From the Renal Division, Brigham and Women's Hospital, Harvard
Medical School, Boston, Massachusetts 02115
The metabolism of Krebs cycle intermediates is of
fundamental importance for eukaryotic cells. In the kidney, these
intermediates are transported actively into epithelial cells. Because
citrate is a potent inhibitor for calcium stone formation, excessive
uptake results in nephrolithiasis due to hypocitraturia. We report the cloning and characterization of a rat kidney dicarboxylate transporter (SDCT1). In situ hybridization revealed that SDCT1 mRNA
is localized in S3 segments of kidney proximal tubules and in
enterocytes lining the intestinal villi. Signals were also detected in
lung bronchioli, the epididymis, and liver. When expressed in
Xenopus oocytes, SDCT1 mediated electrogenic,
sodium-dependent transport of most Krebs cycle
intermediates (Km = 20-60 µM),
including citrate, succinate,
-ketoglutarate, and oxaloacetate. Of
note, the acidic amino acids L- and D-glutamate
and aspartate were also transported, although with lower affinity
(Km = 2-18 mM). Transport of citrate
was pH-sensitive. At pH 7.5, the Km for citrate was
high (0.64 mM), whereas at pH 5.5, the
Km was low (57 µM). This is
consistent with the concept that the
2 form of citrate is the
transported species. In addition, maximal currents at pH 5.5 were 70%
higher than those at pH 7.5, and our data show that the
3 form acts
as a competitive inhibitor. Simultaneous measurements of
substrate-evoked currents and tracer uptakes under voltage-clamp
condition, as well as a thermodynamic approach, gave a Na+
to citrate or a Na+ to succinate stoichiometry of 3 to 1. SDCT1-mediated currents were inhibited by phloretin. This plant
glycoside also inhibited the SDCT1-specific sodium leak in the absence
of substrate, indicating that at least one Na+ binds to the
transporter before the substrate. The data presented provide new
insights into the biophysical characteristics and physiological
implications of a cloned dicarboxylate transporter.
 |
INTRODUCTION |
In kidney proximal tubules, reabsorption of Krebs cycle
intermediates such as citrate, succinate,
-ketoglutarate, malate, and fumarate has been shown to be accomplished by
Na+-coupled transporters (1-9). Numerous studies have been
performed in intact proximal tubules (10), isolated brush border
membrane vesicles (BBMV)1
(1-5, 11, 12), and basolateral membrane vesicles (BLMV) (12-14),
mostly using citrate or succinate as substrates.
In BBMV, succinate uptake was found to be mediated with low affinity
(Km = ~1 mM) (5, 8, 12, 15). Studies on the pH dependence suggested that citrate is transported in its
protonated divalent form (Cit
2) (1, 2, 12), whereas
succinate is transported either in its deprotonated (
2) or protonated
(
1) form (11). In addition, it was shown that the
3 form of citrate
(trivalent form, Cit
3) inhibits transport of
Cit
2 (11). Radiotracer studies revealed that the
cotransport process exhibits a stoichiometry of 2-3 sodium
ions/dicarboxylate molecule (2, 8, 16). On the other hand, experiments
with a voltage-sensitive dye showed that the cotransport was
electrogenic (14, 17), which favors a 3:1 stoichiometry.
In BLMV, succinate transport was pH-sensitive and with high affinity
(Km = ~10 µM; Refs. 12 and 13), and
citrate uptake was hardly pH-sensitive (12), suggesting that both
divalent and trivalent citrate can be transported. The functional
differences between BBMV and BLMV transport suggest that there exist
different transporter isoforms on the apical and basolateral sides.
Also, a possible presence of a trivalent citrate transport system in the basolateral membrane of the proximal tubule cell line from opossum
kidney was proposed (9).
Citrate transport in the proximal tubule is of considerable interest
because it has several implications for the kidney function. Citrate is
metabolized by the kidney via the intramitochondrial tricarboxylic acid
cycle, and this process provides up to 10-15% of renal oxidative
metabolism (18, 19). Urinary citrate is a potent inhibitor of calcium
stone formation by chelating calcium and inhibiting precipitation of
calcium and crystallization of calcium-oxalate crystals (20).
Hypocitraturia is found in about half of patients with renal stone
diseases (21). Low urinary citrate levels are found in many conditions
associated with decreases either in intraluminal or intracellular pH in
the proximal tubules (i.e. systemic acidosis) or with
potassium depletion. These conditions are known to increase citrate
reabsorption (6, 22, 23). Interestingly, hypocitraturia may be found
without apparent cause (idiopathic), but the underlying mechanism is
still undetermined (24).
cDNAs of Na+-dicarboxylate cotransporters have recently
been isolated from rabbit kidney (NaDC-1) (25), human kidney (hNaDC-1) (26), rat intestine (27), and rat kidney (28). Both the rabbit and
human transporters can transport tricarboxylic acid cycle metabolites
with low affinities (29, 30). The expression of these cloned
cotransporters in Xenopus oocytes allowed kinetic analyses
under steady-state and presteady-state conditions and, in contrast to
vesicle studies, with excellent control of membrane potential, external
milieu, and in some cases internal milieu. In the present paper, we
report the characterization, using the two-microelectrode voltage-clamp
technique, and the tissue distribution of a rat dicarboxylate
transporter (SDCT1) that has been recently cloned in our laboratory by
homology screening.
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EXPERIMENTAL PROCEDURES |
Isolation of the Rat SDCT1 Clone--
Sprague-Dawley rat kidney
cortex mRNA was reverse transcribed and used for polymerase chain
reaction with a set of degenerative primers corresponding to the amino
acids 35-40 and 142-137 of rabbit NaDC-1 (25). Polymerase chain
reaction products were used to screen the cDNA library of a rat
kidney cortex in the vector
gt10 at high stringency. A positive
clone 2.4 kilobases in size was subcloned into the EcoRI
site of the pBluescript vector and sequenced.
