Functional characterization of high-affinity Na+/dicarboxylate cotransporter found in Xenopus laevis kidney and heart

Naomi Oshiro and Ana M. Pajor

Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas

Submitted 23 June 2004 ; accepted in final form 5 June 2005


    ABSTRACT
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 ABSTRACT
 METHODS
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 DISCUSSION
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The SLC13 gene family includes sodium-coupled transporters for citric acid cycle intermediates and sulfate. The present study describes the sequence and functional characterization of a SLC13 family member from Xenopus laevis, the high-affinity Na+/dicarboxylate cotransporter xNaDC-3. The cDNA sequence of xNaDC-3 codes for a protein of 602 amino acids that is ~70% identical to the sequences of mammalian NaDC-3 orthologs. The message for xNaDC-3 is found in the kidney, liver, intestine, and heart. The xNaDC-3 has a high affinity for substrate, including a Km for succinate of 4 µM, and it is inhibited by the NaDC-3 test substrates 2,3-dimethylsuccinate and adipate. The transport of succinate by xNaDC-3 is dependent on sodium, with sigmoidal activation kinetics, and lithium can partially substitute for sodium. As with other members of the family, xNaDC-3 is electrogenic and exhibits inward substrate-dependent currents in the presence of sodium. However, other electrophysiological properties of xNaDC-3 are unique and involve large leak currents, possibly mediated by anions, that are activated by binding of sodium or lithium to a single site.

SLC13 gene family; citric acid cycle intermediate; lithium


THE METABOLIC INTERMEDIATES of the citric acid cycle, such as succinate and {alpha}-ketoglutarate, are transported across plasma membranes of mammalian cells by sodium-coupled transporters belonging to the SLC13 gene family (for reviews, see Refs. 16 and 18). Three transporters have been identified to date, including the low-affinity Na+/dicarboxylate cotransporter (NaDC-1), the Na+/citrate transporter that also carries dicarboxylates but at lower affinity (NaCT), and the high-affinity NaDC (NaDC-3). The substrate specificity and tissue distribution of these transporters are distinct: NaDC-1 is primarily expressed in kidney proximal tubule and intestine (17), NaCT is found in the liver, brain, and testis (11), and NaDC-3 is found in the kidney basolateral membrane (9), liver, placenta, and brain (4, 26).

There is relatively little known about NaDCs in nonmammalian vertebrates. A type of NaDC-3 that functionally resembles the NaDC-3 from mammals has been cloned from winter flounder kidney (fNaDC-3) (23). A low-affinity Na+/dicarboxylate cotransporter, xNaDC-2, has also been identified in Xenopus laevis intestine (1). The functional properties of xNaDC-2 resemble those of the NaDC-1 orthologs with the exception of its tissue distribution and cation specificity. NaDC-2 is found only in intestine, not in kidney, and transports succinate equally well in lithium and sodium. In the mammalian members of the SLC13 family, lithium acts as a high-affinity competitor of sodium and low concentrations of lithium produce transport inhibition (20).

In this study, we report the sequence and functional properties of xNaDC-3. The amino acid sequence of xNaDC-3 has been remarkably conserved during evolution. However, the tissue distribution is unique, with mRNA found in the heart, kidney, liver, and intestine. This is the first report of a dicarboxylate transporter from heart. The transport properties of xNaDC-3 in many ways resemble those of other NaDC-3 orthologs: transport is sodium dependent and electrogenic, the transporter has a high affinity for succinate, and the substrate specificity is similar to that of other NaDC-3. However, unlike the other members of the SLC13 family, xNaDC-3 exhibits large leak currents that are activated by lithium or sodium, but may be carried by anions.


    METHODS
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 ABSTRACT
 METHODS
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Identification of xNaDC-3. A Basic Local Alignment Search Tool search against the rabbit NaDC-1 sequence identified a related sequence from Xenopus laevis embryo in the expressed sequence tag (EST) database (Accession no. BG360244, 493 bases). The clone was purchased from the American Type Culture Collection (IMAGE clone 4404882). The plasmid cDNA in pCMV-SPORT6 vector was sequenced in both directions by the UTMB Sequencing Facility (Sealy Center for Molecular Science) and it was found to have a full-length insert of 2.3 Kb. The full sequence has been deposited in GenBank with the accession number AY525098.

