The transport properties of the human renal
Na+- dicarboxylate cotransporter under voltage-clamp
conditions
Xiaozhou
Yao and
Ana M.
Pajor
Department of Physiology and Biophysics, University of Texas
Medical Branch, Galveston, Texas 77555-0641
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ABSTRACT |
The transport properties of the human
Na+-dicarboxylate cotransporter, (hNaDC-1), expressed in
Xenopus laevis oocytes were characterized using the
two-electrode voltage clamp technique. Steady-state succinate-evoked
inward currents in hNaDC-1 were dependent on the concentrations of
succinate and sodium, and on the membrane potential. At
50 mV, the
half-saturation constant for succinate
(K0.5succinate) was 1.1 mM and the
half-saturation constant for sodium
(K0.5sodium) was 65 mM. The Hill coefficient
was 2.3, which is consistent with a transport stoichiometry of 3 Na+:1 divalent anion substrate. The hNaDC-1 exhibits a
high-cation selectivity. Sodium is the preferred cation and other
cations, such as lithium, were not able to support transport of
succinate. The preferred substrates of hNaDC-1 are fumarate
(K0.5 1.8 mM) and succinate, followed by
methylsuccinate (K0.5 2.8 mM), citrate (K0.5 6.8 mM) and
-ketoglutarate
(K0.5 16 mM). The hNaDC-1 may also
transport sodium ions through an uncoupled leak pathway, which is
sensitive to phloretin inhibition. We propose a transport model for
hNaDC-1 in which the binding of three sodium ions is followed by
substrate binding.
sodium; succinate; Xenopus laevis oocytes; electrogenic
cotransport
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INTRODUCTION |
THE EPITHELIAL
CELLS OF THE renal proximal tubule reabsorb filtered Krebs cycle
intermediates, such as succinate,
-ketoglutarate, and citrate, on a
low-affinity Na+-dicarboxylate cotransporter, NaDC-1, found
on the apical membrane (16). A high-affinity
Na+-dicarboxylate cotransporter is found on the basolateral
membrane of proximal tubule cells (16). The low-affinity
NaDC-1 plays an important role in regulating the concentration of
urinary citrate, which acts as a chelator of calcium. Hypocitraturia is
a risk factor for kidney stone formation (19). NaDC-1
belongs to a distinct gene family of sodium-coupled anion transporters
that includes the Na+-sulfate transporter, NaSi-1, and is
not related to any other known families of transport proteins
(16). NaDC-1 orthologs, corresponding to
low-affinity NaDC-1, have been isolated from rabbit, human,
and rat (4, 14, 22). Other
members of this family include a Na+ or
Li+-dependent dicarboxylate transporter from Xenopus
laevis intestine, NaDC-2 (1), and the high affinity
Na+-dicarboxylate cotransporters from rat, NaDC-3 (SDCT2),
and flounder, flNaDC-3 (3, 11,
23).
The human Na+-dicarboxylate cotransporter, hNaDC-1,
is 78% identical in sequence to the transporter from rabbit, rbNaDC-1
(15). In transport assays using radiotracer substrates,
both transporters have low affinities for succinate and both exhibit a
stimulation of citrate transport at acidic pH. However, the two
transporters differ in their affinities for citrate, in cation binding,
and in sensitivity to inhibitors (18). For example,
hNaDC-1 has a much lower affinity for citrate and for sodium than
rbNaDC-1. Furthermore, unlike rbNaDC-1, hNaDC-1 is relatively
insensitive to inhibition by lithium (18). Because the
rabbit and human Na+-dicarboxylate cotransporters differ in
their transport properties, it is likely that their
electrophysiological characteristics are also somewhat different.
In the present study, we characterized the transport properties of
hNaDC-1 expressed in X. laevis oocytes using the
two-electrode voltage-clamp technique. Many of the effects of voltage
on hNaDC-1 are similar to the effects of voltage on rbNaDC-1
(17). For example, the half-saturation constant for sodium
(K0.5sodium) in hNaDC-1 is very
sensitive to membrane potential whereas the half-saturation constant
for succinate (K0.5succinate) is
relatively voltage independent. However, unlike rbNaDC-1, the
substrate-dependent currents in hNaDC-1 were very dependent on sodium,
and no currents were seen in the presence of lithium. Interestingly,
hNaDC-1 also exhibited a phloretin-sensitive leak pathway for the
transport of sodium uncoupled to the movement of substrate, suggesting
that hNaDC-1 could also act as a sodium transport pathway. In
conclusion, this study provides new insights into the functional
properties of the Na+-dicarboxylate cotransporter from
human kidney.
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METHODS |
cRNA transcription.
The hNaDC-1 cDNA in pSPORT1 plasmid was linearized with Xba
I and in vitro cRNA transcription was done using the T7 mMessage mMachine Kit (Ambion) (15).
Oocyte preparation.
