Molecular cloning, chromosomal organization, and functional
characterization of a sodium-dicarboxylate cotransporter from mouse
kidney
Ana M.
Pajor and
Nina N.
Sun
Department of Physiology and Biophysics, University of Texas
Medical Branch, Galveston, Texas 77555
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ABSTRACT |
The sodium-dicarboxylate cotransporter of the renal
proximal tubule, NaDC-1, reabsorbs filtered Krebs cycle intermediates and plays an important role in the regulation of urinary citrate concentrations.1 Low urinary citrate is a risk factor for
the development of kidney stones. As an initial step in the
characterization of NaDC-1 regulation, the genomic structure and
functional properties of the mouse Na+-dicarboxylate
cotransporter (mNaDC-1) were determined. The gene coding for mNaDC-1,
Slc13a2, is found on chromosome 11. The gene is ~24.9 kb
in length and contains 12 exons. The mRNA coding for mNaDC-1 is found
in kidney and small intestine. Expression of mNaDC-1 in Xenopus
laevis oocytes results in increased transport of di- and
tricarboxylates. The Michaelis-Menten constant
(Km) for succinate was 0.35 mM, and the
Km for citrate was 0.6 mM. The transport
of citrate was stimulated by acidic pH, whereas the transport of
succinate was insensitive to pH changes. Transport by mNaDC-1 is
electrogenic, and substrates produced inward currents in the presence
of sodium. The sodium affinity was relatively high in mNaDC-1, with
half-saturation constants for sodium of 10 mM (radiotracer experiments)
and 28 mM at
50 mV (2-electrode voltage clamp experiments). Lithium
acts as a potent inhibitor of transport, but it can also partially
substitute for sodium. In conclusion, the mNaDC-1 is related in
sequence and function to the other NaDC-1 orthologs. However, its
function more closely resembles the rabbit and human orthologs rather
than the rat NaDC-1, with which it shares higher sequence similarity.
citrate; succinate; dicarboxylate transport; sodium
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INTRODUCTION |
THE
NA+-dicarboxylate cotransporter of the apical
membrane of the renal proximal tubule, NaDC-1, reabsorbs Krebs cycle
intermediates, such as succinate and citrate, from the tubular filtrate
(19). This transporter has a broad substrate specificity
for di- and tricarboxylates, but tricarboxylate substrates, such as
citrate, are carried in protonated form. NaDC-1 is electrogenic,
coupling three sodium ions with each divalent anion substrate molecule, with the net transfer of one positive charge (20). The
Na+-dicarboxylate cotransporters belong to a gene family
that includes the Na+-sulfate cotransporters, NaSi-1 and
SUT-1 (6, 15). Three orthologs of NaDC-1 have been
isolated from rabbit (rb), human (h), and rat (r) (17, 18,
28). This gene family also contains the Xenopus
laevis (X. laevis) intestinal NaDC-2, which uses both sodium and lithium to drive dicarboxylate transport (2),
and the high-affinity transporters, NaDC-3, found on the basolateral membrane of kidney proximal tubule and brush-border membrane of placenta (3, 11, 30).
One of the physiological functions of NaDC-1 is to regulate the
concentration of urinary citrate, an endogenous inhibitor of calcium
stone formation (8, 23). Low urinary citrate
concentrations are associated with an increased risk of kidney stone
formation (23). However, there is very little information
on the relationship between NaDC-1 activity and the mechanisms that
produce hypocitraturia. For example, it is not known whether NaDC-1
contributes to hypocitraturia directly, although there are reports of
kidney stone patients with idiopathic hypocitraturia (24,
16). Alternately, the role of NaDC-1 in producing hypocitraturia
could be secondary to another problem, such as metabolic acidosis. The
transport of citrate by NaDC-1 is stimulated by acidic pH (21,
34). One mechanism for this transport activation is an increase
in the concentration of the preferred substrate,
citrate2
, as the pH is decreased. However, chronic
metabolic acidosis also stimulates citrate uptake by inducing
transporter activity (9), which is the result of an
increase in NaDC-1 mRNA and protein (1). Other chronic
conditions, such as K+ deficiency and starvation, also
stimulate citrate transport (1, 29, 33).