RT-PCR. Poly(A+) RNA from female Xenopus laevis was prepared as described previously (1). The RNA, 1.5 µg of each sample, was then used as a template for first-strand cDNA synthesis using the SuperScript preamplification system for First Strand cDNA synthesis (Life Technologies) with random hexamer primers. The RNA template was digested with RNAse H and the samples were stored at –80°C. The PCR reaction was done using the Failsafe PCR kit (Epicenter) with Pre-Mix Buffer C according to manufacturer's directions. The following sequence specific primers were used: sense 5'-CTCCATCTTGTTCCAACTGAAATG-3' and antisense 5'-GGAACGGAACCCAATCAGGAATGG-3'. The annealing temperature was optimized using a temperature gradient. The quality of the cDNA used in RT-PCR was assessed by amplification of GAPDH cDNA using primers specific for the Xenopus sequence (Genbank accession no. XLU41753), sense 5'-TGAGGGACTCATGACAACAGTCC-3', and antisense, 5'-ACAGACTAGCAGGATGGGCGAC-3'.

The following cycles were used for the RT-PCR reactions: 95°C x 5 min, during which the Failsafe enzyme mix was added, followed by 25 cycles of 94°C x 30 s, 60°C x 30 s, and 72°C x 45 s, and finally a 15-min elongation at 72°C. The control group had water instead of cDNA. The PCR products were incubated 10 min with Taq DNA polymerase to add terminal A's and then subcloned into the pCR2.1 vector using the TOPO TA cloning kit (Invitrogen) according to manufacturer's directions. The subcloned PCR products were then sequenced by the UTMB Sequencing facility (Sealy Center for Molecular Science).

Expression of xNaDC-3 in human retinal pigment epithelial cells. The human retinal pigment epithelial (HRPE) cell line, derived from HRPE cells transformed with SV40 (AG 06096, Coriell Institute), was cultured in modified Eagle's medium containing Glutamax and 25 mM HEPES (GIBCO-BRL) supplemented with 15% noninactivated fetal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in 5% CO2. For transient transfections, 1.2 x 105 cells were plated per well of a 24-well plate and each well of cells was transfected with 1.8 µl Fugene 6 (Roche) and 0.6 µg plasmid DNA (9:3 ratio).

Transport assays. Transport assays were carried out 48 h after transfections. The sodium buffer contained (in mM) 120 NaCl, 5 KCl, 1.2 MgSO4, 1.2 CaCl2, 5 D-glucose, and 25 HEPES, pH adjusted to 7.4 with 1 M Tris. Choline (Ch) and lithium (Li) buffers were the same as sodium buffer but contained 120 mM ChCl or LiCl in place of NaCl. For the assays, each well was washed twice with Ch buffer, and then incubated with a sodium buffer containing 14C-succinate (44 mCi/mmol, NEN) for a fixed time period, typically 30 min. For kinetic experiments, 6-min uptakes were used to estimate initial rates; uptakes were linear up to 30 min (results not shown). After the incubation period, the uptakes were stopped and extracellular radioactivity removed with four washes of sodium buffer. The wash volume was 1 ml and the uptake volume was 0.25 ml. Cells were dissolved in 1% SDS, transferred to scintillation vials, and counted on a scintillation counter. For all experiments, uptakes in vector-transfected cells were subtracted from uptakes in experimental plasmid-transfected cells. Statistical analysis with one-way ANOVA or Student's t-test was done using the SigmaStat program (Jandel).