Stage V and VI oocytes were obtained from female X. laevis
frogs (Xenopus I) and collagenase treated as described previously (14, 18). The oocytes were injected with
~46 nl of hNaDC-1 cRNA (0.5 µg/µl) 1 day after isolation and
cultured at 18°C in modified Barth's medium. Culture dishes and
medium were changed daily.
Transport solutions.
Sodium buffer consisted of (in mM) 100 NaCl, 2 KCl, 1 MgCl2, 1 CaCl2, and 10 HEPES, buffered to pH
7.5 using Tris base. Choline buffer, lithium buffer, and cesium buffer
were prepared by replacing NaCl with 100 mM cholineCl, LiCl, or CsCl,
respectively. For transport solutions containing different sodium
concentrations, the NaCl was replaced by cholineCl. Stock solutions of
inhibitors were prepared as follows: 100 mM phloretin in ethanol; 50 mM
tetrodotoxin (TTX), citrate-free (Calbiochem) in 100 mM acetic acid; 50 mM niflumic acid in ethanol; 500 mM amiloride in DMSO; 500 mM DIDS in
DMSO, gadolinium chloride (GdCl3), and 100 mM
tetraethylammonium (TEA) in water. The inhibitors were diluted to their
final concentration in sodium buffer just before use. Control solutions
received vehicle alone (ethanol, DMSO, or acetic acid).
Electrophysiology.
Experiments were performed 3-5 days after cRNA injection using the
two-electrode voltage clamp method with a Geneclamp 500 amplifier (Axon
Instruments), as described (17). The microelectrodes were
filled with 3 M KCl and had resistances between 0.4 and 0.8 M
. Test
voltage pulses were applied for 100 ms between +50 and
150 mV (in
20-mV decrements) at a holding potential of
50 mV, and membrane
currents were recorded. The voltage pulses were controlled with the
pCLAMP version 6.0 program suite (Axon Instruments). For most
experiments, the substrate-dependent currents were determined from the
difference between currents measured in sodium buffer in the absence
and presence of substrate. The substrate was washed away by superfusion
with choline buffer, and experiments were continued only when the
oocytes returned to control condition, usually after 5-10 min. The
sodium leak currents were determined from the difference between
currents measured in choline buffer (0 sodium) and sodium buffer (100 mM sodium).
Data analysis.
Steady-state substrate-dependent currents were fitted to the
Hill/Michaelis-Menten equations using SigmaPlot software (Jandel Scientific): I = Imax*[S]n/ {(K0.5)n + [S]n}, where I is the
substrate-induced current, Imax is the maximum current observed at saturating substrate concentrations,
K0.5 is the substrate concentration at
half-maximal current, and n is the Hill coefficient. For the
Michaelis-Menten equation, n = 1. The error bars for
figures of kinetic data represent errors of the fit. Other experimental
results are expressed as mean ± SE (N = no. of
experiments using different donor frogs). Unless otherwise noted, all
experiments were repeated with oocytes from at least three different frogs.
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RESULTS |
Steady-state inward currents activated by succinate.
The presence of substrate and sodium produced inward currents in
oocytes expressing hNaDC-1. Figure
1A shows typical current tracings from an oocyte superfused with sodium buffer and subjected to
the voltage-pulse protocol, from a holding potential of
50 mV. In the
presence of sodium and succinate, inward currents were produced (Fig.
1B). Pre-steady-state charge movements were not evident
(Fig. 1, A and B). Figure 1C shows the
steady-state currents measured at each voltage in the presence and
absence of succinate. The difference between the currents measured with
and without substrate is the substrate-dependent current (Fig.
1D). Control, uninjected, or water-injected oocytes had very
small currents in the presence of sodium, as seen previously
(17). No inward currents were detected in control oocytes
when succinate was added to the medium (results not shown).

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Fig. 1.
Current traces were obtained from an oocyte expressing
human Na+-dicarboxylate cotransporter (hNaDC-1) before
(A) and after (B) addition of 1 mM succinate to
bathing medium. Experiments were performed on the 4th day after cRNA
injection. The oocyte membrane was held at a holding potential of 50
mV. Pulse protocol is shown. In C is shown steady-state
currents recorded in the absence ( ) and presence
( ) of 1 mM succinate from the experimental data shown
in A and B, respectively. In D the
current-voltage relationship (I-V) curve was
obtained as the difference between currents before and after
application of 1 mM succinate.
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The steady-state inward currents produced by succinate in hNaDC-1 were
concentration and voltage dependent. The currents increased with
increasing concentrations of succinate and also with hyperpolarizing potentials (Fig. 2A). For each
voltage, the currents were plotted against succinate concentration and
fit to the Michaelis-Menten equation (Fig. 2B). The maximal
succinate-induced current, Imaxsuccinate,
increased almost twofold upon hyperpolarization from
10 to
150 mV
(Fig. 2C). K0.5succinate was
relatively unaffected by voltage in the range from
50 to
150 mV and
increased at potentials more positive than
50 mV (Fig.
