From the Department of Physiology, University of
Arizona, Tucson, Arizona 85724 and the ¶ Department of Physiology,
UCLA School of Medicine, Los Angeles, California 90095-1751
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The two-electrode voltage clamp was used to study
the currents associated with transport of succinate by the cloned
Na+/dicarboxylate cotransporter, NaDC-1, expressed in
Xenopus oocytes. The presence of succinate induced inward
currents which were dependent on the concentrations of succinate and
sodium, and on the membrane potential. At 50 mV, the
K0.5succinate was 180 µM and the K0.5Na+ was
19 mM. The Hill coefficient was 2.3, which is consistent with a transport stoichiometry of 3 Na+:1 divalent anion
substrate. Currents were induced in NaDC-1 by a range of di- and
tricarboxylates, including citrate, methylsuccinate, fumarate, and
tricarballylate. Although Na+ is the preferred cation,
Li+ was also able to support transport. The
K0.5succinate was approximately
10-fold higher in Li+ compared with Na+. In the
presence of Na+, however, Li+ was a potent
inhibitor of transport. Millimolar concentrations of
Li+ resulted in decreases in apparent succinate affinity
and in the Imaxsuccinate.
Furthermore, lithium inhibition under saturating sodium concentrations showed hyperbolic kinetics, suggesting that one of the three cation binding sites in NaDC-1 has a higher affinity for Li+ than
Na+. We conclude that NaDC-1 is an electrogenic anion
transporter that accepts either Na+ or Li+ as
coupling cations. However, NaDC-1 contains a single high affinity binding site for Li+ that, when occupied, results in
transport inhibition, which may account for its potent inhibitory
effects on renal dicarboxylate transport.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The active transport of Krebs cycle intermediates, such as
succinate and citrate, is mediated by a specific sodium-coupled transporter found on the apical membrane in epithelial cells of the
kidney proximal tubule (1, 2). The Na+-dicarboxylate
cotransporter reabsorbs a wide range of di- and tricarboxylic acids in
the form of divalent anions. This transporter is sensitive to
inhibition by lithium (3), and patients receiving therapeutic doses of
lithium exhibit increased renal excretion of -ketoglutarate and
glutarate (4). The cDNA coding for the rabbit renal
Na+/dicarboxylate cotransporter,
NaDC-1,1 has been cloned and
sequenced (5), and the protein has been identified in renal brush
border membranes (6). NaDC-1 belongs to a distinct gene family of
sodium-coupled anion transporters that includes the
Na+/dicarboxylate cotransporters, hNaDC-1, from human
kidney (7), and NaDC-2, from Xenopus intestine (8), and the
renal Na+/sulfate cotransporter, NaSi-1 (9).
The transport mechanism of NaDC-1 is thought to involve the ordered binding of four charged substrates: 3 Na+ ions and 1 divalent anion substrate (10-12), resulting in one net inward positive charge across the membrane per cycle. Experiments with rabbit renal brush border membrane vesicles support this hypothesis: sodium-dependent transport of succinate was affected by changes in membrane potential, and transport of succinate also caused a depolarization of membrane potential (12-14). However, the dependence of transport kinetics on membrane voltage is not known, and there have been no direct measurements of currents associated with Na+/dicarboxylate cotransport.
In this study, we have used a two-electrode voltage clamp to study the kinetics of succinate transport by NaDC-1 expressed in Xenopus oocytes. The results show that NaDC-1 is electrogenic, with a stoichiometry of 3 Na+ per succinate. Negative membrane potentials increase succinate transport. The apparent affinity of the transporter for Na+ is increased by negative membrane voltage, whereas succinate binding is relatively voltage-independent. The cation selectivity of NaDC-1 is unique. Although Na+ is the preferred cation, Li+ can support transport. However, succinate transport is inhibited when one of the three cation binding sites in NaDC-1 is occupied by Li+. In conclusion, this study provides new insights into the transport mechanism of NaDC-1 and the inhibitory action of lithium on renal dicarboxylate transport.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Xenopus oocytes-- Stage V and VI oocytes from Xenopus laevis (NASCO) were dissected and defolliculated as described previously (5, 11). The oocytes were injected with 50 nl of NaDC-1 cRNA (0.5 µg/µl) 1 day following isolation. Currents were measured 3-5 days later. The oocytes were maintained at 18 °C in Barth's medium supplemented with 5% heat-inactivated horse serum, 2.5 mM sodium pyruvate, 50 mg/ml gentamicin. For experiments, oocytes were superfused with sodium buffer containing (in mM): 100 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, and 10 HEPES-Tris, pH 7.5. For cation replacement experiments, sodium was replaced with an equimolar concentration of other cations, as chloride salts. The results reported are for single experiments that are representative of experiments repeated with oocytes from at least three donor frogs.