Substrate kinetic measurements were determined by nonlinear regression to the Michaelis-Menten equation: v = Vmax·[S]/(Km + [S]), where v represents the initial rate of uptake at a given substrate concentration, Vmax is the maximal rate of uptake at saturating substrate concentrations, [S] is the concentration of substrate, and Km is the Michaelis-Menten constant representing the substrate concentration that produces 1/2 Vmax. Sodium activation experiments were analyzed by nonlinear regression to the Hill equation: v = (Vmax·[Na]n)/(KNan + [Na]n) + v0, where v0 represents transport of succinate in the absence of sodium, v is the initial rate of uptake at a given sodium concentration, Vmax is the maximal transport rate at saturating Na concentration, n is the Hill coefficient, and KNa is the half-saturation constant for sodium. The nonlinear regressions were calculated using SigmaPlot (Jandel).

Xenopus oocytes. Female Xenopus laevis were obtained from Xenopus I. Stage V and VI oocytes were dissected and treated with collagenase as described previously (17). The defolliculated and sorted oocytes were injected with 46 nl of 1 µg/µl cRNA on the following day. The injected oocytes were cultured at 18°C in Barth's medium supplemented with 5% heat-inactivated horse serum, 2.5 mM pyruvate, 100 mg/ml gentamycin sulfate, and 5 µg/ml ceftazidime (GlaxoSmithKline). Experiments were done 5 days after injection. Culture vials and medium were changed daily.

Electrophysiology. Oocytes expressing xNaDC-3 were used to measure substrate-induced steady-state currents with the use of a two-electrode voltage clamp, as described previously (20). The oocytes were given 100-ms test pulses between +50 and –150 mV with a decrement of 20 mV and a holding potential of –50 mV, with the use of the pCLAMP6 program (Axon instruments). The electrode resistance was between 0.4 and 0.5 M{Omega}. For experiments involving chloride replacement, experiments were done using a 3M KCl agar bridge. The average of three measurements was taken for every trial.

Test solutions of 1 mM succinate were prepared in a buffer containing either (in mM) 100 NaCl, 100 ChCl, or 100 LiCl. The sodium buffer was composed of (in mM) 100 NaCl, 2 KCl, 1 MgCl2, 1 CaCl2, and 10 HEPES, adjusted to pH 7.5 with 1 M Tris. Ch and Li buffers contained 100 mM ChCl or LiCl in place of NaCl. For chloride replacement experiments, the NaCl was replaced with other sodium salts. The solutions also contained (in mM) 6 Cl as 2 KCl, 1 MgCl2, and 1 CaCl2 to minimize liquid junction potentials. In the voltage-clamp experiments, each oocyte was superfused with Na+, Li+, or Ch-containing buffer, and the voltage pulse protocol was applied. Substrate-induced currents were measured in the same cation buffer containing 1 mM succinate. After the voltage pulse protocol, the substrate solution was washed away with Ch buffer and the oocyte was allowed to recover basal currents and resting membrane potential. The difference in steady-state currents in the presence and absence of succinate represents the substrate-dependent current.


    RESULTS
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 RESULTS
 DISCUSSION
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Sequence of xNaDC-3. The sequence of xNaDC-3 is 2345 bases and contains a single open reading frame that codes for a protein of 602 amino acids (Genbank accession no. AY525098). The amino acid sequence alignments of xNaDC-3 with other NaDC-3 orthologs and xNaDC-2 are shown in Fig. 1. There is very high sequence conservation among the NaDC-3 orthologs, and this is the primary reason for naming the cDNA xNaDC-3. For example, the Xenopus and human or mouse NaDC-3 proteins are 73% identical. The flounder NaDC-3 is about 65% identical to xNaDC-3. In contrast, the two Xenopus proteins, xNaDC-3 and xNaDC-2, are only 45% identical in sequence. Other Na+/di- and tricarboxylate transporters from the same gene family, NaDC-1 and NaCT, are ~43–48% identical to xNaDC-3, and the Na+/sulfate cotransporters NaSi-1 are ~39% identical (not shown).



