (LLCPK),
Madin-Darby canine kidney cells, and CaCo-2 cells.
Mouse NaPi-IIb and flounder NaPi-IIb used in the
same study were sorted apically in all the cell lines
used. Molecular components responsible for this difference in sorting have yet to be identified.
Vertebrates other than mammals tend to vary the
GFR to maintain water homeostasis and to secrete
accumulating metabolic by-products (13, 23). Therefore, tubular transport processes are not the primary
R309
R310
INVITED REVIEW
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
(NaPi-5) expressed in Xenopus laevis oocytes. J Membr Biol
160: 9–25, 1997.
Fucentese M, Winterhalter K, Murer H, and Biber J.
Functional expression of rat renal Na/Pi cotransport (NaPi-2) in
Sf 9 cells by the baculovirus system. J Membr Biol 144: 43–48,
1995.
Gamba G, Saltzberg N, Lombardi M, Miyanoshita A, Lytton J, Hediger MA, Brenner BM, and Hebert SC. Primary
structure and functional expression of a cDNA encoding the
thiazide-sensitive, electroneutral sodium-chloride cotransporter. Proc Natl Acad Sci USA 90: 2749–2753, 1993.
Gmaj P and Murer H. Cellular mechanisms of inorganic
phosphate transport in kidney. Physiol Rev 66: 36–70, 1986.
Gupta A and Renfro JL. Control of phosphate transport in
flounder renal proximal tubule primary cultures. Am J Physiol
Regulatory Integrative Comp Physiol 256: R850–R857, 1989.
Hartmann CM, Hweson AS, Kos CH, Hilfiker H, Soumounou Y, Murer H, and Tenenhouse HS. Structure of murine
and human renal type II Na⫹-phosphate cotransporter genes
(Npt2 and NPT2). Proc Natl Acad Sci USA 93: 7409–1744,
1996.
Hartmann CM, Wagner CA, Busch AE, Markovich D,
Biber J, Lang F, and Murer H. Transport characteristics of a
murine renal Na/Pi-cotransporter. Pflügers Arch 430: 830–836,
1995.
Hattenhauer O, Traebert M, Murer H, and Biber J. Regulation of small intestinal Na-P(i) type IIb cotransporter by
dietary phosphate intake. Am J Physiol Gastrointest Liver
Physiol 277: G756–G762, 1999.
Hayes G, Busch A, Loetscher M, Waldegger S, Lang F,
Verrey F, Biber J, and Murer H. Role of N-linked glycosylation in rat renal Na/Pi cotransport. J Biol Chem 269: 24143–
24149, 1994.
Helps C, Murer H, and McGivan J. Cloning, sequence analysis and expression of the cDNA encoding a sodium-dependent
phosphate transporter from the bovine renal epithelial cell line
NBL-1. Eur J Biochem 228: 927–930, 1995.
Hernando N, Sheikh S, Karim-Jimenez Z, Galliker H,
Forgo J, Biber J, and Murer H. Asymmetrical targeting of
type II Na-P(i) cotransporters in renal and intestinal epithelial
cell lines. Am J Physiol Renal Physiol 278: F361–F368, 2000.
Hilfiker H, Hartmann CM, Stange G, and Murer H. Characterization of the 5⬘-flanking region of OK cell type II Na-Pi
cotransporter gene. Am J Physiol Renal Physiol 274: F197–
F204, 1998.
Hilfiker H, Kvietikova I, Hartmann CM, Stange G, and
Murer H. Characterization of the human type II Na/Pi-cotransporter promoter. Pflügers Arch 436: 591–598, 1998.
Hilfiker H, Hattenhauer O, Traebert M, Forster I, Murer
H, and Biber J. Characterization of a murine type II sodiumphosphate cotransporter expressed in mammalian small intestine. Proc Natl Acad Sci USA 95: 14564–14569, 1998.
Hisano S, Haga H, Li Z, Tatsumi S, Miyamoto I, Takeda E,
and Fukui Y. Immunohistochemical and RT-PCR detection of
Na⫹-dependent inorganic phosphate cotransporter (NaPi-2) in
rat brain. Brain Res 772: 149–155, 1997.
Hoffman N, Thees M, and Kinne R. Phosphate transport by
isolated renal brush border vesicles. Pflügers Arch 362: 147–
156, 1976.
Huelseweh B, Kohl B, Hentschel H, Kinne RK, and
Werner A. Translated anti-sense product of the Na/phosphate
co-transporter (NaPi-II). Biochem J 332: 483–489, 1998.