2D). The K0.5succinate in Fig.
2D was 1.1 mM at
50 mV. In five separate experiments, the
K0.5succinate at
50 mV was 1.1 ± 0.1 mM (mean ± SE).

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Fig. 2.
Succinate-dependent steady-state current as a function of
succinate concentration in an oocyte expressing hNaDC-1. A:
steady-state currents due to addition of succinate at different
concentrations between 0.05 and 10 mM were plotted against membrane
potential. B: current-voltage relations as a function of
succinate concentration at different membrane potentials of 10, 50,
90, 130 and 150 mV. Data were fit to the Michaelis-Menten
equation. C: half-saturation constant for succinate
(K0.5succinate) as a function of membrane
potential. D: maximal succinate-induced current
(Imaxsuccinate) as a function of membrane
potential.
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Figure 3 shows the effects of sodium on
the kinetics of succinate-induced currents in hNaDC-1. Decreasing the
sodium concentration resulted in a large decrease in succinate-induced
currents, consistent with our previous study showing that hNaDC-1 has a
very low cation affinity, with an apparent Km
for sodium of ~78 mM (15). The Imaxsuccinate appeared to be unaffected by
sodium concentrations between 25 and 100 mM, although there were large
errors in the data fits at lower sodium concentrations (Fig.
3B). The Imaxsuccinate increased
with more negative voltages at all sodium concentrations (Fig.
3B). In contrast, the
K0.5succinate increased considerably as the
sodium concentration was reduced. The
K0.5succinate at
50 mV was 1.3 mM at 100 mM sodium, 4.2 mM at 75 mM sodium, and 11.4 mM at 25 mM sodium (Fig.
3C). The differences in
K0.5succinate at different sodium
concentrations were more pronounced at more positive membrane
potentials whereas no differences in
K0.5succinate were evident at very negative
membrane potentials (Fig. 3D).

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Fig. 3.
Substrate activation of currents. A: inward
succinate-dependent currents in a single oocyte were measured at 25, 75, and 100 mM sodium at a holding potential of 50 mV. B:
Imaxsuccinate as a function of sodium
concentration. Error bars represent SE of the fit. C:
K0.5 succinate plotted as a
function of sodium concentration. D: voltage dependence of
K0.5succinate at different sodium
concentrations. It was not possible to obtain a reliable fit of data
from voltages more positive than 30 mV.
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Substrate specificity.
To examine substrate specificity, oocytes expressing hNaDC-1 were
superfused with 10 mM concentrations of various substrates and
steady-state currents were recorded (Fig.
4). The currents were expressed as a
percentage of the succinate-induced current. Similar to the substrate
specificity of rbNaDC-1 (17), the currents recorded in the
presence of fumarate in hNaDC-1 were larger than those measured in
succinate (Fig. 4). Methylsuccinate, dimethylsuccinate, citrate, and
-ketoglutarate induced <50% of the succinate-induced currents in
the human NaDC-1 (Fig. 4). In contrast, the largest currents in
rbNaDC-1 were seen with methylsuccinate, citrate, and tricarballylate
(17). In hNaDC-1, small currents of <20% of the
succinate-induced currents were observed with tricarballylate and
glutarate, whereas sulfate, lactate, and L-glutamate
produced currents that were <5% of control. Pyruvate did not induce
any measurable inward current. None of the substrates induced inward currents in water-injected control oocytes (data not shown).

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Fig. 4.
Substrate specificity of hNaDC-1. Steady-state
currents induced by various substrates (10 mM) at 50 mV were
expressed as a percentage of steady-state current evoked by succinate
in same oocytes. Data are reported as mean ± SE
(n = 4-7). Me-succinate, methylsuccinate; -KG,
-ketoglutarate; DMS, dimethylsuccinate. Most of the substrates were
free acids, but fumarate, -KG, lactate, and pyruvate were added as
sodium salts.
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To determine whether the differences in substrate-induced currents were
due to differences in Imax or
K0.5, kinetic measurements were made.
Although the K0.5 is lowest for succinate
(1.1 mM), the transport capacity (Imax) for
fumarate is greater than for succinate (Table
1). The Imax
/ K0.5 ratio for fumarate is more than
twofold greater than that for succinate, indicating that it is
transported more efficiently. The rank order of substrate preference in
hNaDC-1 is fumarate, followed by succinate, methylsuccinate, citrate,
and
-ketoglutarate (Table 1). However, it should be noted that the
preferred species of citrate transported by hNaDC-1 is
citrate2
, which accounts for only ~1.3% of the
total citrate at pH 7.5 (pKa 5.62) (2,
18). Therefore, the K0.5 of
citrate2
in hNaDC-1 is ~88 µM, although the possible
inhibition of transport by citrate3
(18)
could affect this value.