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Fig. 1. Alignments between the predicted amino acid sequence of the high-affinity Na+/dicarboxylate cotransporter from Xenopus laevis (xNaDC-3; Genbank accession no. AY525098) with the sequences of other NaDC-3 orthologs from mouse (accession no. AF306491), human (accession no.AF154121), and flounder (accession no. AF102261), and the intestinal low-affinity NaDC from Xenopus laevis (xNaDC-2; accession no. U87318). Amino acids that are identical in all 5 sequences are shaded in black, and amino acids identical in 4 of 5 sequences are in gray.

 
Tissue distribution of xNaDC-3. RT-PCR reactions using xNaDC-3 specific primers were used to determine the tissue distribution of xNaDC-3. Poly(A+) RNA from adult female Xenopus laevis frogs was used as a template for cDNA synthesis. As shown in Fig. 2, there were positive PCR reactions with cDNA prepared from liver, heart, intestine and kidney. No signals were seen in PCR reactions done using stomach cDNA, despite a positive signal using the xGAPDH control primers. This tissue distribution is very different from that of xNaDC-2, which is found only in intestine (1). The PCR products were cloned into the pCR2.1 vector and sequenced. The amino acid sequences of the PCR products were identical to xNaDC-3, although there were some nucleotide differences that did not affect the amino acid sequence. This result verifies that the transporter message is found in both embryos and adults because the original cDNA was prepared from stage 17/19 embryo (Genbank accession no. BG360244).



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Fig. 2. Tissue distribution of xNaDC-3. Ethidium bromide-stained agarose gel of RT-PCR products from Xenopus laevis tissues amplified with sequence-specific primers (as outlined in METHODS) for xNaDC-3 (top) or xGAPDH (bottom). DNA size standards (in Kb) are shown at left. Positive reactions with xNaDC-3 primers producing the expected 1.2-Kb product were seen with liver, heart, intestine, and kidney mRNA but not in control (water only) or stomach samples.

 
Transport properties of xNaDC-3: substrates. The plasmid coding for xNaDC-3 was expressed in HRPE cells. As shown in Fig. 3A, there was increased transport of the dicarboxylates succinate, malate, and glutarate, with very low background transport in cells transfected with pCMV-SPORT6 vector alone. The succinate transport was increased ~63-fold and the glutarate transport was ~140-fold above background. In contrast, the transport of citrate was increased only 2.7-fold above background (Fig. 3A). The affinity of xNaDC-3 for succinate is high. In the experiment shown in Fig. 3B, the Km for succinate is 4 µM and in three separate experiments the average Km for succinate is 5 ± 1 µM (means ± SE).



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Fig. 3. A: substrates of xNaDC-3. Uptakes of 25 µM 14C-radiolabeled substrates were measured in human retinal pigment epithelial (HRPE) cells transfected with plasmid pCMV-SPORT6 plasmid-containing xNaDC-3 cDNA or with vector alone. Fifteen-minute uptakes were measured in sodium-containing buffer. The bars represent means ± SE of 2–3 experiments. The transport in all xNaDC-3 groups, including citrate, is significantly greater than in controls (P < 0.05). B: kinetics of succinate transport in HRPE cells expressing xNaDC-3. Six-minute uptakes of 14C-succinate were measured at concentrations ranging from 0.8 to 66 µM in sodium buffer. Each point represents the means ± SE of 3 wells of a 24-well plate, corrected for counts in parallel groups of vector-transfected cells. The Km for succinate is 4 µM and the maximal rate of uptake (Vmax) is 10.9 pmol/well per min.

 
The substrate specificity of xNaDC-3 was further tested by measurement of the uptake of 10 µM 14C-succinate with or without 1 mM test inhibitors (Fig. 4). Transport was strongly inhibited by succinate, methylsuccinate, {alpha}-ketoglutarate, malate, adipate, and 2,3-dimethylsuccinate. Adipate, {alpha}-ketoglutarate, and 2,3-dimethylsuccinate are good substrates for the high affinity NaDC-3 transporters and poor substrates of NaDC-1 orthologs. Flufenamate inhibits the human and rabbit NaDC-1 with an IC50 of ~1 mM in human and 250 µM in rabbit (21) and 1 mM flufenamate produced ~50% inhibition of xNaDC-3. There was some inhibition by aspartate, which also interacts with some of the NaDC-3 transporters (12).