As a first step in studying the regulatory mechanisms in NaDC-1 and
preparatory to the production of transgenic or "knockout" mice, the
gene structure and functional properties of the
Na+-dicarboxylate cotransporter in mouse were studied. We
find that the gene structure in mice is remarkably similar to that in
humans and that the NaDC-1 found in mouse kidney has functional
similarities to the other NaDC-1 orthologs. However, there are some
species differences in substrate selectivity and in interaction with
lithium, suggesting that mouse Na+-dicarboxylate
cotransporter (mNaDC-1) may also be useful for studying
structure-function relationships in this family of transporters.
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METHODS |
mNaDC-1 cloning.
The cDNA coding for the mNaDC-1 was amplified from reverse-transcribed
mouse kidney mRNA by PCR. The primers were designed based on sequence
alignments of the rabbit Na+-dicarboxylate cotransporter
(rbNaDC-1) with the mouse genomic sequence of chromosome 11 (GenBank AC002324) and were designed to amplify the coding region and
part of the 3' untranslated region of mNaDC-1. The sense primer
included the start codon (in bold): 5'-GACAGGCTGGTCCTCACCATGGC-3' and the antisense primer was:
5'-CCTGAACACCGGGAAACACACCC-3'. The PCR was done using the Advantage
cDNA PCR kit (Clontech) containing a proofreading DNA polymerase,
Klentaq, to minimize mutations. The PCR product was cloned into the
pCRII vector using the TopoTA cloning kit (In Vitrogen). The pCRII
vector contains EcoR I sites on both sides of the insertion
site of the PCR product. This allowed the excision of the insert from
the pCRII vector using EcoR I and subcloning into the
EcoR I sites of a construct of pSPORT I containing the 5'
untranslated region (UTR) and part of the 3' UTR and
poly(A+) tail of rbNaDC-1, as described previously
(18). Both strands of the mNaDC-1 cDNA were sequenced at
the Sealy Center for Molecular Science (University of Texas Medical
Branch, Galveston, TX). The sequence was assembled and analyzed, and
the gene was mapped, using programs from the Genetics Computer Group
package. Sequence comparisons were run using the BLAST server at the
National Center for Biotechnology Information. The secondary structure
model was prepared based on Kyte-Doolittle hydropathy analysis of the
sequence (13).
X. laevis oocytes and transport measurements.
Stage V and VI oocytes from X. laevis were dissected and
injected as described previously (17). Transport of
[3H]succinate (DuPont-NEN) and [14C]citrate
(Moravek) was measured between 4 and 7 days after injection, also as
described (17). The sodium transport buffer contained (in
mM) 100 NaCl, 2 KCl, 1 MgCl2, 1 CaCl2, and 10 HEPES, buffered to pH 7.5 with Tris base. For choline or lithium
transport buffers the NaCl was replaced by 100 mM choline Cl or LiCl,
respectively. In the transport assays, the oocytes were rinsed briefly
with choline buffer to remove sodium and serum. Transport was initiated by replacement of the choline rinse with 0.4 ml of the appropriate transport buffer as described in the figure legends. Transport was
stopped by the addition of 4 ml ice-cold choline buffer followed by
removal of extracellular radioactivity with three additional washes in
cold choline buffer. Individual oocytes were transferred to
scintillation vials and dissolved in 0.25 ml 10% SDS. Scintillation cocktail was added, and radioactivity was counted. Counts in control uninjected oocytes were subtracted from the counts in cRNA-injected oocytes. Data are presented as means ± SE, except for kinetic constants for which the error represents the error of the fit. Kinetic
constants were calculated by nonlinear regression to the Michaelis-Menten and Hill equations, using SigmaPlot 5.0 software (Jandel Scientific).
Electrophysiology experiments.
Measurements of substrate-induced inward currents in X. laevis oocytes expressing mNaDC-1 were made using the
two-electrode voltage clamp technique, as described (20).
The pulse protocol was controlled using pClamp6 software (Axon
instruments) and consisted of 100-ms voltage steps from a holding
potential of
50 mV between +50 and
150 mV in 20-mV decrements.
Substrate-dependent currents were determined from the difference
between currents measured in sodium buffer with and without substrate.
Northern blot.