Ishizuya-Oka A, Stolow MA, Ueda S, and Shi Y. Temporal
and spatial expression of an intestinal Na/Pi cotransporter
correlates with epithelial transformation during thyroid hormone-dependent frog metamorphosis. Dev Genet 20: 53–66,
1997.
Jehle AW, Hilfiker H, Pfister MF, Biber J, Lederer E,
Krapf R, and Murer H. Type II Na-Pi cotransport is regulated
transcriptionally by ambient bicarbonate/carbon dioxide tension in OK cells. Am J Physiol Renal Physiol 276: F46–F53,
1999.
Johann SV, Gibbons JJ, and O’Hara B. GLVR1, a receptor
for gibbon ape leukemia virus, is homologous to a phosphate
Downloaded from http://ajpregu.physiology.org/ by 10.220.32.246 on June 14, 2017
13. Braun EJ and Dantzler WH. Vertebrate renal system. In:
Handbook of Physiology. Comparative Physiology. Bethesda,
MD: Am Physiol Soc., 1997, sect. 13, vol. I, chapt. 8, p. 481–576.
14. Busch A, Waldegger S, Herzer T, Biber J, Markovich D,
Hayes G, Murer H, and Lang F. Electrophysiological analysis of Na⫹/Pi cotransport mediated by a transporter cloned
from rat kidney and expressed in Xenopus oocytes. Proc Natl
Acad Sci USA 91: 8205–8208, 1994.
15. Busch AE, Wagner CA, Schuster A, Waldegger S, Biber J,
Murer H, and Lang F. Properties of electrogenic Pi transport
by a human renal brush border Na⫹/Pi transporter. J Am Soc
Nephrol 6: 1547–1551, 1995.
16. Bun-Ya M, Nishimura M, Harashima S, and Oshima Y.
The PHO84 gene of Saccharomyces cerevisiae encodes an inorganic phosphate transporter. Mol Cell Biol 11: 3229–3238,
1991.
17. Chien ML, Foster JL, Douglas JL, and Garcia JV. The
amphotropic murine leukemia virus receptor gene encodes a
71-kilodalton protein that is induced by phosphate depletion.
J Virol 71: 4564–4570, 1997.
18. Clayton RA, White O, Ketchum KA, and Venter JC. The
first genome from the third domain of life. Nature 387: 459–
462, 1997.
19. Collins JF, Bulus N, and Ghishan FK. Sodium-phosphate
transporter adaptation to dietary phosphate deprivation in
normal and hypophosphatemic mice. Am J Physiol Gastrointest
Liver Physiol 268: G917–G924, 1995.
20. Collins JF and Ghishan FK. Molecular cloning, functional
expression, tissue distribution, and in situ hybridization of the
renal sodium phosphate (Na⫹/P(i)) transporter in the control
and hypophosphatemic mouse. FASEB J 8: 862–868, 1994.
21. Custer M, Loetscher M, Biber J, Murer H, and Kaissling
B. Expression of Na-Pi cotransport (NaPi-2) in rat kidney:
localization by RT-PCR and immunohistochemistry. Am J
Physiol Renal Fluid Electrolyte Physiol 266: F767–F774, 1994.
22. Danisi G and Murer H. Inorganic phosphate absorption in
small intestine. In: Handbook of Physiology. The Gastrointestinal System. Intestinal Absorption and Secretion. Bethesda, MD:
Am Physiol Soc., 1991, sect. 6, vol. IV, chapt. 12, p. 323–336.
23. Dantzler WH. Comparative aspects of renal function. In: The
Kidney, Physiology and Pathophysiology, edited by Seldin DW
and Giebisch G. New York: Raven, 1992, p. 885–942.
24. Database Services, elegans C [online]. The Sanger Centre.
http://www.sanger.ac.uk [2000 June 21].
25. De la Horra C, Hernando N, Lambert G, Forster I, Biber
J, and Murer H. Molecular determinants of pH sensitivity of
the type IIa Na/P(i) cotransporter. J Biol Chem 275: 6284–
6287, 2000.
26. Elger M, Werner A, Herter P, Kohl B, Kinne RK, and
Hentschel H. Na-P(i) cotransport sites in proximal tubule and
collecting tubule of winter flounder (Pleuronectes americanus).
Am J Physiol Renal Physiol 274: F374–F383, 1998.