The kinetic measurements for citrate shown in Table 1 were made in the
presence of 100 µM niflumic acid to inhibit currents through an
endogenous hemi gap-junction channel that is seen in some batches of
uninjected oocytes. As reported previously, the currents through this
hemi gap-junction channel are very large (0.5-1 µA), outwardly
directed, and they are activated by the removal of calcium
(28). We have observed these currents in some batches of
uninjected oocytes in the presence of large concentrations of citrate
or in the presence of 5 mM EDTA, suggesting that the effect of citrate
is the chelation of calcium (results not shown). Niflumic acid does not
affect succinate or citrate transport or currents in hNaDC-1 (results
not shown).
Sodium effects on succinate-dependent inward currents.
The dependence of succinate-induced steady-state currents on external
sodium concentration in oocytes expressing hNaDC-1 is illustrated in
Fig. 5. There was no measurable current
at 5 mM sodium, but above this concentration the succinate-induced
currents increased with increasing concentrations of sodium (Fig.
5A). The succinate-induced currents were sigmoidal functions
of sodium concentration (Fig. 5B) and could be fit by the
Hill equation. The maximum succinate-induced current at saturating
sodium concentrations, Imaxsodium, increased with
hyperpolarizing potentials, ~1.8-fold between
50 and
150 mV (Fig.
4C). The apparent affinity constant for sodium,
K0.5sodium, was very sensitive to voltage,
decreasing from 68 mM at
50 mV to 45 mM at
150 mV (Fig.
4D). In three separate experiments, the mean
K0.5sodium measured at
50 mV was 65 ± 7 mM. The apparent Hill coefficient, n, was 2.2 at
50
mV and was relatively independent of voltage (Fig. 4E). The
mean n was 2.3 ± 0.3 (N = 3 experiments).

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Fig. 5.
Effect of sodium concentration on succinate-induced
steady-state inward currents. A: typical
I-V curves obtained from an oocyte expressing
hNaDC-1 at different sodium concentrations. B: steady-state
currents obtained from an hNaDC-1-expressing oocyte at 10, 50,
90, 130, and 150 mV plotted as a function of sodium
concentration. Data were fit to the Hill equation. C:
dependence of maximal sodium-induced current
(Imaxsodium) on membrane
potential. D: dependence of
K0.5sodium on membrane potential.
E: the apparent Hill coefficients were plotted against
membrane potential. Error bars represent SE of fit. Concentration of
succinate was 10 mM in these experiments. It was not possible to obtain
a reliable fit of data measured at +50 mV.
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The effect of succinate concentration on sodium-activation of currents
in hNaDC-1 was also examined. However, the succinate-induced currents
did not reach saturation in many of the experiments when the succinate
concentrations were reduced below 10 mM and it was not possible to
obtain reliable fits of the data to the Hill equation. In only one of
the seven experiments, it was possible to fit the data at higher
succinate concentrations (Fig. 6). In
this experiment, the K0.5sodium was similar
at the two higher succinate concentrations (54 mM in 5 mM succinate vs.
59 mM in 1 mM succinate) (Fig. 6). The sodium-activation curve measured
with 0.5 mM succinate did not reach saturation although there appeared
to be a shift in K0.5sodium to the right
(Fig. 6). The Imaxsodium was
lower at 1 mM succinate (
72 nA) compared with 5 mM succinate (
160
nA) (Fig. 6).

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Fig. 6.
Sodium activation of succinate-dependent currents in an
oocyte expressing hNaDC-1. Inward currents at increasing sodium
concentrations are plotted for succinate concentrations of 0.5, 1, and
5 mM. Holding potential was 50 mV. Kinetic constants are
K0.5 sodium 54 ± 11 mM (5 mM
succinate); 59 ± 19 mM (1 mM succinate);
Imaxsodium 160 ± 32 nA (5 mM
succinate); 72 ± 19 nA (1 mM succinate); Hill coefficient,
n, 2.4 ± 0.7 (5 mM succinate); 1.9 ± 0.5 (1 mM
succinate).
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Cation specificity of hNaDC-1.
Substrate-induced currents in oocytes expressing hNaDC-1 were very
specific for sodium (Fig. 7). No
substrate-induced currents were seen in hNaDC-1 when sodium was
replaced by lithium, choline (pH 7.5), or cesium. In a single
experiment, no currents were observed in potassium and there was also
no chloride dependence (results not shown). Small succinate-dependent
inward currents, ~8% of those seen in sodium, were observed in
choline at pH 5.5 but only at membrane potentials more negative than
70 mV (Fig. 7). In contrast, our previous study of rbNaDC-1 found
that lithium could substitute for sodium, and lithium produced up to
25% of the current seen in sodium (17).

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Fig. 7.