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Fig. 4. Inhibition of succinate transport in HRPE cells expressing xNaDC-3. Fifteen-minute uptakes of 10 µM 14C-succinate were measured in the presence and absence of 1 mM test substrate. Data are expressed as a percentage of control without inhibitor, 8.7 ± 1.2 pmol/min-well (means ± SE, n = 6). The inhibition by citrate and lactate was not statistically significant, all other test substrates showed significant inhibition (P < 0.05). Me-succinate, 2-methylsuccinate; aKG, {alpha}-ketoglutarate; 2,3-DMS, 2,3-dimethylsuccinate.

 
Transport properties of xNaDC-3: cations. The transport of succinate by xNaDC-3 was measured in the presence of different cations. Unlike xNaDC-2, which had similar rates of transport in sodium and lithium (1), xNaDC-3 resembled more closely the mammalian and fish NaDC-3 orthologs. There was some transport, ~5%, in Ch and ~25% in Li compared with sodium buffer (Fig. 5). Lithium did not inhibit transport of succinate in the presence of sodium, however. By comparison, in the rabbit NaDC-1, 5 mM lithium added to 95 mM sodium produces >50% transport inhibition (21).



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Fig. 5. Cation specificity of xNaDC-3 expressed in HRPE cells. The uptake of 14C-succinate (25 µM) was measured over 15 min in buffers containing 120 mM sodium or other cations in place of sodium. The inhibition by lithium was tested by comparing uptakes in 95% Na:5% choline (Na:Ch) or 95% sodium:5% lithium (Na:Li). The uptakes are expressed as a percentage of uptakes in sodium (control). The bars represent means ± SE, n = 3 experiments. The error bars in the sodium group represent the SE of uptake rates in sodium among 3 experiments.

 
The activation of succinate transport by increasing concentrations of sodium is shown in Fig. 6A. There is a sigmoidal relationship between sodium concentration and succinate transport, with an apparent Hill coefficient of 2.5 and, in three experiments, a mean Hill coefficient of 1.8 ± 0.4 (means ± SE). This indicates that a minimum of two to three sodium ions are involved in the transport of succinate by xNaDC-3. KNa was 50 mM in the single experiment shown in Fig. 6A, and, in three experiments, 55 ± 12 mM (means ± SE). The transport of succinate in the absence of sodium is ~6% of the transport in 120 mM sodium. Because succinate transport in xNaDC-3 occurs in solutions in which sodium is completely replaced by lithium, we also measured the activation of succinate transport by lithium. As shown in Fig. 6B and in a second experiment (not shown), the transport activation appeared sigmoidal but did not saturate at the highest concentration used in this study, 120 mM. Therefore, it was not possible to fit the data to the Hill equation. We also tried to measure the Km for succinate in lithium buffer. Because of low transport activity it was difficult to obtain high quality data. In one experiment, the Km for succinate in lithium buffer was 60 µM (not shown).



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Fig. 6. A: sodium activation of succinate transport in xNaDC-3 expressed in HRPE cells. Six-minute uptakes of 10 µM [14C]succinate were measured in buffers containing 0–120 mM sodium, with the sodium replaced by choline. There was some uptake in the absence of sodium, 0.2 pmol/well per min. The Hill coefficient is 2.53 and the half-saturation constant for sodium, K is 50 mM. B: activation of succinate transport by lithium in HRPE cells expressing xNaDC-3. Six-minute uptakes of 10 µM 14C-succinate were measured in lithium concentrations ranging from 0 to 120 mM (choline replacement). Each point represents the mean ± SE of 3 wells from a 24-well plate, corrected for counts in parallel groups of vector-transfected cells.