27. Elvin CM, Dixon NE, and Rosenberg H. Molecular cloning
of the phosphate (inorganic) transport (pit) gene of Escherichia
coli K12. Identification of the pit⫹ gene product and physical
mapping of the pit-gor region of the chromosome. Mol Gen
Genet 204: 477–484, 1986.
28. Field JA, Zhang L, Brun KA, Brooks DP, and Edwards
RM. Cloning and functional characterization of a sodium-dependent phosphate transporter expressed in human lung and
small intestine. Biochem Biophys Res Commun 258: 578–582,
1999.
29. Fire A. RNA-triggered gene silencing. Trends Genet 15: 358–
363, 1999.
30. Forster I, Hernando N, Biber J, and Murer H. The voltage
dependence of a cloned mammalian renal type II Na/Pi cotransporter. J Gen Physiol 112: 1–18, 1998.
31. Forster I, Loo DF, and Eskandari S. Rat and flounder renal
type II Na-Pi cotransporters have identical stoichiometry but
different Na binding cooperativity. Am J Physiol Renal Physiol
276: F644–F649, 1999.
32. Forster IC, Wagner CA, Busch AE, Lang F, Biber J, Hernando N, Murer H, and Werner A. Electrophysiological
characterization of the flounder type II Na⫹/Pi cotransporter
INVITED REVIEW
52.
53.
54.
55.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70. Medveczky N and Rosenberg H. Phosphate transport in
Escherichia coli. Biochim Biophys Acta 241: 494–506, 1971.
71. Meyer A and Schartol M. Gene and genome duplications in
vertebrates: the one-to-four (-to-eight in fish) rule and the
evolution of novel gene functions. Curr Opin Cell Biol 11:
699–704, 1999.
72. Miyamoto K, Segawa H, Morita K, Nii T, Tatsumi S,
Taketani Y, and Takeda E. Relative contributions of Na⫹dependent phosphate co-transporters to phosphate transport in
mouse kidney: RNase H-mediated hybrid depletion analysis.
Biochem J 327: 735–739, 1997.
73. Miyamoto K, Tatsumi S, Sonoda T, Yamamoto H, Minami
H, Taketani Y, and Takeda E. Cloning and functional expression of a Na-dependent phosphate cotransporter from human
kidney: cDNA cloning and functional expression. Biochem J
305: 81–85, 1995.
74. Miyamoto K, Tatsumi S, Yamamoto H, Katai K, Taketani
Y, Morita K, and Takeda E. Chromosome assignments of
genes for human Na(⫹)-dependent phosphate co-transporters
NaPi-3 and NPT-1. Tokushima J Exp Med 42: 5–9, 1995.
75. Mulroney SE, Woda CB, Bishop EK, Schulkin J, Haramati A, and Levi M. Central effects of low dietary phosphate
(Pi) on Pi appetite and brain and kidney NaPi-2 transporters
(Abstract). FASEB J 14: 472.28, 2000.
76. Murer H and Biber J. Molecular mechanisms of renal apical
Na/phosphate cotransport. Annu Rev Physiol 58: 607–618,
1996.
77. Murer H, Forster I, Hernando N, Lambert G, Traebert M,
and Biber J. Posttranscriptional regulation of the proximal
tubule NaPi-II transporter in response to PTH and dietary P(i).
Am J Physiol Renal Physiol 277: F676–F684, 1999.
78. Murer H, Werner A, Reshkin S, Wuarin F, and Biber J.
Cellular mechanisms in proximal tubular reabsorption of inorganic phosphate. Am J Physiol Cell Physiol 260: C885–C899,
1991.
79. Nalbant P, Böhmer C, Dehmelt L, Wehner F, and Werner
A. Functional characterization of a Na/Pi cotransporter (NaPiII) from zebrafish and identification of related transcripts.
J Physiol (Lond) 520: 79–89, 1999.
80. O’Hara B, Johann SV, Klinger HP, Blair DG, Rubinson H,
Dunn KJ, Sass P, Vitek SM, and Tobins T. Characterization
of a human gene conferring sensitivity to infection by gibbon
ape leukemia virus. Cell Growth Differ 1: 119–127, 1990.
81. Olah Z, Lehel C, Anderson WB, Eiden MV, and Wilson CA.
The cellular receptor for gibbon ape leukemia virus is a novel
high affinity sodium-dependent phosphate transporter. J Biol
Chem 269: 25426–25431, 1994.
82. Ottosen PD. Ultrastructure and segmentation of microdissected kidney tubules in the marine flounder, Pleuronectes
platessa. Cell Tissue Res 190: 27–45, 1978.