Oocyte Preparation--
Stage V and VI oocytes were extracted
from female Xenopus laevis frogs and prepared as described
previously (31). Capped cRNA of rat SDCT1 was synthesized by in
vitro transcription from cDNAs in pBluescript
SK
. Defolliculated oocytes were injected with 25-50 ng
of cRNA or water at the same day or the following day after
defolliculation and maintained in Barth's solution (88 mM
NaCl, 10 mM HEPES, 1.8 mM MgCl2, 1 mM KCl, 0.82 mM CaCl2, 0.82 mM MgSO4, and 0.33 mM Ca(NO3)2, pH 7.4 by Tris-base or HEPES)
supplemented with 2.5 mM sodium pyruvate, 50 µg/ml
gentamicin, 10 units/ml penicillin, and 10 µg/ml streptomycin at
18 °C.
In Situ Hybridization--
Digoxigenin-labeled antisense and
sense run-off transcripts were synthesized using a Genius kit
(Boehringer Mannheim) from a linearized expression plasmid (pBluescript
SK
) containing the complete SDCT1 coding sequence, using
T3 and T7 RNA polymerases, respectively. Transcripts were
alkali-hydrolyzed to an average length of 200-400 nucleotides.
In situ hybridization was performed on cryosections (10 µm) of fresh-frozen tissue as described previously (32). The
hybridization buffer consisted of 50% formamide, 5× SSC, 2% blocking
reagent (Boehringer Mannheim), 0.02% SDS, and 0.1%
N-laurylsarcosine. Probe concentrations were ~200 ng/ml.
Sections were immersed in slide mailers in hybridization solution and
hybridized at 68 °C for 18-68 h. Sections were then washed three
times in 2× SSC and for 2 × 30 min in 0.2× SSC at 68 °C. The
hybridized labeled probes were visualized using anti-digoxigenin Fab
fragments (Boehringer Mannheim) and 5-bromo-4-chloro-indolyl phosphate/nitroblue tetrazolium chloride substrate (32). Sections were
developed in substrate solution for 20-44 h and then rinsed in 100 mM Tris, 100 mM NaCl, 1 mM EDTA at
pH 9.5 and coverslipped with Vectashield (Vector).
Radiotracer Transport Measurements--
Uptake experiments were
performed 3-6 days following injection. 8-10 oocytes were incubated
in 0.5 ml of modified ND96 Barth's solution (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, pH 5.5-9.5
by Tris-base or HEPES) containing specific tracer substrate
([14C]citrate or succinate) and terminated by washing
five times with the ice-cold Barth's solution containing 1 mM citrate. Individual oocytes were then dissolved in 250 µl of 10% SDS and mixed with 2.5 ml of scintillation mixture.
Electrophysiology--
The two-microelectrode voltage-clamp
technique was used to perform experiments in conjunction with a
commercial amplifier (Clampator One, model CA-1B, Dagan Co.,
Minneapolis, MN). Solutions used for extracellular perfusion (at
approximately 1.5 ml/min) contained 100 mM NaCl + choline-chloride, 10 mM HEPES, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2,
0-5 mM citrate, pH 5.5-9.5 by Tris-base or HEPES. For
experiments using Cl
-free solutions, gluconate-(Na, K,
Ca, Mg) was used to replace NaCl, KCl, CaCl2 or
MgCl2, and an agar bridge, composed of 3 M KCl
and 2% agar, was used to connect the bath solution with the grounding
electrodes. After 5 min of membrane potential stabilization following
microelectrode impalements, the membrane potential was clamped at the
holding potential (Vh) of
50 mV. 90-ms voltage pulses between
160 and +60 mV were then applied, and steady-state currents were obtained by averaging signals 70-85 ms after initiation of voltage pulses. The substrate-evoked currents were evaluated as the
difference between currents recorded before and after substrate addition. Experimental results were expressed in the form of mean ± S.E. (N), where N indicates the number of
oocytes obtained from at least two different frogs. The curve-fitting
procedures were performed using SigmaPlot (version 4.00, San Rafael,
CA), and each fitted parameter is associated with an error that
represents the error in the fitting estimates.
Determination of Charge to Tracer Uptake Ratio--
One of the
approaches to determine the Na+ to citrate (or succinate)
stoichiometry consists of simultaneously measuring citrate- or
succinate-evoked currents and [14C]citrate (or succinate)
uptake under voltage-clamp conditions. Currents were monitored and
recorded after an oocyte was clamped at
60 mV and perfused with
substrate-free solution (control solution). The perfusion was stopped
before adding to the chamber 10 µl of extracellular solution
containing 20-fold concentrated cold and hot substrate. For
measurements at pH 7.5, cold citrate and succinate were 20 and 2 mM, respectively. Upon substrate addition, the solution in
the chamber was gently mixed until the substrate-evoked currents start
to stabilize. After 4-5 min, washing was started by perfusing the
oocyte with the control solution, and the current usually came back to
the original base line. The oocyte was removed from the chamber for
further washing with ice-cold extracellular solution containing 1 mM citrate before proceeding with scintillation counting. The charge moved during clamping was calculated by integrating the
substrate-evoked current over the uptake period. If there was a slight
base line shift during the uptake period, a linear shift was assumed.
Charge was converted into pmol to compare with radiolabeled substrate
uptake. The volume of the oocyte chamber was estimated to be about 200 µl; thus the final substrate concentration ([S]) in the chamber was
approximately 20-fold diluted (i.e. ~1 mM
citrate or ~0.1 mM succinate). Because cold and hot
substrates were premixed, the ratio of specific current to the
radiolabeled uptake will not be affected by spatial and temporal
variations in substrate concentration within the chamber.