A mouse multiple-tissue Northern blot, purchased from Origene
Technologies, contained 2 µg of poly(A+) RNA in each
lane. The blot was probed with the full-length mNaDC-1 insert
(EcoR I fragment) at high stringency in 50% formamide
at 42°C (17). High-stringency washes in 0.1× SSC, 0.1%
SDS at 55°C were also done as described previously (17).
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RESULTS |
Cloning of mNaDC-1.
A routine search of the GenBank database revealed a genomic sequence of
a portion of mouse chromosome 11 (GenBank accession no. AC002324) that
is very similar in sequence to other NaDC-1-type Na+-dicarboxylate cotransporters (17, 18).
Gene-specific primers were designed based on the sequence alignment
with rabbit NaDC-1. PCR amplification of mouse kidney cDNA using the
gene-specific primers produced a cDNA of 2,200 bases containing a
single open reading frame. The mNaDC-1 cDNA sequence codes for a
protein of 586 amino acids (Fig. 1).
There are two consensus sequences for N-glycosylation,
Asn570 and Asn580, both located at the COOH
terminus, a glycosylation pattern that is similar to other members of
this gene family (19). The sequence also contains two
potential protein kinase C phosphorylation sites (Ser211 and Ser435), of which only
Ser435 is predicted to be located in an intracellular loop.
The alignment of the amino acid sequence of mNaDC-1 with other NaDC-1
orthologs is also shown in Fig. 1. The closest relative of mNaDC-1 is
the rat NaDC-1, (rNaDC-1), which is 92% identical in sequence. The mNaDC-1 is 77% identical to human Na+-dicarboxylate
cotransporter (hNaDC-1) and 75% identical to rbNaDC-1. The mNaDC-1 is
~47% identical to the NaDC-3 orthologs (from rat and flounder) and
45% identical to the Na+-sulfate cotransporter, NaSi-1
(4, 15, 28, 30).

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Fig. 1.
Alignment of Na+-dicarboxylate cotransporter
type 1 (NaDC-1) amino acid sequences from mouse (GenBank accession no.
AF201903), rat (AF058714), human (U26209), and rabbit (U12186). The
alignment was made by using the Genetics Computer Group software
package (GCG) program Pileup. The amino acids that are identical in all
of the NaDC-1 orthologs are shown in the bold consensus line (Cons).
The locations of the 11 predicted transmembrane domains are indicated
by lines above the sequences.
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Mouse Slc13a2 gene.
The human gene coding for hNaDC-1 has been assigned the name SLC13A2,
which represents the solute carrier family 13, member 2 (member 1 of
this family is NaSi-1). Therefore, using the nomenclature for mouse
genes, the mNaDC-1 gene is called Slc13a2. The intron-exon organization of the Slc13a2 gene, shown in
Table 1, was determined by sequence
alignment between the mNaDC-1 cDNA and the sequence of mouse chromosome
11 (GenBank AC002324). The total length of the gene is ~24.9 kb, and
it is divided into 12 exons (Fig. 2). The
sequence of the mNaDC-1 cDNA and the exons in the gene are identical.
The gene sequence also contains a TATA box located 93 bases upstream of
the start codon, suggesting that the translation start site is likely
to be between 53-68 bases upstream of the start codon.

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Fig. 2.
Top: secondary structure model of mouse
Na+-dicarboxylate cotransporter (mNaDC-1) with 11 transmembrane domains and two N-glycosylation sites at the
COOH terminus (Y). The amino acids encoded by each of the 12 exons in the Slc12a2 gene are shown as alternate shaded and
nonshaded circles. Bottom: intron-exon organization of the
Slc13a2 gene. The gene coding for mNaDC-1 contains 12 exons and a total
size of ~24.9 kb. The sequence of the entire Slc13a2 gene
is found in GenBank AC002324.
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The predicted secondary structure model of mNaDC-1 (Fig. 2) contains 11 transmembrane domains and an extracellular COOH terminus, similar to
the structure of the other NaDC-1s (19). The exons in the
Slc13a2 gene code for different transmembrane domains, and
the boundaries often occur near the interface between the membrane- and
aqueous-exposed portions of the protein (Fig. 2). The intron-exon
boundaries in the mouse gene are identical to those of the
corresponding human gene, SLC13A2 [the human gene was mapped by
comparing the hNaDC-1 sequence with the sequence of chromosome 17 (GenBank AC005726), results not shown]. The human gene is found on
chromosome 17 p11.1-q11.1 (14, 18).