83. Paquin J, Vincent E, Dugre A, Xiao Y, Boyer CJ, and
Beliveau R. Membrane topography of the renal phosphate
carrier NaPi-2: limited proteolysis studies. Biochim Biophys
Acta 1431: 315–328, 1999.
84. Persson BL, Petersson J, Fristedt U, Weinander R, Berhe
A, and Pattison J. Phosphate permeases of Saccharomyces
cerevisiae: structure, function and regulation. Biochim Biophys
Acta 1422: 255–272, 1999.
85. Pfister MF, Hilfiker H, Forgo J, Lederer E, Biber J, and
Murer H. Cellular mechanisms involved in the acute adaptation of OK cell Na/Pi-cotransport to high- or low-Pi medium.
Pflügers Arch 435: 713–719, 1998.
86. Pfister MF, Ruf I, Stange G, Ziegler U, Lederer E, Biber J,
and Murer H. Lysosomal degradation of renal type II Na/Pi
cotransporter, a novel principle in the regulation of membrane
transport. Proc Natl Acad Sci USA 45: 1909–1914, 1998.
87. Quabius ES, Murer H, and Biber J. Expression of proximal
tubular Na-Pi and Na-SO4 cotransporters in MDCK and LLCPK1 cells by transfection. Am J Physiol Renal Fluid Electrolyte
Physiol 270: F220–F228, 1996.
88. Roa NN and Torriani A. Molecular aspects of phosphate
transport in Escherichia coli. Mol Microbiol 4: 1083–1090,
1990.
Downloaded from http://ajpregu.physiology.org/ by 10.220.32.246 on June 14, 2017
56.
permease of Neurospora crassa and is expressed at high levels
in the brain and thymus. J Virol 66: 1635–1640, 1992.
Kavanaugh MP and Kabat D. Identification and characterization of a widely expressed phosphate transporter/retrovirus
receptor family. Kidney Int 49: 959–963, 1996.
Kavanaugh MP, Miller, Zhang W, Law W, Kozak SL, Kabat D, and Miller AD. Cell-surface receptors for gibbon ape
leukemia virus and amphotropic murine retrovirus are inducible sodium-dependent phosphate symporters. Proc Natl Acad
Sci USA 91: 7071–7075, 1994.
Kempson SA, Loetscher M, Kaissling B, Biber J, Murer H,
and Levi M. Parathyroid hormone action on phosphate transporter mRNA and protein in rat proximal tubules. Am J Physiol
Renal Fluid Electrolyte Physiol 268: F784–F791, 1995.
Kido S, Miyamoto K, Mizobuchi H, Taketani Y, Ohkido I,
Ogawa N, Kaneko Y, Harashima S, and Takeda. Identification of regulatory sequences and binding proteins in the type
II sodium/phosphate cotransporter NPT2 gene responsive to
dietary phosphate. J Biol Chem 274: 28256–28263, 1999.
Kohl B, Herter P, Hulseweh B, Elger M, Hentschel H,
Kinne RK, and Werner A. Na-Pi cotransport in flounder:
same transport system in kidney and intestine. Am J Physiol
Renal Fluid Electrolyte Physiol 270: F937–F944, 1996.
Kohl B, Wagner CA, Huelseweh B, Busch AE, and Werner
A. The Na/Pi cotransport system (NaPi-II) with a cleaved backbone: implications on function and membrane insertion.
J Physiol (Lond) 508: 341–350, 1998.
Kos CH, Tihy F, Econs MJ, Murer H, Lemieux N, and
Tenenhouse HS. Localization of a renal sodium-phosphate
cotransporter gene to human chromosome 5q35. Genomics 19:
176–177, 1994.
Kos CH, Tihy F, Murer H, Lemieux N, and Tenenhouse
HS. Comparative mapping of Na⫹-phosphate cotransporter
genes, NPT1 and NPT2, in human and rabbit. Cytogenet Cell
Genet 75: 22–24, 1996.
Lambert G, Traebert M, Hernando N, Biber J, and Murer
H. Studies on the topology of the renal type II NaPi cotransporter. Eur J Physiol 437: 972–978, 1999.
Lenburg ME and O’Shea EK. Signaling phosphate starvation. Trends Biochem Sci 21: 383–387, 1996.
Levi M, Kempson SA, Loetscher M, Biber J, and Murer H.