Thermodynamic Determination of Stoichiometry--
This procedure
consists of measuring the reversal potential
(Vr), i.e. the membrane potential
where the inhibitor-sensitive current is zero. In the present study,
succinate was used as the substrate at 20 and 200 µM, and
phloretin at 0.5 mM was used as the inhibitor. Because at
pH 7.5 succinate is predominantly in its divalent (
2) form and
assuming that n sodium ions are coupled to one succinate
molecule, the relationship between Vr and
succinate concentration ([Succinate]) at 22 °C is (33) as
follows.
|
(Eq. 1)
|
where C is a constant if it is assumed that bilateral
Na+ and intracellular substrate concentrations remained
unchanged during measurements. C can be eliminated when the
Vr shift is determined by changing succinate
from 20 to 200 µM.
Formulae Describing Inhibition of Cit
2 Transport by
Cit
3--
Assuming that Cit
3 is a
competitive inhibitor for Cit
2 (with an inhibition
constant of
Ki
3) and that
the Na+:citrate stoichiometry is 3:1, the observed currents
can be expressed as follows (34).
|
(Eq. 2)
|
where, N and e indicate the number of
transporters and the elementary charge, respectively, and
[Cit
2] and [Cit
3] denote the
concentrations of Cit
2 and Cit
3,
respectively.
Km
2 is the
affinity constant for Cit
2. The presence of SDCT1 might
affect the protonation state of citrate during substrate-protein
interaction. Assuming that citrate is either in its
2 or
3 form at
pH
5.5 and that the equilibrium constant is K,
then
|
(Eq. 3)
|
where [Cit] is the concentration of citrate. Equation 2 can
also be rewritten in the Michaelis-Menten form.
|
(Eq. 4)
|
where the affinity constant for citrate
(KmCit), and the
maximal current for citrate
(ImaxCit) are functions of
[H+]:
|
(Eq. 5)
|
 |
RESULTS |
Sequence Homology and in Situ Hybridization--
The SDCT1 clone
encoded a 587-amino acid-residue protein that has 75% identity to the
rabbit NaDC-1 (25) and 77% to the human kidney hNaDC-1 (26). During
this study, rat intestinal and renal Na+-dicarboxylate
cotransporters were also identified (27, 28) and showed 98% amino acid
identity to SDCT1 respectively. However, functional characterization of
these proteins has not been reported. High stringency in
situ hybridization experiments demonstrated that SDCT1 is
predominantly localized in S3 segments of proximal tubules in the outer
stripe of outer medulla (Fig.
1a) and in a small subset of
tubular cells in the outer part of inner medulla (not shown). In
duodenum, SDCT1 is strongly expressed by enterocytes lining the lower
three-quarters of the intestinal villi but not in lower crypt cells
(Fig. 1b). SDCT1 expression was also observed in ileum (not
shown). In liver, a small subpopulation of cells, possibly hepatocytes,
was strongly labeled for SDCT1 message (Fig. 1d). These
SDCT1-positive cells did not form a particular pattern and were
scattered throughout the liver. In lung, SDCT1 message was expressed by
cells in the bronchiole epithelium (Fig. 1f) and by cells in
the alveolar epithelium (Fig. 1h). Finally, SDCT1 mRNA
was expressed in the tubular epithelium of epididymis: high levels of
SDCT1 mRNA were present in epithelial cells in the initial segment
and more moderate levels in proximal segments of the epididymis head
(Fig. 1j). In more distal segments of epididymis head (Fig. 1l) or in segments of epididymis tail (not shown), SDCT1
labeling was negative.

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Fig. 1.
Distribution of SDCT1 mRNA in rat tissues
as detected by nonradioactive in situ hybridization using
digoxigenin-labeled cRNA probes. a, in kidney, SDCT1
labeling displays the characteristic pattern of S3 proximal tubule
segments in the outer stripe of outer medulla. CO, cortex;
OS, outer stripe; IS, inner stripe;
IM, inner medulla. b, in duodenum, SDCT1 is
strongly expressed in enterocytes lining the intestinal villi.
M, muscle layer; V, villus; L, lumen.
c, control experiment shows the absence of labeling in
duodenum with sense probe. d, in liver, SDCT1 is relatively
strongly expressed by a small subset of cells (arrows).
e, control experiment shows the absence of labeling in liver
with sense probe. f-i, in lung, SDCT1 is expressed in cells
of the bronchiole epithelium (f, arrow) and in
cells of the alveolar epithelium (h, arrows).
g and i, sense signals in bronchiole and alveolar
epithelium. j, in epididymis, SDCT1 is strongly expressed in
cells in the initial segment of the tubular epithelium. k,
sense signal in epididymis initial segment. l, in the
epididymis head, more proximal segments (arrow) of the
tubular epithelium express moderate SDCT1 levels, whereas more distal
segments express very low levels. Bars, 2 mm for
a, 200 µm for b and c, and 100 µm
for d-l.
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Expression in Xenopus Oocytes--
When mRNA of the SDCT1 was
injected into Xenopus oocytes, 25- and 50-fold increases in
[14C]citrate and succinate uptakes were obtained,
respectively, compared with H2O-injected oocytes (Fig.
2a). Using the
two-microelectrode voltage-clamp technique, SDCT1-mediated transport
was shown to be electrogenic and sodium-dependent, as no
significant currents were observed upon citrate addition when NaCl was
substituted by choline-chloride (Fig. 2b). Citrate-evoked
inward currents were pH-sensitive and stimulated by increasing the
proton concentration in the solution (Fig. 2c). In contrast,
succinate-evoked cotransport was not pH-sensitive between pH 5.5 and pH
7.5, although it was slightly reduced at pH 8.5 (Fig. 2d).
Because succinate has a pK value (pK2) of 5.6, these data suggest that
it can be transported either in its
1 or
2 form. When chloride was
replaced by gluconate, no remarkable difference in current was observed
at various potentials (Fig. 2e), demonstrating that
SDCT1-mediated transport is chloride-independent.

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Fig. 2.