Functional characterization of mNaDC-1: substrates.
X. laevis oocytes injected with cRNA coding for mNaDC-1 had
sodium-dependent dicarboxylate transport activity, whereas control oocytes do not express dicarboxylate or tricarboxylate transporters (17, 20). As shown in Fig.
3, the Michaelis-Menten constant (Km) for succinate in mNaDC-1 was 318 µM, and
in a second experiment the Km was 373 µM. The
Km for citrate was 732 µM (Fig. 3), and in
three experiments the mean Km for citrate was
612 ± 132 µM (means ± SE). The mNaDC-1 substrate
affinities are more similar to those of the rbNaDC-1 (17),
despite being more closely related in sequence to rNaDC-1, which has a
Km for succinate of ~25 µM (4,
27). Also, there was no evidence of substrate inhibition by high
concentrations of citrate or succinate in mNaDC-1, as reported for the
rNaDC-1 (SDCT1) (4). As shown previously for other NaDC-1
orthologs, the transport of succinate by mNaDC-1 was unaffected by
changes in pH whereas the transport of citrate was markedly stimulated
at acidic pH (Fig. 4). This result
supports the hypothesis that tricarboxylates are preferentially carried in protonated form and also shows that mNaDC-1 is functionally related
to other NaDC-1s. The response to pH in NaDC-2 and the NaDC-3s are
different from those of the NaDC-1 orthologs (2, 3, 11).

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Fig. 3.
Substrate kinetics in oocytes expressing mNaDC-1. Five-minute
uptakes were measured in sodium-containing buffers. A:
succinate transport as a function of increasing concentrations of
succinate in the medium. The Michaelis-Menten constant
(Km) for succinate is 318 ± 37 µM (the
error represents the SE of the regression), and the maximum velocity
(Vmax) is 18 ± 1 nmol · oocyte 1 · h 1.
B: kinetics of citrate transport. The
Km for citrate is 732 ± 217 µM, and the
Vmax is 19 ± 2 nmol · oocyte 1 · h 1.
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Fig. 4.
Effect of pH on transport of succinate and citrate by
mNaDC-1. Transport of 100 µM [3H]succinate or
[14C]citrate was measured in mNaDC-1-injected
Xenopus laevis oocytes for 15 min. Transport solutions
contained 100 mM sodium buffered to pH 7.5 or 5.5. Data shown are
means ± SE; n = 5 oocytes.
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The substrate specificity of mNaDC-1 was determined by inhibition of
succinate transport and by measurement of substrate-induced currents.
The most potent inhibitor of succinate transport by mNaDC-1 was
2,2-dimethylsuccinate (DMS) (Fig. 5), a
substrate previously thought to be specific for the high-affinity
transporter, NaDC-3, found on the renal basolateral membrane
(32). Other substrates that produced >50% inhibition of
transport include fumarate, succinate, glutarate, tricarballylate, and
-ketoglutarate (Fig. 5). The substrate selectivity of mNaDC-1 was
further tested by measurement of substrate-induced currents using the
two-electrode voltage clamp technique. As is the case for other members
of this family, transport of succinate by mNaDC-1 is electrogenic,
producing inward currents in the presence of 1 mM substrate and 100 mM
sodium. Figure 6A shows the
voltage-dependence of succinate-induced currents in mNaDC-1. The
currents produced by test substrates at
50 mV expressed as a
percentage of the succinate-induced current are shown in Fig.
6B. As predicted by the transport inhibition profile, large
currents were produced by dicarboxylates (fumarate,
-ketoglutarate, and dimethylsuccinate) and tricarboxylates (citrate and
tricarballylate). Smaller currents, 10-30% of the
succinate-induced currents, were seen in the presence of the acidic
amino acids, aspartate and glutamate, and no currents were produced by
the monocarboxylate, pyruvate.

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Fig. 5.