 
Electrophysiological measurements. The function of xNaDC-3 was also assessed by two-electrode voltage clamp of Xenopus oocytes injected with xNaDC-3 cRNA. In the absence of substrate, sodium and lithium-dependent leak currents were measured in oocytes expressing xNaDC-3. Fig. 7, AC, show currents in an individual oocyte expressing xNaDC-3 in the presence of different cation-containing solutions, with or without 1 mM succinate. The summary results from five different experiments are shown in Fig. 7, D and E. The cation-evoked currents are largest in lithium, followed by sodium (Fig. 7D). The difference between currents measured in sodium or Li vs. Ch (INaICh or ILiICh) represents the cation-induced leak current. The leak currents are not seen in control, uninjected oocytes (Fig. 8A), similar to what we reported previously (28). The total succinate-dependent currents in different cations are shown in Fig. 7E (xNaDC-3-injected oocytes) and Fig. 8B (control oocytes). These currents are calculated from the difference in the presence and absence of substrate for a given cation (for example, Isuccinate+NaINa). The small outward current seen in Ch with substrate is also present in control oocytes, and thus probably represents succinate inhibition of an endogenous current. There were outward-directed substrate-dependent currents in lithium in xNaDC-3. The corresponding currents in control oocytes are much smaller. We reported similar outward currents in substrate and lithium in oocytes expressing mouse NaDC-3 (19). These lithium and substrate-dependent outward currents probably represent inhibition of the lithium-induced leak current by substrate, although this remains to be tested in more detail. There may also be a small Li+/succinate cotransport current but it is obscured by the larger leak current. The substrate-dependent measurements in sodium probably represent a combination of the activation of Na+/succinate cotransport currents and inhibition of the Na+-leak current. This produces an inward current that appears independent of voltage (Fig. 7E). However, a more accurate estimate of Na+/substrate cotransport current is shown in Fig. 9A. The substrate-dependent current in xNaDC-3 was determined from the difference between current in sodium + succinate and the current in choline. Because control oocytes have similar currents in sodium and choline, the current measured in choline in xNaDC-3 injected oocytes probably represents the baseline current. This current-voltage relationship resembles those of the other members of the SLC13 family (2, 20). The magnitude of the leak currents induced by sodium or lithium are a linear function of the amount of expression of xNaDC-3 (Fig. 9B), which supports the hypothesis that the leak currents are a property of xNaDC-3.



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Fig. 7. Voltage dependence of substrate and cation-dependent currents in Xenopus oocytes expressing xNaDC-3. AC: currents in the presence and absence of 1 mM succinate (S) in a single oocyte expressing xNaDC-3 superfused with choline (A), lithium (B), or sodium (C) buffers. D: summary of currents in oocytes expresing xNaDC-3 superfused with choline-, lithium-, or sodium-containing buffers. E: total succinate-dependent currents in oocytes expressing xNaDC-3, calculated from the difference between currents in the presence and absence of 1 mM succinate in buffers containing choline, lithium, or sodium. Data are means ± SE from 5 separate batches of oocytes.

 


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Fig. 8. Cation and substrate-dependent currents in control, uninjected oocytes. A: steady-state currents in oocytes superfused with choline, lithium, or sodium (compare with Fig. 7D). B: succinate-dependent currents in control oocytes, calculated from the difference between currents in the presence and absence of 1 mM succinate in buffers containing choline, lithium, or sodium. Data are means ± SE, n = 3.

 


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Fig. 9. A: voltage dependence of succinate-dependent currents in oocytes expressing xNaDC-3. Because of the large leak current activated by sodium, the succinate-dependent currents are calculated from the difference between currents in sodium + 1 mM succinate and currents in choline, which should have the smallest leak current. Data are means ± SE of oocytes from 5 batches of oocytes. B: relationship between substrate-dependent currents and Na+-induced leak current at –150 mV in control oocytes or oocytes expressing xNaDC-3.