Citrate- or succinate-evoked cotransport
under various conditions. a,
[14C]succinate (0.1 mM) and citrate (1 mM) uptakes were measured and averaged from 8-10
SDCT1-injected (filled bars) and H2O-injected
(open bars) oocytes in each group. b,
citrate-generated current was continuously recorded at the holding
potential of 50 mV in the presence of 100 mM
Na+ or in the absence of Na+. Application of
citrate (2 mM) is indicated by solid bars.
c, pH dependence of inward current generated by 200 µM citrate (solid bars) is demonstrated by
superperfusing oocytes with solution at pH 7.5 or 6.5 (Vh = 50 mV). d, currents evoked by
addition of 20 µM succinate were obtained at different pH
and normalized to the current obtained at pH 5.5 (= 49.3 ± 2.8 nA, n = 3). e, Na+-citrate
cotransport currents were detected at different potentials in the
presence (filled bars) or absence (open bars) of
chloride.
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|
Ion and Substrate Specificity--
In addition to sodium,
potassium can also drive substrate transport. At
50 mV, 50 mM K+, and pH 7.5, 1 mM citrate
stimulated currents averaging 20 ± 3% (n = 5) of
those evoked in the presence of 100 mM Na, under the same
conditions. In contrast, when 1 mM succinate was used in
place of 1 mM citrate, the K+-coupled current
was only 10% of the Na+-coupled current, and no detectable
currents were observed with 50 µM succinate. These data
indicate that K+ couples to substrates with a lower
efficiency than Na+. When 1 mM citrate or 50 µM succinate was added to solution containing 100 mM Li+ in place of Na+, no
detectable currents were stimulated, indicating that lithium cannot
drive SDCT1-mediated cotransport. In fact, lithium had a significant
inhibitory effect. In the presence of 50 µM succinate, when 3 mM Li+ was added to solutions containing
100 and 20 mM Na+, the Na+-coupled
currents were reduced to 50 ± 2 and 28 ± 1%
(n = 3), respectively. Because higher Li+
inhibition was observed at lower Na+ concentration, these
data suggest that Li+ can compete with Na+ for
binding but is not itself translocated.
Currents evoked by citrate addition were voltage-dependent
(Figs. 3a and
4a). At
50 mV and pH 7.5, the apparent affinity for citrate was 0.64 ± 0.01 mM
(n = 5) (Fig. 3b). Currents elicited by
application of substrates (1 mM) to the same oocytes ranked in the following order: fumarate > L-malate > succinate >
-ketoglutarate > oxaloacetate > citrate > L-aspartate > L-glutamate > D-aspartate > D-glutamate (Fig. 3d). On the other hand,
neutral and positively charged amino acids, maleic acid, amiloride,
dimethylsuccinate, furosemide, and monocarboxylates
(L-lactate, pyruvate, nicotinate, acetoacetate, and
-hydroxybutyrate), all at 1 mM levels, did not evoke
detectable currents (Vh =
50 mV). Likewise,
these substances had no inhibitory effects on 1 mM
citrate-evoked currents, which is similar to results obtained from
tracer measurements for human NaDC-1 (29). These results indicate that
monocarboxylates are not transported by SDCT1. The Krebs cycle
metabolites succinate,
-ketoglutarate, and oxaloacetate stimulated
SDCT1-mediated currents with high affinities and high efficiencies,
whereas both L-/D-glutamate and aspartate
generated currents with low affinities and low efficiencies (Table
I).

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Fig. 3.
I-V curves of citrate-evoked currents and pH
dependence of the apparent affinity constants and maximal currents for
citrate. a, currents due to addition of citrate ranging
from 0.02 to 0.5 mM at pH 5.5 were plotted against the
membrane potential. b, concentration dependence of
SDCT1-mediated currents at Vh = 50 mV and pH
7.5. For these representative data, a Michaelis-Menten fit (Equation 4)
gave Km = 0.70 ± 0.10 mM and
Imax = 58.6 ± 3.4 nA. The average
Km was 0.64 ± 0.01 (n = 5).
c, currents recorded at pH 5.5 yielded Km = 49 ± 4 µM and Imax = 90.6 ± 2.4 nA. d, to obtain the substrate
specificity, currents due to application of various substrates at 1 mM were compared in the same oocytes (n = 3-8).
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Fig. 4.
Sodium dependence of SDCT1-mediated currents
at pH 7.5. a, I-V curves at various [Na+]
were obtained as the difference between currents before and after
application of 2 mM citrate. b, at 50 mV, the
sigmoidal relationship of the cotransport current versus
[Na+] was fitted to the following Hill equation
|
(Eq. 6)
|
yielding
KmNa = 17.6 ± 0.7 mM, nH = 1.9 ± 0.1, and
ImaxNa = 202 ± 4 nA.
The averaged
KmNa and
nH from 6 oocytes were 19.5 ± 0.7 mM and 2.07 ± 0.09, respectively. c,
voltage dependence of
KmNa,
nH, and
ImaxNa was obtained by
fitting the data shown in panel a to the Hill
equation.
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Table I
Substrate specificity of SDCT1
Substrate affinity constants (Km) and maximal
currents (Imax) were measured under voltage-clamp
conditions (Vh = 50 mV). The ratio
Imax/Km is equal to the initial
slope of the current versus [S] curve and indicates the transport
efficiency of a substrate. Solution contained 100 mM
Na+, and the pH was 7.5. Averages were obtained from 3-8
oocytes.
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The apparent affinity constant for sodium was 19.5 ± 0.7 mM, and the Hill coefficient (nH)
was 2.07 ± 0.09 (n = 6) at 2 mM citrate, Vh =
50 mV, and pH 7.5 (Fig.
4b). This suggests that at least 2 sodium ions are coupled
to each citrate molecule. In 4 oocytes from different batches, the
maximal current for sodium (ImaxNa) increased about
3-fold upon hyperpolarization from 0 to
160 mV, and the apparent
affinity constant for sodium
(KmNa) decreased
50% from
40 to
160 mV (Fig. 4c).