Substrate specificity of mNaDC-1. Transport of 10 µM
[3H]succinate was measured for 15 min in the presence or
absence of 1 mM test inhibitors. The transport in the presence of
inhibitor is expressed as a percentage of control measured in the
absence of inhibitor. DMS, 2,2-dimethylsuccinate; -KG,
-ketoglutarate. Data shown are means ± SE; n = 3 experiments. There was significant inhibition (P < 0.05) by all substrates except for aspartate and pyruvate.
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Fig. 6.
A: voltage dependence of succinate-induced currents in
an oocyte expressing mNaDC-1. The pulse protocol was as described in
METHODS. Vm, membrane voltage.
B: substrate-dependent inward currents (I)
measured at 50 mV expressed as a percentage of the currents measured
with succinate (Isuccinate). Data shown are
means ± SE; n = 3 experiments.
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Functional characterization: cations.
The mouse NaDC-1 is a sodium-dependent transporter. Replacement of
sodium with choline resulted in complete abolishment of transport (Fig.
7 and 8). Increasing the concentrations
of sodium in the transport buffers resulted in a sigmoid activation of
succinate transport (Fig. 7). The half-saturation constant for sodium
(KNa) was 9.5 mM, and the apparent Hill
coefficient, nH, was 1.48 (Fig. 7). In a
second experiment (not shown) the apparent KNa
was 11 mM and nH was 1.87.

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Fig. 7.
Sodium activation of succinate transport in oocytes
expressing mNaDC-1. The transport of 100 µM
[3H]succinate was measured for 5 min in buffers
containing 0-100 mM Na+ (Na+ replaced by
choline). The data were fit by the Hill equation. The half-saturation
constant for sodium (KNa): 9.5 ± 1.5 mM,
Vmax: 1,359 ± 83 pmol · oocyte 1 · h 1, Hill
coefficient (nH) = 1.48 ± 0.35 (± SE of
regression). Each data point represents the means ± SE of 5 oocytes.
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Fig. 8.
Sodium activation of succinate-induced currents in an oocyte
expressing mNaDC-1 measured with the 2-electrode voltage clamp method.
A: succinate-dependent inward currents as a function of
sodium concentration. The succinate concentration was 100 µM. The
curves at four of the voltages tested are shown. B: the
effect of voltage on Imax (top), the
half-saturation constant for sodium
(K0.5Na; middle), and
apparent nH (bottom), determined from
nonlinear regression to the Hill equation. The error bars represent
errors of the fit.
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The sodium-activation experiment was also done using the two-electrode
voltage clamp technique (Fig. 8). The
substrate-dependent currents in oocytes expressing mNaDC-1 were
measured at different sodium concentrations. Similar to the radiotracer
uptake experiment, there was a sigmoidal relationship between sodium
concentration and substrate-dependent inward currents in mNaDC-1 (Fig.
8A). The half-saturation constant for sodium
(K0.5sodium) was 28 mM at
50 mV
[in 3 experiments, K0.5sodium at
50 mV was 22.5 ± 3.1 mM, means ± SE]. The
K0.5sodium decreased as the membrane
voltage became more negative (Fig. 8B), indicating that
sodium binding is a voltage-sensitive step. The maximum current
observed at saturating substrate concentrations, (Imax), became larger with more negative
membrane potentials. The nH under voltage clamp
conditions was ~2 (between 1.9-2.1) at all voltages tested (Fig.
8B). In three experiments, the nH at
50 mV was 2.1 ± 0.04.
In the rbNaDC-1, lithium competes with sodium with high affinity at one
of the three sodium binding sites, resulting in transport inhibition
with an apparent inhibition constant of 2 mM (20). However, in the absence of sodium, lithium can drive transport of
succinate in rbNaDC-1, although the succinate Km
measured in lithium is very high, around 3 mM (20).
Therefore, we examined the effects of lithium in mNaDC-1. As shown in
Fig. 9A, lithium can partially
substitute for sodium, producing ~30% of the transport rate seen in
sodium. The inhibition by lithium was lower than that seen in the
rbNaDC-1, however. In 5 mM lithium the inhibition was only ~20%
(Fig. 9B).

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Fig. 9.