Electrophysiology--
Oocyte currents were measured using the
two-electrode voltage clamp method at 22 °C (15, 16). The
microelectrodes were filled with 3 M KCl and had
resistances <1 megohm, usually between 0.4 and 0.8 megohm. The voltage
pulses were controlled with the pClamp6 program suite (Axon
Instruments). Current-voltage relationships were obtained from a pulse
protocol consisting of test voltages applied for 100 ms between +50 and
150 mV (in 20-mV decrements), with a holding potential of
50 mV.
The results of three runs were averaged for each trial.
Data Analysis-- Steady state substrate-dependent currents were fitted to the Hill/Michaelis-Menten equations using SigmaPlot software (Jandel Scientific)
![]() |
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Succinate-induced Currents in NaDC-1-- Current traces obtained in an oocyte injected with NaDC-1 cRNA are shown in Fig. 1. The addition of substrate in the presence of sodium induced inward currents (Fig. 1B) compared with sodium buffer alone (Fig. 1A). Inward currents of up to 3000 nA were measured in oocytes expressing NaDC-1, but not in control, uninjected oocytes (not shown). In Fig. 1C, the steady state currents from Fig. 1, A and B, are plotted as a function of membrane potential. Fig. 1D shows the substrate-dependent currents in NaDC-1, which are calculated from the difference between the currents in the presence and absence of succinate. In this experiment, the I-V curves were linear, although the I-V curves often showed saturation with more negative membrane potentials. In response to step changes in membrane voltage, NaDC-1 also exhibited pre-steady state charge movements (Fig. 1A), which were reduced in the presence of substrate (Fig. 1B). However, the pre-steady state charge movements in NaDC-1 were very rapid (time constants approximately 2-6 ms) and did not show saturation at the positive potentials tested. The pre-steady state charge movements in NaDC-1 were observed only in oocytes exhibiting very high expression of the transporter.
|
Kinetics of Activation by Succinate--
The magnitude of the
steady state currents in NaDC-1 was dependent on the concentration of
succinate (Fig. 2, A and
B). For each voltage, the data were fit to Equation 1, with
n = 1. The maximal current,
Imaxsuccinate, increased and
saturated at hyperpolarizing membrane potentials (Fig. 2C).
At negative membrane potentials, the
K0.5succinate was
voltage-independent (Fig. 2D). For example, at 50 mV, the K0.5succinate was 180 µM and at
150 mV the
K0.5succinate was 140 µM (Fig. 2D). However, there was an increase
in K0.5succinate to 270 µM at
10 mV.
|
Substrate Specificity of NaDC-1--
The substrate specificity of
NaDC-1 was examined by measuring currents in the presence of 10 mM concentrations of test substrates. As shown in Fig.
3, four of the substrates tested produced
larger currents than did succinate: methylsuccinate, citrate, fumarate, and tricarballylate. The currents seen in the presence of
-ketoglutarate and glutarate were about 50% of those reported for
succinate, which could reflect a lower affinity of NaDC-1 for these
substrates. For example, the Km for glutarate in
NaDC-1 is approximately 6 mM (11), close to the 10 mM substrate concentration used in these studies. Small
currents were measured in dimethylsuccinate, sulfate and pyruvate (Fig.
3). Finally, there were no detectable substrate-dependent
currents with lactate, consistent with previous uptake studies (5).
|
Sodium Activation of Succinate-dependent
Currents--
Na+ is thought to be an essential activator
of NaDC-1 (10, 12). Sodium activation of
succinate-dependent currents was measured under
voltage-clamp conditions. The succinate-dependent currents in NaDC-1 were sigmoidal functions of Na+ concentration and
showed saturation (Fig. 4A).
The maximum succinate-dependent current at saturating
Na+ concentrations,
ImaxNa+, was dependent on
membrane potential, becoming larger and saturating at more negative
membrane potentials (Fig. 4B). The
K0.5Na+ was strongly affected by
membrane potential, decreasing from 19 mM at 50 mV to 9.7 mM at
150 mV (Fig. 4C). The apparent Hill coefficient, n, was insensitive to voltage; n was
2.3 at
50 mV and 2.4 at
150 mV (Fig. 4D).
|
Cation Selectivity of NaDC-1--
The largest
succinate-dependent currents in NaDC-1 were seen in the
presence of Na+ (Fig. 5).