Proton Dependence of Citrate and Glutamate Transport--
At
50
mV, the apparent affinity for citrate increased about 10-fold to
57 ± 8 µM (n = 4) when pH decreased
from 7.5 to 5.5 (Fig. 3, b and c). These
observations are consistent with previous findings on rabbit and human
NaDC-1 (29) and renal membrane vesicles (2, 11). In contrast,
succinate-evoked currents were pH-independent (Fig. 2d). The
proton affinity constant
(KmH) was
determined at [Cit] = 1 mM (Fig.
5a) and averaged 62 ± 14 nM (corresponding to pH 7.2 ± 0.1) from 3 oocytes.
The pH dependence of the [14C]citrate uptake was also
determined and exhibited a Michaelis-Menten relationship with a similar
KmH value (pH
7.2 ± 0.3, see Fig. 5b). The uptake vanished at high pH, indicating that trivalent citrate was not remarkably transported. Because proton translocation is not associated with transport of
succinate (and other Krebs cycle intermediates, e.g.
-ketoglutarate) (data not shown), protons are unlikely to be
coupling ions for citrate uptake. We propose that protons serve to
protonate the trivalent form of citrate and that the divalent form is
the predominant form transported.

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Fig. 5.
H+ sensitivities
obtained from current and/or tracer measurements. a,
under voltage-clamp conditions (Vh = 50
mM, 1 mM citrate), average currents (for pH
from 5.5 to 8.5 with increment of 0.5) obtained from 3 oocytes were
fitted to the Hill equation with
KmH = 59.7 ± 16.7 nM (corresponding to pH 7.3 ± 0.3),
nH = 0.67 ± 0.08, and
ImaxH = 392 ± 21 nA. b, [14C]citrate uptakes at 1 mM level were obtained and averaged from 8-10 oocytes at
each pH value (from 5.5 to 9.5 with increment of 1.0). From the
Michaelis-Menten fit,
KmH = 62 ± 20 nM (or pH 7.2 ± 0.3) and
ImaxH = 653 ± 46 pmol/10 min/oocyte. c, currents evoked by 2 mM
L-glutamate at pH 5.5, 6.5, 7.5, and 8.5 are illustrated.
The solid line represents the Michaelis-Menten fit plus a
constant c with
KmH = 8.1 ± 2.4 nM (or pH 8.1 ± 0.3),
ImaxH = 233 ± 20 nA, and c = 9.0 ± 4.6 nA. The dashed
line is the best fit assuming that only the 2 form of glutamate
is transported (I = Imax·KmH/(KmH + [H+]), with
KmH = 10.8 ± 2.7 nM (or pH 8.0 ± 0.2) and
ImaxH = 228 ± 18 nA.
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For pH
5.5, citrate is both in the Cit
2 and
Cit
3 form, and their relative proportion in solution is
described by the equilibrium constant pK3 (= 6.4). If
Cit
3 had no effect on Cit
2 transport, then
we would expect to have the same maximal currents for citrate
(ImaxCit) at different pH.
However, measurements performed in the same oocytes revealed that
ImaxCit at pH 5.5 was 70%
higher than ImaxCit at pH
7.5 (see example in Fig. 3), indicating that Cit
3
inhibits Cit
2 transport. If inhibition was
noncompetitive, currents at high substrate or proton concentrations
would be expected to decrease (34). If inhibition was uncompetitive,
the current as a function of [H+] would be expected to be
sigmoidal and to drop at high substrate concentrations. Because both of
these predictions are not supported by our data, it is reasonable to
assume competitive inhibition of Cit
2 transport by
Cit
3. Under this assumption and using Equation 5, we
found that K = 1.0 ± 0.1 µM, which
corresponds to a pK value of 6.0 ± 0.1, Km
2 = 33 ± 4 µM and
Ki
3 = 1.5 ± 0.2 mM (n = 3). The obtained pK value is
close to pK3 of citrate, indicating that citrate
protonation is likely to be determined by bulk solution. This result
shows that the apparent affinity constant for divalent citrate is in
fact high and close to Km for (total) citrate at pH
5.5 and that the trivalent citrate is a relatively low efficiency
inhibitor. Because divalent citrate is equivalent to a dicarboxylate in
terms of the number of negative charges, our data show that SDCT1
transports only dicarboxylates.
Interestingly, glutamate transport exhibited a pH dependence opposite
to that of citrate transport. Currents resulting from addition of 2 mM L-glutamate at pH 7.5, 6.5, and 5.5 represented 31.8, 9.6, and 4.4% of that at pH 8.5 (Fig.
5c). Because glutamate has a pK2 of 9.67 for the
amino group, this result indicates that glutamate is largely
transported in its
2 form (Glu
2) and that the affinity
for Glu
2 is in fact high. However, if only the
2 form
was transported, we would predict, based on the Michaelis-Menten fit
(dashed curve in Fig. 5c), a much lower current
at pH 6.5 and no current at pH 5.5, contrary to the observed data
(solid circles in Fig. 5c). This suggests that
the predominant
1 form is also transported, although at a much lower
transport rate. The pH dependence of glutamate and citrate transport
supports our concept that protons affect SDCT1-mediated transport
through substrate protonation or deprotonation. Selective transport of
either Cit
2 or Glu
2 would result in pH
changes on both trans and cis sides of the membrane.
Voltage-dependent Steps--
To obtain information on
the voltage dependence of individual steps, we determined maximal
currents Imax as a function of membrane
potential. At high Na+,H+ and substrate
concentration, the binding processes are fast enough so that
translocation of the loaded transporter or relocation of the free
transporter across the membrane becomes rate-limiting and determines
Imax. We determined the voltage dependence of
Imax for citrate, sodium, and proton (Fig.
6a), all of which exhibited a
3-fold increase upon hyperpolarization of Vm from
20 to
160 mV. This indicates that translocation of either the loaded or the
free transporter or both are voltage-dependent.

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Fig. 6.