Cation dependence of mNaDC-1. A: transport of 100 µM
[3H]succinate was measured in transport buffers
containing 100 mM concentrations of sodium, lithium, or choline (as
chloride salts). B: transport of 100 µM
[3H]succinate was measured in transport buffers
containing either 95 mM Na+:5 mM choline or 95 mM
Na+:5 mM Li+. Fifteen-minute uptakes were
measured. Data shown are means ± SE of 5 oocytes.
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The cation selectivity of currents induced by mNaDC-1 is shown in Fig.
10. There are no inward currents in the
presence of choline. Interestingly, the substrate-dependent currents in
lithium are ~8% of the currents measured in sodium, which is lower
than the uptake measurements in lithium (Fig. 9A). In three
experiments, the currents measured in lithium at
50 mV and 1 mM
succinate were 10 ± 1.5% (means ± SE) of the currents
measured in sodium.

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Fig. 10.
Cation specificity of mNaDC-1 measured using the
2-electrode voltage clamp technique. Steady-state substrate-dependent
currents measured in 100 mM sodium, lithium, or choline are plotted as
a function of membrane potential. The succinate concentration was 1 mM.
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Northern blot.
The tissue distribution of mNaDC-1 mRNA is shown in Fig.
11. The predominant hybridization
signal was seen at 2.4 kb in kidney and small intestine although there
was a less abundant message of ~5 kb. There was no hybridization
signal with brain, heart, stomach and skeletal muscle, similar to the
tissue distributions of other NaDC-1 orthologs (19).

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Fig. 11.
Multiple tissue Northern blot of mouse
poly(A+) RNA probed at high stringency with mNaDC-1 cDNA.
The major hybridization signal was at 2.4 kb in kidney and small
intestine. Sk muscle, skeletal muscle.
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DISCUSSION |
The mouse low-affinity Na+-dicarboxylate
cotransporter, called mNaDC-1, is found in the kidney and small
intestine. The Slc13a2 gene, coding for mouse NaDC-1, is
located on chromosome 11, and has been mapped to ~10 kb upstream of
the nude gene, whn (25). Slc13a2 is
divided into 12 exons, each of which codes for approximately one
transmembrane domain of mNaDC-1, which resembles the gene structure of
other membrane transporters, such as the Na+-glucose
cotransporter, hSGLT1 (31). Remarkably, the gene structure of Slc13a2 is identical to the structure of the human gene,
SLC13A2, which is found on chromosome 17 (14, 18).
The functional properties of mNaDC-1 resemble those of other NaDC-1
orthologs, although there are some interesting species differences.
Transport by mNaDC-1 is sodium dependent, electrogenic, and the
preferred substrates appear to be divalent anions, as seen in the other
members of this family (19). The mNaDC-1 transporter has a
broad substrate specificity for a range of di- and tricarboxylates, including fumarate, succinate, and citrate. Interestingly, mNaDC-1 handles DMS as a substrate. Although DMS was previously thought to be
specific for the high-affinity Na+-dicarboxylate
cotransporter (NaDC-3) found on the basolateral membrane in kidney
proximal tubule (32), some of the low-affinity (NaDC-1)
transporters, such as rNaDC-1, also carry DMS (20).
The coupling coefficient in mNaDC-1 is likely to be 3 Na+:1
divalent anion substrate, similar to the other NaDC transporters (19). Inward currents were measured in oocytes expressing
mNaDC-1 in the presence of substrate and sodium, suggesting that there is a net movement of positive charge into the cells. Citrate transport was stimulated by acidic pH, which supports the idea that citrate is
carried in divalent form. There was no effect of pH on succinate transport, suggesting that both succinate2
and
succinate1
may be substrates of the transporter,
although succinate exists predominantly as a divalent anion at
physiological pH. Finally, the apparent nH of
1.5-2.1 in mNaDC-1 is also consistent with three sodium binding
sites. The nH indicates the minimal number of
sodium binding sites, and is affected by the strength of interaction or
cooperativity between the sites (26).