Cation specificity of hNaDC-1. Substrate-dependent
steady-state inward currents were recorded in choline (pH 7.5), choline
(pH 5.5), cesium, lithium, and sodium buffer in an oocyte expressing
hNaDC-1. Currents were plotted as a function of membrane potential.
Concentration of succinate was 10 mM.
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Sodium-dependent leak currents in hNaDC-1.
In oocytes expressing hNaDC-1, external sodium produced an inward
current in the absence of substrate, the sodium-dependent leak current,
which represented ~20% of the total maximal current measured in
saturating concentrations of succinate (Fig.
8A). The mean leak current was
17 ± 1% (mean ± SE, N = 15 oocytes, 8 frogs). The sodium-dependent leak current was a linear function of the
amount of expression of hNaDC-1 whereas uninjected or water-injected oocytes had low-sodium currents of approximately
7 nA at
50 mV
(Fig. 8B). The current-voltage relationship of the
substrate-independent sodium currents is shown in Fig. 8C.
The currents were measured at sodium concentrations between 5 and 100 mM. The currents were inwardly directed at all voltages tested, and
there was a steep response to voltage between
50 and
150 mV. In
addition, the sodium-dependent leak currents in hNaDC-1 were saturable
with increasing concentrations of sodium. The currents at
50 mV from Fig. 8C were replotted as a function of sodium
concentration. In the experiment shown in Fig. 8D, the
half-saturation constant for leak
(K0.5leak) was 186 mM. In three
separate experiments, the
K0.5leak was 193 ± 11 mM
(mean ± SE). The substrate-independent sodium currents were also
seen in rbNaDC-1 and represented ~13% of the maximal
substrate-induced currents (results not shown).

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Fig. 8.
Sodium-dependent leak currents associated with hNaDC-1
expression. A: membrane currents in an oocyte expressing
hNaDC-1 were continuously recorded in choline buffer, sodium buffer,
and sodium buffer with 10 mM succinate at holding potential of 50 mV.
B: leak current relative to total succinate-induced current.
Control, water-injected oocytes are shown in (N = 11 oocytes from 5 frogs) and hNaDC-1-expressing
oocytes are represented by (N = 15 oocytes from 8 frogs). The line is a linear regression through
only (slope=0.14, intercept= r2 = 0.81). C: steady-state sodium-dependent currents in 5.9
nA, an oocyte expressing hNaDC-1 measured in different sodium
concentrations and plotted as a function of voltage. D:
kinetics of the sodium-dependent leak current at 50 mV in an oocyte
expressing hNaDC-1. Steady-state sodium-dependent leak currents from
C are plotted as a function of sodium concentration.
K0.5leak was 185 ± 85 mM and
Imaxleak was 74 ± 24 nA.
Hill coefficient was 1.01 ± 0.27 (mean ± SE of the fit).
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The sodium-dependent leak current in oocytes expressing hNaDC-1 was
sensitive to inhibition by 0.5 mM phloretin, which was more pronounced
at hyperpolarized membrane potentials (Fig.
9A). The effect of phloretin
was completely reversible after a 15-min washout with choline buffer.
However, phloretin had no effect on substrate-dependent currents in
hNaDC-1, either at 10 mM succinate (Fig. 9B) or at
concentrations of succinate as low as 50 µM (results not shown).
Phloretin also had no effect on currents in water-injected oocytes
(data not shown). The concentration dependence of phloretin inhibition
of the sodium current in hNaDC-1 at
50 mV is shown in Fig.
9C. The IC50 in this experiment was 0.2 mM, and
the maximal inhibition was 59%. In three experiments, the
IC50 for phloretin was 0.2 ± 0.05 mM, and the maximal
inhibition was 58 ± 9% (mean ± SE).

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Fig. 9.
Effect of phloretin on hNaDC-1-mediated currents, plotted
as a function of membrane potential. A: effect of 0.5 mM
phloretin on steady-state sodium leak currents in an oocyte expressing
hNaDC-1. B: succinate-dependent steady-state currents in the
same hNaDC-1-expressing oocyte, measured with and without 0.5 mM
phloretin. Concentration of succinate was 10 mM. C:
concentration dependence of phloretin inhibition of the sodium leak
current. IC50 was 0.2 mM and maximal inhibition was 59%.
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To test whether activation of endogenous sodium channels in the oocyte
membranes could potentially account for the sodium-dependent leak
currents in oocytes expressing hNaDC-1, the sensitivity of the currents
to inhibitors was measured. There have been reports that heterologous
expression of channels or transporters in X. laevis oocytes
can induce the expression of a hyperpolarization-activated cation-selective current that is blocked by DIDS and TEA
(24). However, the sodium-dependent leak pathway in
oocytes expressing hNaDC-1 was insensitive to 1 mM DIDS or 100 µM TEA
(data not shown). X. laevis oocytes also express
nonselective mechanosensitive channels that are inhibited by amiloride
or Gd3+ (25). However, neither 500 µM
amiloride nor 100 µM Gd3+ had any effect on the
sodium-dependent leak pathway in our experiments (data not shown).