Voltage dependence of maximal currents and
affinity constants. a, maximal currents were normalized
to Imax at 160 mV. The maximal current for
citrate (ImaxCit, , = 361 nA at 160 mV) was obtained at 100 mM
Na+ and pH 7.5. The maximal current for proton
(ImaxH, , = 225 nA at
160 mV) was obtained at 100 mM Na+ and 1 mM citrate. The maximal current for sodium
(ImaxNa, , = 404 nA at
160 mV) was obtained at 2 mM citrate and pH 7.5. b, affinities were obtained in the same oocytes used to
determine Imax shown in a and were
normalized to Km at 160 mV. In this example, the
affinity constants at 160 mV for citrate, proton, and Na+
were 1.07 mM, 116 nM (or pH 6.94), and 11.2 mM, respectively.
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To gain insight into the voltage dependence of the
Na+,H+ and substrate binding processes, we
plotted the apparent affinity constants Km as a
function of Vm (Fig. 6b). The citrate affinity decreased with hyperpolarization, which is consistent with
unfavorable binding of negative charge at hyperpolarized Vm. The sodium affinity increased with
hyperpolarization, consistent with preferred binding of positive charge
at negative Vm. Interestingly, despite the
positive charge of H+, its affinity had the same
Vm dependence as that of citrate. This
paradoxical Vm dependence can be explained if
H+ and Cit
3 react to form Cit
2
before being transported, resulting in a proton-binding affinity that
is characterized by binding of the negatively charged
Cit
2. Thus H+, unlike Na+, is
likely not a coupling ion but serves to protonate citrate before
binding to the transporter.
Charge:Uptake Ratio--
We measured the citrate or
succinate-evoked current under voltage-clamp conditions and, at the
same time, [14C]citrate or [14C]succinate
uptake (see "Experimental Procedures"). The charge moved during
uptake is equal to the time integral of the current (Fig.
7a). At pH 7.5, the
charge:uptake ratio in the presence of 1 mM
[14C]citrate was found to be 0.97 ± 0.06, averaged
from 3 oocytes. In the presence of 0.1 mM
[14C]succinate, the ratio averaged 1.04 ± 0.05 from
4 oocytes. When combining the data from all measurements, a linear fit
with a slope (charge:uptake ratio) equal to 1.03 ± 0.03 was
obtained (Fig. 7b). At pH 7.5, succinate is mainly in its
2 form, and our data presented above showed that citrate is also
transported in its
2 form. It follows that both the sodium:citrate
and the sodium:succinate stoichiometries are 3:1 for SDCT1.

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Fig. 7.
Stoichiometry determination using
simultaneous measurements of substrate-evoked currents and tracer
uptakes under voltage-clamp conditions (Vh = 60 mV). a, representative example of currents
generated by 100 µM succinate (cold + hot). The charge
moved was calculated by integrating the succinate-evoked current over
the uptake period. b, the charge moved was converted to pmol
and plotted against uptake. For experiments using succinate ( ) the
incubation time was 5 min, whereas for those using citrate ( ) the
incubation time was 4 min. When both data are plotted together, the
slope of the linear fit, which is equal to the charge:uptake ratio, is
1.03 ± 0.03.
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Inhibition of SDCT1 Transport and Reversal Potentials--
In
oocytes expressing SDCT1 but not in H2O-injected oocytes,
external Na+ in the absence of external substrate inhibited
outward currents at positive potentials (Fig.
8, a, b, and
e). These SDCT1-specific outward currents are likely to
correspond to the reversed transport generated by intracellular
dicarboxylates. In contrast, succinate (200 µM) or
citrate (2 mM) did not significantly inhibit outward currents in the absence of extracellular Na+ (see
curve corresponding to 0 Na+ in Fig.
4a). Phloretin (0.5 mM) inhibited both inward
and outward currents obtained in the presence of 20 mM
external Na+ and 200 µM succinate (Fig. 8,
c, d, and e) with an estimated inhibition constant (Ki) of 40 µM at
50 mV. No significant phloretin-inhibitable currents were detected in
H2O-injected oocytes. In the absence of external substrate
but in the presence of sodium, a small phloretin-inhibitable inward
current (at Vm <
90 mV, Fig.
9) along with a phloretin-inhibitable
outward current (at Vm >
90 mV) was observed.
This indicates that an SDCT1-mediated uncoupled sodium leak exists and
that at least one sodium ion binds first to the transporter. Thus, the
small currents at Vm <
100 mV in Fig.
8e (open circles) arise from both the
SDCT1-specific sodium leak and SDCT1-independent sodium currents.

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Fig. 8.
Na+ and phloretin effects on
SDCT1-specific currents. a, time course of
transmembrane currents recorded in the absence of external
Na+ upon voltage jumps from Vh of
50 mV to final potentials ranging between 160 and +60 mV, each
separated by 20 mV. For clarity, only currents corresponding to
Vm of 160, 120, 80, 20, +20, and +60 mV
are shown. b, currents at 100 mM extracellular
Na+ were recorded with the same oocyte as in a.
Outward currents at positive potentials were inhibited by
Na+ addition. c, currents were obtained in the
presence of 200 µM succinate and 20 mM
extracellular Na+. d, both inward and outward
currents were inhibited by application of 0.5 mM phloretin
(Pt) to the same oocyte as in c. e,
currents inhibited by Na+ and phloretin were obtained as
the difference between those shown in a and b
(open circles) and between those in c and
d (solid circles), respectively.
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Fig. 9.
Phloretin-sensitive currents versus
Vm at different external succinate
concentrations. [Phloretin] = 0.5 mM and
[Na+] = 20 mM. In the absence of external
substrate ( ), phloretin inhibited an inward sodium leak with a
reversal potential equal to 91 mV. When 20 µM succinate
was applied, Vr shifted to 17.5 mV. Between 20 ( ) and 200 µM ( ) external succinate, the
Vr shift was 57.5 mV in this example.