When Na+ was replaced by choline or Cs+, the
inward currents were abolished. However,
substrate-dependent inward currents were observed in
Li+ (approximately 6-25% of the currents seen in
Na+, depending on membrane potential), suggesting that
Li+ supports succinate transport. This result verifies
previous studies done with renal brush border membrane vesicles (3).
H+ appears to be a very poor substitute for Na+
since only small (<30 nA) substrate-induced currents were observed in choline at pH 5.5 (Fig. 5). There was no difference between the
succinate-induced currents measured in Na+ at pH 7.5 and
5.5 (results not shown).
|
Succinate Kinetics Measured in Lithium--
The succinate-induced
currents in lithium were measured as a function of increasing succinate
concentrations (Fig. 6). As seen in
Na+, there was saturation of the substrate-induced lithium
currents with increasing concentrations of succinate. For comparison,
the currents produced by 1 mM succinate in Na+
in the same oocyte were 185 nA at
150 mV and
140 nA at
50 mV
(not shown). As seen with Na+, the
Imaxsuccinate for
succinate-dependent currents in lithium increased with more negative membrane potentials. However, the effect of voltage on the
Imaxsuccinate measured in lithium
was more pronounced than in Na+ (see Fig. 2). Rather than
saturating with hyperpolarizing membrane potentials (see Fig. 2), the
Imaxsuccinate in Li+
was supralinear (Fig. 6C). The apparent affinity of NaDC-1
for succinate was lower in Li+ compared with
Na+. The
K0.5succinate measured in
Li+ was approximately 3 mM (Fig. 6D)
compared with 0.18 mM in Na+ (Fig. 2). The
K0.5succinate in Li+
was relatively insensitive to changes in voltage (Fig.
6D).
|
Effect of Lithium on Succinate Kinetics--
Lithium is both an
activator and an inhibitor of succinate transport by NaDC-1 (3, 5),
consistent with competition for the same cation binding site. As shown
in Fig. 7, the addition of 5 mM Li+ resulted in a decrease in the
Imaxsuccinate and an increase in
the K0.5succinate. At 50 mV,
for example, the Imaxsuccinate
was
280 nA in the absence of Li+ and
200 nA in the
presence of Li+ (Fig. 7A). There was a much
larger effect of Li+ on the apparent affinity for
succinate; the K0.5succinate
(
50 mV) increased from 180 to 750 µM after the addition
of 5 mM Li+ (Fig. 7B).
|
Effect of Lithium at Saturating Sodium and Succinate
Concentrations--
The inhibition of succinate-induced currents by
millimolar concentrations of Li+ suggests that NaDC-1 has
at least one cation binding site with a high affinity for
Li+. Therefore, inhibition of succinate-induced currents by
increasing concentrations of Li+ was measured at saturating
succinate and Na+ concentrations (10 mM
succinate and 100 mM Na+). Li+
caused a concentration dependent inhibition of succinate-induced currents, which was slightly more pronounced at more positive membrane
potentials, 54% maximal inhibition at 150 mV compared with 76%
maximal inhibition at
10 mV (Fig. 8).
The apparent Ki for lithium was slightly larger at
more negative membrane potentials, around 4 mM at
150 mV
and 2.9 mM at
50 mV (Fig. 8). The apparent Hill
coefficient was
1 at all membrane potentials tested and insensitive
to voltage (not shown).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The overexpression of NaDC-1 in Xenopus oocytes has provided a means of characterizing its electrophysiological properties. In oocytes expressing NaDC-1, the presence of substrate induced inward currents that were dependent on the concentrations of substrate and sodium, and on the membrane potential. Although Na+ is the preferred cation, Li+ can also support transport in NaDC-1. However, the poor activation of transport by Li+ produces inhibition when Na+ is present. In many respects, the electrophysiological characteristics of substrate-induced currents in NaDC-1 resemble those of transporters of neutral substrates, including the Na+/glucose cotransporter, SGLT1 (15).