Molecular regulation of renal phosphate transport. J Membr
Biol 154: 1–9, 1996.
Levi M, Loetscher M, Sorribas V, Custer M, Arar M,
Kaissling B, Murer H, and Biber J. Cellular mechanisms of
acute and chronic adaptation of rat renal Pi transporter to
alterations in dietary Pi. Am J Physiol Renal Fluid Electrolyte
Physiol 267: F900–F908, 1994.
Loetscher M, Kaissling B, Biber J, Murer H, and Levi M.
Role of microtubules in the rapid regulation of renal phosphate
transport in response to acute alterations in dietary phosphate
content. J Clin Invest 99: 1302–1312, 1997.
Loetscher M, Scarpetta Y, Levi M, Wang H, Zajicek HK,
Biber J, Murer H, and Kaissling B. Rapid downregulation of
rat renal Na/Pi cotransporter in response to parathyroid hormone: role of microtubule rearrangement. J Clin Invest 104:
483–494, 1999.
Lolkema JS, Speelmans G, and Konings WN. Na⫹-coupled
versus H⫹-coupled energy transduction in bacteria. Biochim
Biophys Acta 1187: 211–215, 1994.
Magagnin S, Werner A, Markovich D, Sorribas V, Biber J,
and Murer H. Expression cloning of human and rat renal
cortex Na/Pi-cotransport. Proc Natl Acad Sci USA 90: 5979–
5983, 1993.
Martinez P and Persson BL. Identification, cloning and characterization of a derepressible Na⫹-coupled phosphate transporter in Saccharomyces cerevisiae. Mol Gen Genet 258: 628–
638, 1998.
McPherson JD, Krane MC, Wagner-McPherson CB, Kos
CH, and Tenenhouse HS. High resolution mapping of the
renal sodium-phosphate cotransporter gene (NPT2) confirms its
localization to human chromosome 5q35. Pediatr Res 41: 632–
634, 1997.
R311
R312
INVITED REVIEW
103. Van Veen HW, Abee T, Kortstee GJ, Konings WN, and
Zehnder AJ. Translocation of metal phosphate via the phosphate inorganic transport system of Escherichia coli. Biochemistry 33: 1766–1770, 1994.
104. Van Zeijl M, Johann SV, Closs E, Cunningham J, Eddy R,
Shows TB, and O’Hara B. A human amphotropic retrovirus
receptor is a second member of the gibbon ape leukemia virus
receptor family. Proc Natl Acad Sci USA 91: 1168–1172, 1994.
105. Vayro S, Kemp R, Beechey RB, and Shirazi-Beechey S.
Preparation and characterization of basolateral plasma-membrane vesicles from sheep parotid glands. Mechanisms of phosphate and D-glucose transport. Biochem J 279: 843–848, 1991.
106. Verri T, Markovich D, Perego C, Norbis F, Stange G,
Sorribas V, Biber J, and Murer H. Cloning of a rabbit renal
Na-Pi cotransporter which is regulated by dietary phosphate.
Am J Physiol Renal Fluid Electrolyte Physiol 268: F626–F633,
1995.
107. Vinogradov AE. Intron-genome size relationship on a large
evolutionary scale. J Mol Evol 49: 376–384, 1999.
108. Wanner BL. Signal transduction in the control of phosphateregulated genes of Escherichia coli. Kidney Int 49: 964–967,
1996.
109. Werner A, Dehmelt L, and Nalbant P. Na⫹-dependent phosphate cotransporters: the NaPi protein families. J Exp Biol 201:
3135–3142, 1998.
110. Werner A, Kempson SA, Biber J, and Murer H. Increase of
Na/Pi-cotransport encoding mRNA in response to low Pi diet in
rat kidney cortex. J Biol Chem 269: 6637–6639, 1994.
111. Werner A, Murer H, and Kinne RK. Cloning and expression
of a renal Na-Pi cotransport system from flounder. Am J
Physiol Renal Fluid Electrolyte Physiol 267: F311–F317, 1994.
112. Werner A, Nalbant P, and Dehmelt L. The intestinal
Na⫹-Pi co-transporter IIb: a novel keyplayer in phosphate
homeostasis (Abstract). J Physiol (Lond) 520P: S127, 1999.
113. White KE, Biber J, Murer H, and Econs MJ. Chromosomal
localization of two human genes involved in phosphate homeostasis: the type IIb sodium-phosphate cotransporter and
stanniocalcin-2 Somat. Cell Mol Genet 24: 357–362, 1998.