Niflumic acid was tested as a potential inhibitor of the hemi-gap
junction channel (28), but it also had no effect on the
sodium-dependent leak pathway in oocytes expressing hNaDC-1 (data not
shown). Finally, we tested TTX because the endogenous sodium channels
of X. laevis oocytes are voltage-gated and sensitive to
micromolar TTX (12). However, 10
4 M TTX did
not affect the sodium-dependent leak current in hNaDC-1 (data not
shown). Furthermore, none of these agents affected the substrate-dependent currents in hNaDC-1 (data not shown).
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DISCUSSION |
The coupled transport of succinate and sodium by hNaDC-1 is
electrogenic and produces an inwardly directed current. The
voltage-sensitive steps in transport by hNaDC-1 appear to be sodium
binding and substrate turnover, whereas the binding of substrate
appears to be relatively independent of voltage. The response to
voltage in hNaDC-1 is similar to that of many sodium-dependent
transporters, including rbNaDC-1 (17), the
Na+-glucose cotransporter, SGLT1 (20), the
Na+-iodide symporter, NIS (5), and the
Na+-phosphate cotransporter, NaPi-5 (7).
Therefore, despite differences in structure between different families
of sodium-coupled transporters, many of these transporters share
similarities in mechanism.
Substrate kinetics.
The kinetic constants for hNaDC-1 from the two-electrode voltage clamp
studies agree quite well with previous data from radiotracer uptake
experiments. The K0.5succinate was 1.1 mM,
similar to the Km for succinate of 0.8 mM
(18), verifying that hNaDC-1 has a relatively low affinity
for substrates. In transport experiments, the Km
for citrate was ~7 mM (18), which would account for the
relatively small citrate-induced currents, <50% of the
succinate-induced currents, in this study. Interestingly, although most
sodium-coupled transporters, including hNaDC-1, show no effect of
voltage on K0.5substrate at potentials more
negative than
50 mV, the rat ortholog of the
Na+-dicarboxylate cotransporters rNaDC-1 (SDCT1) had an
increase in the K0.5citrate with more
negative membrane potentials (4).
The Na+-dicarboxylate cotransporters have broad,
overlapping substrate specificities for 4-carbon, terminal dicarboxylic
acids in the trans-configuration, including many Krebs cycle
intermediates, such as succinate,
-ketoglutarate, and citrate
(27). However, there are species differences in preferred
substrates among the NaDC-1 orthologs. For example, the largest
currents in hNaDC-1 were induced by fumarate and succinate, whereas the
currents induced by
-ketoglutarate and citrate were only ~50% of
the succinate-induced currents. The rabbit NaDC-1 differs from hNaDC-1
primarily in having large citrate-induced currents, likely because
rbNaDC-1 has a higher affinity for citrate (17,
18). In contrast, the rNaDC-1 has small citrate-induced
currents like hNaDC-1 (4, 22). However, the
rNaDC-1 substrate selectivity differs from that of hNaDC-1 mainly in
the
-ketoglutarate-induced currents, which were almost as large as
the currents produced by succinate (4, 22).
Therefore, it is likely that these closely related transporters contain
subtle differences in the structures of their substrate binding sites
that distinguish between similar substrate structures. In addition,
there are differences in relative Imax between substrates, suggesting differences in translocation or intracellular release of the substrates.
Sodium.
hNaDC-1 has a relatively low affinity for sodium, with a
K0.5sodium of 68 mM, which agrees with the
value of 78-150 mM from radiotracer uptake studies
(10, 18). The
K0.5sodium in hNaDC-1 decreased with
membrane hyperpolarization, indicating that the binding of sodium is
likely to be voltage dependent. Sodium activation experiments with
hNaDC-1 under voltage-clamp conditions resulted in an apparent Hill
coefficient of 2.3, compared with 2.1-2.5 in radiotracer uptake
experiments (10, 18). The Hill coefficient in
hNaDC-1 is consistent with a coupling stoichiometry of 3 sodium ions:1
divalent anion substrate.
hNaDC-1 has a very strong preference for sodium as the coupled cation.
Lithium was not able to substitute for sodium, consistent with our
previous studies that showed that hNaDC-1 is insensitive to inhibition
by lithium (18). Interestingly, there appear to be species
and isoform differences in lithium handling in the family related to
NaDC-1. rbNaDC-1 is very sensitive to inhibition by millimolar
concentrations of lithium, which competes with sodium at one of the
three cation binding sites (17). At higher concentrations, lithium can drive transport in rbNaDC-1, although the
K0.5succinate is very large, ~30 mM
(17). In contrast, lithium inhibition but not substitution
is seen in rNaDC-1 (SDCT1) and in the high-affinity Na+-dicarboxylate cotransporter, rNaDC-3 (SDCT2)
(3, 4, 11). The X. laevis
intestine Na+-dicarboxylate cotransporter, NaDC-2, is
driven equally well by either lithium or sodium, and lithium does not
inhibit transport in the presence of sodium (1). The
differences in lithium handling suggest that there are differences in
the structures of the cation binding sites among the members of this family.