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Determination of the Stoichiometry Using the Thermodynamic
Method--
Upon external addition of 20 or 200 µM
succinate, the phloretin-sensitive currents exhibited reversal
potentials Vr that shifted toward more positive
potentials (Fig. 9). The effects of phloretin on SDCT1 are similar to
that of phlorizin on the high affinity Na+-glucose
cotransporter SGLT1 expressed in oocytes (31, 33, 35, 36). From 20 to
200 µM succinate, the Vr shift
averaged 54.5 ± 1.9 mV (n = 4), which is
equivalent to an n value of 3.08 ± 0.04 (see Equation 1). This value confirms the result from tracer determinations that the
sodium:substrate stoichiometry is 3:1.
Significance of SDCT1-mediated Uncoupled Sodium Currents--
In
the absence of external substrate and at 20 µM succinate,
reversal potentials obtained from phloretin-inhibited currents averaged
85 and
4 mV, respectively. From these data, the characteristic constant Kc that quantitatively describes the
significance of the sodium leak with respect to the cotransport current
(33) can be estimated to be 1 µM. When [Succinate] = KC, the Na+-succinate cotransport
current is equal to the uncoupled sodium leak current (33). This means
that the sodium leak is small and equivalent to the current generated
by 1 µM succinate. Because Km for
succinate is 24 µM, the sodium leak in the absence of
extracellular substrate represents approximately 4% of the maximal
current for succinate (see Equation 3 in Ref. 37). At low substrate
concentration (comparable with its Kc), SDCT1
does not function in the proper coupling mode because of the sodium leak. At [S]
Kc, where the leak is negligible (33),
the sodium:substrate coupling is stoichiometric.
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DISCUSSION |
Localization--
In the present study, we have described a high
affinity Na+-coupled dicarboxylate transporter, SDCT1, from
rat kidney, its localization, and a number of biophysical
characteristics. Our in situ hybridization studies
demonstrated that SDCT1 is localized in the late portion of proximal
tubules (S3 segments). Previous studies using membrane vesicles
prepared from renal cortex revealed low affinity transport in BBMV
(Km for succinate = ~1 mM) and
high affinity transport in BLMV (Km for
succinate = ~10 µM). In BBMV, citrate but not
succinate transport was highly dependent on extracellular pH. In BLMV,
extracellular pH had a remarkable effect on succinate and
methylsuccinate uptake but little effect on citrate uptake (12, 13). In
the present study, using oocytes expressing SDCT1, succinate transport
was with high affinity and pH-independent (Fig. 2d), and
citrate transport increased about 4-fold when pH was decreased from 8.5 to 5.5 (Fig. 5). Thus, the pH dependence of SDCT1-mediated transport
suggests that SDCT1 is an apical transporter. It is possible that the
previously studied low affinity system in BBMV has a high capacity and
masked the high affinity transport of SDCT1 in S3 segments. In fact,
tubule perfusion studies in rabbit kidney indicated that dicarboxylate reabsorption occurs in S3 segments with low capacity compared with the
early part (i.e. S1 segments) (10, 38). It is likely that
high affinity low capacity SDCT1 participates in final reabsorption of
dicarboxylates that escape the early part of proximal tubules where the
low affinity transporters rabbit and human NaDC-1 are expected to be
expressed (25, 26). Similar situations have been demonstrated for the
reabsorption of other solutes such as glucose.
The strong expression of SDCT1 mRNA in the small intestine supports
the view that it plays an important role in intestinal absorption of
dietary dicarboxylates, including citrate and other Krebs cycle
intermediates. Absorbed citrate is mainly utilized in the liver and the
kidney, but little is known about metabolism in other organs. In the
initial segment of the epididymis, SDCT1 mRNA message and the
citrate and glutamate levels are high (39, 40). Based on micropuncture
studies, glutamate concentrations reach 50, 20, and 0.5 mM
in the initial (caput), middle (corpus), and distal (cauda) segments of
the epididymis, respectively (39). This massive glutamate decrease is
paralleled by a decrease in luminal Na+ concentration (110, 60, and 20 mM in caput, corpus, and cauda, respectively),
glutamate transport activity, and
-glutamyl transpeptidase levels
(39, 41) (
-glutamyl transpeptidase may partially provide glutamate
by hydrolyzing glutathione). These distributions are in good agreement
with that of SDCT1 mRNA in epididymis, suggesting the involvement
of SDCT1 in contributing to the low pH environment in the head of the
epididymis and the nutritional needs of sperms.
Selectivity of Driving Cations--
Based on the observed
K+-dependent SDCT1-mediated currents and on the
Li+-inhibited sodium currents, we have suggested that both
K+ and Li+ can compete with Na+ for
binding to SDCT1. Similar interactions between monovalent cations were
reported previously in SGLT1 where both Li+ and
H+ can be driving ions (42). In the amino acid transporter
KAAT1 cloned from lepidopteran insect larvae, both Na+ and
K+ are good driving ions (43). This might be due to a
similarity in ionic structure among monovalent cations. K+
at 2 mM did not drive any significant currents in the
absence of Na+ (Fig. 4a). Also we did not
observe any difference in 100 mM Na+-coupled
currents between solutions with or without 2 mM
K+. In contrast, because intracellular K+
concentrations, under physiological conditions, are much higher than
intracellular Na+ concentrations
([Na+]i), the K+-coupled reversed
transport might significantly contribute to the observed outward
currents mediated by SDCT1. SDCT1 might provide an important
dicarboxylate exit pathway through reversed transport.
Stoichiometry--
Previous vesicle studies indicated a
sodium:substrate stoichiometry of 2:1 to 3:1. For SDCT1, a 3:1
stoichiometry was obtained using both the voltage-clamp tracer method
and the thermodynamic method. The voltage-clamp condition was critical
in these stoichiometry studies to accurately determine specific charge
accumulation in oocytes. Currents were continuously recorded during the
entire radioisotope uptake. This is important as currents remarkably change during uptake (Fig. 7a). The 3:1 stoichiometrical
ratio has physiological implications. Firstly, it can create higher dicarboxylate gradients across the cell membrane than a 2:1 coupling mechanism. Secondly, the 3:1 stoichiometry of dicarboxylate transport results in an electrogenic transport that can utilize the existing membrane potential as a driving force for substrate accumulation. The
high stoichiometry and high affinity ensure efficient reabsorption of
trace amounts of dicarboxylates that escaped the early proximal kidney
tubules.