114. Willsky GR and Malamy MH. Characterization of two genetically separable inorganic phosphate transport systems in
Escherichia coli. J Bacteriol 144: 356–365, 1980.
115. Zamore PD, Tuschl T, Sharp PA, and Bartel DP. RNAi:
double-stranded RNA directs the ATP-dependent cleavage of
mRNA at 21 to 23 nucleotide intervals. Cell 101: 25–33,
2000.
116. Zhang XX, Tenenhouse HS, Hewson AS, Murer H, and
Eydoux P. Assignment of renal-specific Na(⫹)-phosphate cotransporter gene Slc17a2 to mouse chromosome band 13B by in
situ hybridization. Cytogenet Cell Genet 77: 304–305, 1997.
117. Zhao N and Tenenhouse HS. Npt2 gene disruption confers
resistance to the inhibitory action of parathyroid hormone on
renal sodium-phosphate cotransport. Endocrinology 141: 2159–
2165, 2000.
Downloaded from http://ajpregu.physiology.org/ by 10.220.32.246 on June 14, 2017
89. Renfro JL and Gupta A. Comparative physiology of phosphate transport across renal plasma membranes. In: Comparative Aspects of Sodium Cotransport Systems, edited by Kinne
RKH. Basel: Karger, 1990, p. 216–240.
90. Roomans GM, Blasco F, and Borst-Pauwels GW. Cotransport of phosphate and sodium by yeast. Biochim Biophys Acta
467: 65–71, 1977.
91. Roomans GM and Borst-Pauwels GW. Interaction of cations
with phosphate uptake by Saccharomyces cerevisiae. Effects of
surface potential. Biochem J 178: 521–527, 1979.
92. Rosenberg H, Russell LM, Jacomb PA, and Chegwidden
K. Phosphate exchange in the pit transport system in Escherichia coli. J Bacteriol 149: 123–130, 1982.
93. Shachaf C, Skorecki KL, and Tzukerman M. Role of AP2
consensus sites in regulation of rat Npt2 (sodium-phosphate
cotransporter) promoter. Am J Physiol Renal Physiol 278:
F406–F416, 2000.
94. Shirazi-Beechey SP, Penny JI, Dyer J, Wood IS, Tarpey
PS, Scott D, and Buchan W. Epithelial phosphate transport
in ruminants, mechanisms and regulation. Kidney Int 49: 992–
996, 1996.
95. Skulachev VP. The laws of cell energetics. Eur J Biochem 208:
203–209, 1992.
96. Skulachev VP. The latest news from the sodium world. Biochim Biophys Acta 1187: 216–221, 1994.
97. Sonnhammer ELL, Heijne G, and Krogh A. A hidden
Markov model for predicting transmembrane helices in protein
sequences. In: Proceedings of Sixth International Conference on
Intelligent Systems for Molecular Biology, edited by Glasgow
EJ, Littlejohn T, Major F, Lathrop R, Sankoff D, and Sensen C.
Menlo Park, CA: AAAI, 1998, p. 175–182.
98. Sorribas V, Markovich D, Hayes G, Stange G, Forgo J,
Biber J, and Murer H. Cloning of a Na/Pi cotransporter from
opossum kidney cells. J Biol Chem 269: 6615–6621, 1994.
99. Taketani Y, Segawa H, Chikamori M, Morita K, Tanaka
K, Kido S, Yamamoto H, Lemori Y, Tatsumi S, Tsugawa N,
Okano T, Kobayashi T, Miyamoto K, and Takeda E. Regulation of type II renal Na⫹-dependent inorganic phosphate
transporters by 1,25-dihydroxyvitamin D3. Identification of a
vitamin D-responsive element in the human NAPi-3 gene.
J Biol Chem 273: 14575–14581, 1998.
100. Taketani Y, Miyamoto K, Tanaka K, Katai K, Chikamori
M, Tatsumi S, Segawa H, Yamamoto H, Morita K, and
Takeda E. Gene structure and functional analysis of the human Na⫹/phosphate co-transporter. Biochem J 324: 927–934,
1997.
101. Tenenhouse HS, Roy S, Martel J, and Gauthier C. Differential expression, abundance and regulation of Na-phosphate
cotransporter genes in murine kidney. Am J Physiol Renal
Physiol 275: F527–F534, 1998.
102. The C. elegans Sequencing Consortium. Genome sequence
of the nematode C elegans: a platform for investigating biology.
Science 282: 2012–2018, 1998.