The apparent affinity constants for succinate in NaDC-1 measured under
voltage-clamp conditions agree well with those obtained from
radiotracer uptake studies in oocytes. The Km for succinate transport in NaDC-1 is around 0.5 mM (5, 11). In this study, the K0.5succinate for
succinate-induced currents was approximately 0.2 mM at 50 mV. Although there was a marked effect of voltage on
K0.5succinate at more positive
membrane potentials, the
K0.5succinate at negative
membrane potentials was relatively insensitive to voltage, indicating
voltage independence of succinate binding. This result was somewhat
surprising considering that succinate is transported as a divalent
anion. However, similar results have been reported for other
sodium-coupled transporters, irrespective of substrate charge,
including the Na+/glucose cotransporter, SGLT1 (15), the
Na+/I
cotransporter, NIS (17), and the
Na+/phosphate cotransporter, NaPi-5 (18). This voltage
independence of substrate binding may be a general feature of some
sodium-coupled transporters and suggests that the substrate binding
site does not sense the electric field. The steep voltage dependence at depolarizing potentials could be a consequence of
voltage-dependent steps in Na+ binding. In
contrast, the Na+/Cl
/
-aminobutyric acid
transporter, GAT-1, and Na+/glutamate transporter, HEAAC1,
exhibit increases in K0.5 with more negative
membrane potentials (19, 20) indicating that the substrate binding site
senses membrane potential in this group of anion transporters. The
stimulation of Imax with more negative membrane
potentials is seen in all of the sodium-coupled transporters studied to
date, including NaDC-1 (15, 17, 20), suggesting that
voltage-dependent steps in substrate turnover are a general property of sodium-coupled transporters.
The effects of voltage on sodium binding in NaDC-1 were more pronounced
than on substrate binding. The
K0.5Na+ decreased by 50% between
50 and
150 mV. As suggested for other sodium-coupled transporters,
this effect of voltage on sodium binding supports an "ion-well"
hypothesis (15). Activation by cations in NaDC-1 is also affected by
voltage since the ImaxNa+ from
sodium activation experiments increased with more negative membrane
potentials. The apparent Hill coefficient in NaDC-1 was unaffected by
changes in membrane potential. Therefore, the voltage sensitivity of
Na+ binding and translocation in NaDC-1 also resembles the
members of the SGLT family (15, 17, 21). In contrast, the
ImaxNa+ of the
Na+/Cl
/
-aminobutyric acid transporter is
independent of voltage (19). The results of the experiments presented
here support the model of NaDC-1 stoichiometry in which 3 Na+ ions are coupled to 1 divalent anion substrate. The
Hill coefficient provides an estimate of the minimum number of cations
involved in transport, assuming cooperativity of cation binding (22). Based on the Hill coefficient of about 2.4 in this study, the minimum
number of strongly cooperative sodium binding sites in NaDC-1 is 3, which agrees with results using brush border membrane vesicles
(10).
NaDC-1 has a relatively broad substrate specificity and the preferred substrates are di- and tricarboxylic acids. Based on effects of pH on citrate transport, but a lack of pH effect on succinate transport, it is thought that NaDC-1 carries predominantly divalent anion substrates (11, 23). This model is supported by the results of these studies in which inward currents were measured in the presence of tricarboxylic acids. If the transported species were trivalent anions, there should be no measurable currents unless the coupling stoichiometry is greater than 3 Na+:1 substrate. Overall, the magnitude of the substrate-dependent currents in NaDC-1 agrees well with uptake studies in oocytes and in native membranes (5, 11). The largest currents were seen with methylsuccinate and citrate. Since the substrate concentration used in these experiments was 10 mM, the currents probably represent the Imax for many of the substrates tested. In transport experiments, the Km for citrate in NaDC-1 expressed in oocytes was 0.9 mM (11). Therefore, the rate of substrate turnover of methylsuccinate and citrate is probably faster than that of succinate. The presence of sulfate produced small inward currents, approximately 5% of the currents generated by succinate. NaDC-1 and NaSi-1 belong to the same gene family and have 43% sequence identity (5, 9). In our previous studies, the two transporters had no overlaps in substrate selectivity (24). However, under voltage-clamp conditions, NaDC-1 and NaSi-1 may have some shared substrates that are carried with low affinity or slow substrate turnover.
The preferred cation carried by NaDC-1 is Na+. Unlike other
sodium-coupled transporters, such as the Na+/glucose
cotransporter (25) and the Na+/Cl/serotonin
cotransporter (26), protons are not effective activators of succinate
transport in NaDC-1. However, Li+ can substitute for
Na+ to produce substrate-dependent currents, which
also indicates that the stoichiometry of
Li+-dependent succinate transport is likely to
be 3:1. Li+ is a poor activator of succinate transport,
most likely because binding of Li+ to NaDC-1 produces a
less favorable conformation for substrate binding compared with binding
of Na+. The apparent substrate affinity in NaDC-1 is about
15-fold lower in lithium
(K0.5succinate 3 mM)
compared with sodium
(K0.5succinate 0.2 mM), consistent with the idea that cations are essential activators of transport by producing an increased affinity for substrate. The exact conformational change produced by cation binding
is determined by the identity of the cation. The steep voltage
dependence of Imaxsuccinate in
Li+ suggests that hyperpolarizing membrane potentials have
a greater effect on one or more steps in the transport cycle in the
presence of Li+ compared with Na+.