Inhibition of SDCT1-mediated Currents--
In oocytes expressing
SDCT1, addition of 100 mM Na+ without external
substrate evoked a large inward current (~120 nA; Fig. 2b). This current is SDCT1-specific because
H2O-injected oocytes only showed small currents (less than
20 nA). The current is not a sodium leak because 1) the total
conductance of the oocyte at 100 mM Na+ is
lower than that in the absence of Na+ and 2) the current
amplitude decreased with hyperpolarization (Fig. 8, a,
b, and e). This indicates that addition of 100 mM Na+ reduces outward currents at positive
membrane potentials (trans-inhibition). Trans-inhibition by Na+ was also observed for
sodium-dependent succinate transport in renal BBMV (4). The
small currents at Vm <
120 mV may be
attributed to the SDCT1-mediated sodium leak and SDCT1-independent
sodium fluxes. Phloretin (as well as Li+) were found to
inhibit both inward and outward currents mediated by SDCT1 (Fig. 8,
c and d). At
50 mV and without external
substrate, the phloretin-sensitive SDCT1 currents were generally
outwardly directed with an Vr average of
85.3
mV. This observation validates the concept that Na+
inhibits SDCT1-specific outward currents.
Endogenous SDCT1 Substrates in Xenopus Oocytes--
The
Vr values can be used to estimate the
concentration of intracellular substrate for SDCT1 in oocytes. Assuming
that [Na+]i is 10 mM (44) and using
Equation 1, the intracellular substrate concentration is equivalent to
~100 µM succinate. If all of the intracellular
substrate was citrate, this would be equivalent to an endogenous
citrate concentration in the mM range, the same order of
magnitude as in the renal tissue (45).
Substrate Binding Order and Kinetic Model--
Although it is well
established now that 3 sodium ions are stoichiometrically coupled to
one substrate molecule, the order by which these 3 sodium ions and the
substrate bind to SDCT1 remains to be resolved. Assuming that
Na+ and substrate bind to the protein in an orderly
fashion, there are four possibilities: SNNN, NSNN, NNSN, and NNNS,
where N = Na+ and S = substrate. The presence of
a phloretin-inhibitable sodium leak indicates that sodium ions bind
before the substrate, eliminating SNNN. On the other hand, because
external Na+ alone but not external substrate alone
trans-inhibits outward currents, this suggests that
Na+ and not substrate binds last to SDCT1, as can be
explained by the King and Altman algorithm: in the absence of external
Na+, NNNS (but not NNSN or NSNN) predicts a term containing
[S]o in the denominator of the expression for the outward
current (Io) (34, 46). When [S]o is high,
Io will decrease (i.e.
Io is trans-inhibited by [S]o),
which contradicts our experimental observation. In contrast, using the above algorithm, both NNSN and NSNN predict a
trans-inhibition by [Na+]o in the
absence of external substrate, as observed, because
[Na+]o appears in the denominator of the
expression for Io. On the other hand, if the
substrate was the last to bind, then the electroneutral exchange
between external tracer substrate and internal cold substrate would be
expected to be remarkable, resulting in an underestimation of charge to
substrate uptake ratio, even under voltage-clamp conditions. However,
the stoichiometry obtained by the tracer method was the same as that
obtained by the thermodynamic method. Thus, our data are consistent
with the models where the binding order for SDCT1 is NNSN or NSNN (Fig. 10).

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Fig. 10.
Symmetrical ordered kinetic model for
SDCT1. T and T' indicate the free
transporter facing the extracellular and intracellular environments,
respectively. N = Na+ and S = substrate. × (= 1 or 2) denotes the number of sodium ions that bind
prior to substrate binding. The transition between the conformation
states T (TN3S) and T' (T'N3S) describes the
free (loaded) carrier translocation across the membrane. The sodium
leak pathway is described by the transition between states
TNx and T'Nx.
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Pathophysiological Implications--
The importance of
dicarboxylate reabsorption in the proximal tubules has been emphasized
as the major determinant of urinary excretion of citrate, the potent
inhibitor of calcium salt crystallization (20, 38). Hypocitraturia is
therefore an important risk factor for kidney stone formation. Among
many factors modulating renal citrate excretion, the most important is
systemic acid-base status and K+ depletion (6). In
metabolic alkalosis, proximal tubular citrate reabsorption is
decreased, whereas it is increased in metabolic acidosis and chronic
K+ depletion, the conditions associated with intracellular
acidosis in the proximal tubular cells. Reduction of intracellular pH
results in decreased citrate levels in the cytoplasm by increasing
citrate entry into the mitochondria via proton-coupled tricarboxylate transport, followed by oxidative phosphorylation (6), and possibly by
increasing cytosolic citrate utilization through ATP citrate lyase
(47). This change stimulates citrate uptake into the cells, and citrate
clearance decreases. Although it has been inferred that the key
determinant of hypocitraturia is intracellular acidosis and changes in
citrate metabolism, the significance of extracellular (luminal) pH in
the alteration of citrate reabsorption was also emphasized (38). Our
studies clearly confirm the concept that the pH sensitivity of citrate
transport mediated by SDCT1 is due to changes in the proportion between
the transported form (Cit
2) and the inhibitory form
(Cit
3).
There is evidence that apical citrate uptake is regulated by chronic
adaptations. Brush border membrane vesicles from chronically K+-depleted rats demonstrate increases in the maximal rate
of the Na+-coupled citrate transport without changes in the
affinities for sodium or citrate (48). Chronic metabolic acidosis in
rats also resulted in enhanced citrate transport in brush border
membrane vesicles when compared with control rats (23). Future
experiments will be needed to determine the regulation of SDCT1 in
chronic adaptations.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF058714.