The m- and rNaDC-1 orthologs are ~92% identical in amino acid
sequence. Despite the sequence similarity, some of their functional properties are very different. For example, the apparent
Km for succinate in mNaDC-1 is around 0.35 mM,
similar to the Km of the rb- and hNaDC-1
(21). Similarly, previous studies using rabbit brush-border membrane vesicles showed that the
Km for succinate is around 0.5 mM (12,
35). In contrast, rNaDC-1 has a higher apparent substrate
affinity, with a Km for succinate of 25 µM for
the cloned transporter expressed in X. laevis oocytes
(27, 4) and a Km of ~150 µM in
rat renal brush-border membrane vesicles (5, 29).
There are also differences between m- and rNaDC-1 in their sensitivity
to inhibition by lithium. The mNaDC-1 is relatively insensitive to
inhibition by lithium, with only ~20% inhibition in the presence of
5 mM lithium. Furthermore, lithium can partially substitute for sodium
in mNaDC-1. In contrast, the rNaDC-1 is sensitive to inhibition by
lithium, with an IC50 of ~3 mM, and lithium cannot
substitute for sodium (4). Because lithium competes with
sodium at one of the three cation binding sites, the identification of
residues contributing to lithium binding or inhibition may provide
information on differences in the structures of cation binding sites.
Chimeras made between h- and rbNaDC-1 transporters show that
transmembrane domain 11 and the COOH-terminal tail contain the residues
that determine their differences in lithium sensitivity (10). The rat and mouse sequences only differ by 8%, and
in exon 12, which codes for the last transmembrane domain, there are
only 4 amino acid differences.
One of the physiological functions of NaDC-1 is to reabsorb filtered
citrate from the proximal tubule lumen, thus helping to regulate the
concentration of urinary citrate. Citrate forms soluble complexes with
calcium, which prevents the precipitation of calcium in the form of
calcium oxalate or calcium phosphate stones. Urinary citrate
concentrations are low in approximately one-half of patients with renal
stones (23). However, there is still very little
information on the relationship between NaDC-1 and hypocitraturia. To
produce hypocitraturia, the activity of NaDC-1 has to increase, which
could be a consequence of several events. For example, in many kidney
stone patients, there are disturbances in acid-base balance. Chronic
metabolic acidosis leads to an increased Vmax
for citrate, mediated through increased NaDC-1 mRNA and protein
concentrations (1). However, there are also reports of
kidney stone patients with idiopathic hypocitraturia or low urinary
citrate in the absence of other disorders (16, 24). One
possible mechanism that could produce low urinary citrate would be an
increase in NaDC-1 activity by direct effects on the transporter
properties. In humans the fractional excretion of citrate is quite
high, ~30%, compared with ~2% in other species (8).
This difference may be due to species differences in citrate Km that are approximately tenfold between
hNaDC-1 and rNaDC-1 (21). Our mutagenesis studies have
shown that a single mutation in NaDC-1 could potentially produce a
large change in Km (7). An
alternate mechanism of activation of NaDC-1 could involve second messenger systems, producing an increase in turnover number. There is
some evidence that NaDC-1 activity can be modulated by increasing the
activity of protein kinases. Treatment of oocytes with phorbol esters
results in a decreased activity by NaDC-1, of which ~30% can be
accounted for by endocytosis of the transporter from the plasma
membrane, suggesting that the remaining decrease in activity is due to
an effect on the transporter itself (22).
In conclusion, we have presented the sequence, chromosomal
organziation, and tissue distribution of mNaDC-1. The mNaDC-1 is a
low-affinity Na+-dicarboxylate cotransporter with broad
substrate selectivity, found in kidney and intestine. This report is
the first characterization of the NaDC-1 from mouse and provides
fundamental information about the transporter as a basis for production
of transgenic or knockout animals. Furthermore, the functional
differences between the mouse and other species identified in this
study should also be useful for future structure-function studies.
 |
ACKNOWLEDGEMENTS |
This study was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grants (DK-46269 and DK-02429).
 |
FOOTNOTES |
Address for reprint requests and other correspondence: A. M. Pajor, Univ. of Texas Medical Branch, Dept. of Physiology and Biophysics, Galveston, TX 77555 (E-mail: ampajor{at}utmb.edu).
1
The nucleotide sequence reported in this paper has
been submitted to the GenBank/EMBL data bank with accession number
AF201903.
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. §1734 solely to indicate this fact.
Received 10 December 1999; accepted in final form 13 April 2000.
 |
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