Lithium also acts as a potent inhibitor of succinate transport by NaDC-1 when Na+ is present, with an apparent Ki of 2 mM (3, 11). In humans and rodents, treatment with Li+ leads to rapid increases in urinary concentrations of Krebs cycle intermediates (4, 27). In this study, Li+ behaved as a mixed-type inhibitor of NaDC-1. Succinate-dependent currents in NaDC-1 were inhibited by millimolar concentrations of Li+, with a decrease in Imaxsuccinate and an increase in K0.5succinate. Mixed-type inhibition is characterized by a combination of competitive inhibition, which would be seen as an increased K0.5succinate due to the mutual exclusion of substrate and inhibitor (i.e. Na+ and Li+), and noncompetitive inhibition, which would be seen as a decrease in Imaxsuccinate due to the production of an inactive intermediate (22). The proportional change in K0.5succinate is much greater than the change in Imaxsuccinate, in the voltage range used in our studies. The smaller effect on Imaxsuccinate could explain why studies with brush border membrane vesicles reported an increase in Km for succinate in the presence of 2 mM Li+ but no significant effect on Vmax (3).
The inhibitory effects of Li+ on NaDC-1 occur at relatively
low concentrations, even in the presence of saturating Na+
concentrations, which confirms previous suggestions that at least one
of the cation binding sites in NaDC-1 has a high affinity for
Li+ (3). The hyperbolic kinetics of inhibition by
Li+ are also consistent with one cation binding site which
has a higher affinity for Li+. This result shows that the
multiple sodium binding sites in NaDC-1 are not identical. The apparent
lithium Ki of 2.5 mM seen at 10 mV is
similar to the Ki measured in transport experiments
in oocytes (11).
One difference between NaDC-1 and other Na+-coupled
transporters is the difficulty in measuring pre-steady state charge
movements in response to voltage jumps. Although pre-steady state
currents were observed in oocytes expressing NaDC-1, it was only
possible to measure them in oocytes exhibiting high transport
expression. The fast time constants, close to the limit of resolution
of the two-electrode voltage clamp, and the lack of saturation of
charge movements in the voltage range used in our studies made it
difficult to obtain reliable fits of the data to the Boltzmann equation in order to estimate Qmax. Interestingly, the
properties of the charge movements in NaDC-1 appear to resemble those
seen in the unrelated Na+/phosphate cotransporter, NaPi-5
(18). The maximum time constant, max, in NaPi-5 is
around 5 ms, compared with time constants between 9 and 150 ms in other
sodium-dependent transporters (28). Pre-steady state charge
movements are thought to represent movement of the transporter in
response to changes in the electric field, probably representing
reorientation of charges or dipoles in the protein or binding and
release of Na+ (28). If either of these steps is
rate-limiting in NaDC-1, the rapid pre-steady state charge movements
could indicate that the turnover number will be relatively high,
although this remains to be tested.
In conclusion, NaDC-1 expressed in Xenopus oocytes generates
substrate-induced currents that are dependent on concentrations of
substrate, sodium, and membrane potential. Although NaDC-1 belongs to a
distinct gene family, in some respects it behaves very much like other
Na+-coupled transporters, including the
Na+/glucose cotransporter (15), the
Na+/I cotransporter (17), and the
Na+/phosphate cotransporter (18). The cation selectivity of
NaDC-1 is unique, however, with a single high affinity binding site for lithium that, when occupied, results in transport inhibition. This
study provides new insights into the mechanism of transport by NaDC-1
and should allow us to design models of NaDC-1 function.
![]() |
ACKNOWLEDGEMENTS |
---|
We are indebted to Dr. Ernest Wright for the generous use of his electrophysiology equipment during the early portions of this study and for critical review of this manuscript. We also thank Ning Sun for preparation and injection of oocytes and Dr. Lucie Parent for the initial pilot experiments demonstrating that NaDC-1 is electrogenic.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants DK46269 and DK02429 (to A. M. P.), GM52094 (to B. A. H.), and NS25554 (to D. D. F. L.).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.
§ To whom correspondence should be addressed: Dept. of Physiology, University of Arizona, College of Medicine, Tucson, AZ, 85724-5051. Tel.: 520-621-9778; Fax: 520-626-2383; E-mail: Pajor{at}biosci.arizona.edu.
1 The abbreviations used are: NaDC-1, rabbit renal Na+/dicarboxylate cotransporter; NaSi-1, Na+/sulfate cotransporter; SGLT1, rabbit Na+/glucose cotransporter.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|