Leak currents.
The results presented in this study suggest that hNaDC-1 may be able to
transport sodium by an uncoupled substrate-independent mechanism. In
the absence of substrate, a sodium-dependent inward current that was
sensitive to inhibition by phloretin was observed in the
hNaDC-1-injected oocytes, but not in control uninjected or
water-injected oocytes. Phloretin did not affect the
substrate-dependent current in hNaDC-1. The magnitude of the sodium
leak current in hNaDC-1 was proportional to the amount of expression of
hNaDC-1. The leak current in hNaDC-1 was also saturable with increasing concentrations of sodium, indicating a low-affinity carrier-mediated pathway (K0.5leak 191 mM at
50
mV). It should be noted, however, that the hyperbolic kinetics of the
leak pathway do not necessarily rule out channel-like activity. For
example, sodium currents through the acetylcholine channel also exhibit
saturation kinetics that can be modeled by the Michaelis-Menten
equation, with an apparent Km for sodium of 102 mM (9). However, the leak current in oocytes expressing hNaDC-1 was insensitive to inhibitors of ion channels that have been
observed previously in X. laevis oocytes, including
amiloride, tetrodotoxin, Gd3+, tetraethylammonium, niflumic
acid, and DIDS (12, 24, 25, 28). Therefore, the substrate-independent sodium current
is likely to be a property of hNaDC-1 rather than the result of
activation of an endogenous channel in the oocytes.
There is evidence that some neurotransmitter transporters, such as the
GABA transporter, exhibit significant efflux activity, which would be
particularly important in neurons during an action potential
(13). Therefore, an alternate hypothesis to account for
the sodium currents seen in the absence of substrate in hNaDC-1 would
be the inhibition of outward currents by addition of extracellular sodium. In this hypothesis, hNaDC-1 would operate in efflux mode when
the cells were bathed in choline, and the addition of extracellular sodium would cause a trans-inhibition of the efflux, which
would appear as an inward current relative to the current measured in choline. However, this hypothesis is not adequately supported by our
experimental results with hNaDC-1. For example, the
substrate-independent sodium current in hNaDC-1 exhibits hyperbolic
kinetics with low affinity
(K0.5leak 191 mM), and
saturation is not reached at the highest sodium concentration used in
these experiments, 100 mM. This result is not consistent with a simple
inhibition of efflux, which should saturate at a much lower sodium
concentration. In experiments with rabbit renal brush-border membrane
vesicles, trans-sodium concentrations inhibited
Na+-succinate uptake with sigmoid kinetics (Hill
coefficient of 1.75) and a K0.5 of 23 mM
(26). Second, the size of the substrate-independent sodium
current is too large to be only efflux, considering that the sodium
concentration inside the oocytes is likely to be 6-10 mM
(4, 28). In experiments measuring uptakes of
radiotracer substrates or inward substrate-dependent currents in
hNaDC-1, there is almost no uptake at sodium concentrations below 25 mM, even with substrate concentrations as high as 10 mM
(18). Therefore, the simplest explanation for the
substrate-independent sodium current in hNaDC-1 is a sodium leak
current (or slippage), and this is our present working hypothesis.
However, further experiments with hNaDC-1 in which the intracellular
and extracellular sodium and substrate concentrations can be controlled
are clearly needed.
Sodium-dependent leak currents in the absence of substrate have also
been reported for other transporters including SGLT1 (20),
NIS (5), and NaPi-2 (6), and the magnitude of
the leak current relative to the substrate-induced current depends on
the transporter. For example, the leak current in SGLT1 is ~15% of
the substrate-induced current (20), whereas NIS exhibits a
leak current of ~35% of the substrate-induced current
(5). rNaDC-1, or SDCT1, also exhibits a small sodium leak,
~4% of the maximal succinate-induced current and both the
succinate-dependent and -independent currents are inhibited by
phloretin (4).
Transport model.
Figure 10 shows an ordered binding
model of hNaDC-1 function, based on the results of this study, which is
very similar to a previous model proposed for the
Na+-dicarboxylate cotransporter in rabbit renal
brush-border membrane vesicles (26). The stoichiometry of
Na+-dicarboxylate cotransport in hNaDC-1 is three sodium
ions for each divalent anion substrate molecule. The presence of the
substrate-independent sodium leak indicates that at least one of the
sodium ions binds before the substrate. The Hill coefficient of one for
the sodium leak current indicates that the leak or slippage occurs
after a single sodium ion has bound to the transporter. However, the Imaxsuccinate was the same at different
sodium concentrations (Fig. 3B), providing evidence for an
ordered binding model in which succinate binds last because increasing
concentrations of succinate can overcome the effect of decreased sodium
concentration. Therefore, in the presence of substrate, the first step
in the model proposed for hNaDC-1 function is the cooperative binding
of three sodium ions, which increases the affinity of the transporter
for substrate. The substrate then binds to the transporter, and the
fully-loaded transporter undergoes a conformational change that exposes
the substrate and cation binding sites to the inside of the cell. The
substrate and cations are released on the inside of the cell after
which the empty carrier reorients its binding sites to face the outside
of the cell. This model is very similar to the models proposed for
SGLT1 (21) and NIS (5). However, it differs from the model describing NaPi-2 function, in which two sodium ions
bind before the substrate and one sodium ion binds last
(6).

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|
Fig. 10.
Model of Na+-dicarboxylate cotransport by
hNaDC-1. C represents carrier and S represents
substrate, ' refers to outside of cell, and " refers to
inside of cell. In this model, Na+ binds to transporter
before substrate. In the absence of substrate, the transporter carries
a single sodium ion as a facilitated diffusion carrier in a leak or
slippage pathway. However, in the presence of substrate, three sodium
ions bind before substrate binds. The fully-loaded carrier reorients so
that the binding sites are exposed to the inside of cell, and the
substrate and sodium are then released on the inside. Another
conformational change occurs which reorients empty substrate and cation
binding sites to outside.
|
|
The K0.5sodium in hNaDC-1 is very sensitive
to voltage. In contrast, the K0.5succinate
is relatively insensitive to voltage changes at negative membrane potentials. However, at low-sodium concentrations, the
K0.5succinate is affected by voltage (Fig.
3D). One explanation for the results is that the binding of
sodium is voltage sensitive whereas the binding of substrate is voltage
independent. At low-sodium concentrations, the transporter does not
have the optimal configuration for substrate binding (as shown by the
larger K0.5succinate, Fig. 3C).
However, changes in membrane potential at low-sodium concentrations
could affect substrate binding indirectly by affecting sodium binding.
The effects of low sodium can be overcome at high enough substrate concentrations.
The direction of transport by hNaDC-1, as in other sodium-coupled
transporters, depends on the direction of the electrochemical gradients
of sodium and succinate. It has been estimated that intracellular
concentrations of sodium in X. laevis oocytes are between 6 and 10 mM, and intracellular succinate is ~100 µM (4, 28). Therefore, a reversal of the substrate-dependent
current in hNaDC-1 would be expected under the appropriate conditions, for example, if the extracellular concentrations of succinate and
sodium were low enough or at positive membrane potentials. However, we
do not see significant outward currents by hNaDC-1, suggesting that the
rates of the outward fluxes may be slower than the rates of the inward
fluxes. This observation is supported by previous kinetic experiments
with rabbit renal brush-border membrane vesicles that showed that the
Vmax for succinate efflux is much lower
than the Vmax for influx (8).
Furthermore, the rate of transport by hNaDC-1 is very low at the sodium
concentrations (6-10 mM) that would be found inside the oocytes
(Fig. 5, Ref. 18). It is likely that, in addition to allowing transport
against a larger electrochemical gradient, the coupling of three sodium ions in hNaDC-1 also prevents significant efflux of substrate from the
renal proximal tubule cells during substrate accumulation.
Conclusion.
In conclusion, the succinate-dependent steady-state inward currents
associated with hNaDC-1 expressed in X. laevis oocytes were
analyzed by using the two electrode voltage-clamp technique. The
substrate-induced currents in hNaDC-1 are dependent on membrane potential and on the concentrations of succinate and sodium. The K0.5sodium decreases with hyperpolarizing
potentials, whereas the K0.5succinate is
relatively insensitive to membrane potential. The only cation that is
able to support succinate transport in hNaDC-1 is sodium. Finally, a
phloretin-sensitive leak pathway for sodium was observed in the oocytes
expressing hNaDC-1. We propose an ordered binding model in which three
sodium ions bind before the substrate. The results provide new
information for clarifying the functional role of the
Na+-dicarboxylate cotransporter in the human kidney.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Owen Hamill for discussions regarding the
Ca2+-sensitive current in oocytes.
 |
FOOTNOTES |
This study was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-46269 and DK-02429, and by a
John Sealy Memorial Endowment Fund Award 2526-99-99.
Address for reprint requests and other correspondence:A.
M. Pajor, Dept. of Physiology and Biophysics, Univ. of Texas Medical Branch, Galveston, TX 77555-0641
(E-mail:ampajor{at}utmb.edu).
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 14 July 1999; accepted in final form 23 February 2000.
 |
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