 
We then tested the concentration dependence of the Na+-activated (Fig. 10) and Li+-activated (Fig. 11) leak currents. The leak currents were determined from the difference between currents in Na+ or Li+ and choline (see total currents in Fig. 7D). The reversal potentials in both sodium and lithium were independent of cation concentration, although the magnitude of the currents changed with cation concentration (Figs. 10A and 11A). The reversal potentials were approximately –5 to –10 mV. This result suggests that the cations activate a current but do not carry it. The kinetics of Na+-activated and Li+-activated leak currents were hyperbolic (Figs. 10B and 11B), rather than sigmoidal, suggesting that there is a single binding site for leak current activation. In contrast to the low affinity for sodium or lithium activation of transport (see Fig. 6), the Km for activation of the leak current was ~20 mM for Na+ and 15 mM for Li+. The Km for the activation was relatively insensitive to voltage, but the maximum current induced by cations was sensitive to voltage (Figs. 10C and 11C).



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Fig. 10. Sodium-dependent leak currents associated with xNaDC-3 expression in oocytes. A: voltage and concentration dependence of sodium-activated currents in a single oocyte expressing xNaDC-3. B: the data from A replotted as a function of sodium concentration. C: voltage dependence of Km (top) and maximum current (Imax; bottom) for currents activated by sodium in oocytes expressing xNaDC-3. Data are means ± SE, n = 5.

 


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Fig. 11. Lithium-dependent leak currents associated with xNaDC-3 expression in oocytes. A: voltage and concentration dependence of lithium-activated currents in a single oocyte expressing xNaDC-3. B: data from A replotted as a function of lithium concentration. C: voltage-dependence of Km (top) and Imax (bottom) for currents activated by lithium in oocytes expressing xNaDC-3. Data are means ± SE, n = 5.

 
We then tested whether the leak currents in xNaDC-3 are sensitive to Cl replacement. The reversal potential for Cl in Xenopus oocytes in 96 mM extracellular Cl is approximately –22 to –25 mV (6). Note that the total currents in different anions were determined, rather than the difference between sodium and choline as in Fig. 10 because different choline salts are not available. As shown in Fig. 12, replacement of NaCl by Na+-gluconate had no effect on the inward currents but reduced the outward currents by ~50%, suggesting that at least part of the leak current may be carried by anions. A similar result was seen with replacement of LiCl by Li-acetate (not shown). Substitution of NaCl with NaBr, NaI, NaNO3, and NaSCN resulted in a negative shift of the reversal potential, leading to a general anion selectivity sequence of SCN > NO3 = I > Br > Cl > Gluconate (Fig. 12).



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Fig. 12. Anion selectivity of currents in oocytes expressing xNaDC-3. Oocytes were superfused with bath solutions containing 100 mM sodium salts of different test anions (shown to the right of each curve). Each solution also contained a total of 6 mM Cl as KCl, MgCl2, and CaCl2. The data points are means ± SE [n = 2 (gluconate) or 3 different donor frogs].

 

    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study reports the functional characterization and sequence of a high-affinity Na+/dicarboxylate cotransporter from the African clawed frog, Xenopus laevis. The transporter has been given the name xNaDC-3 because of its sequence and functional similarities with other NaDC-3 orthologs. The NaDC-3 protein is surprisingly well conserved through evolution and the frog sequence is >70% identical to the mammalian sequences. The transport properties of the xNaDC-3 transporter are similar to those of other NaDC-3 orthologs. However, the electrophysiological properties of the NaDC-3 transporter are unique in that cations can activate large leak currents, possibly involving anions.

The substrate specificity and affinity of xNaDC-3 is similar to that of other NaDC-3 orthologs. The Km for succinate in xNaDC-3 is ~5 µM, which falls into the range of 2–15 µM measured for rat NaDC-3 in different expression systems (4, 12). NaDC-3 transporters from other species have lower affinities for succinate, with Km values ranging from 30 to 180 µM (19, 23, 26). The substrates of NaDC-3 transporters include 2,3-dimethylsuccinate, with substitutions at the second and third carbons of succinate, and dicarboxylates with more than four carbons, such as {alpha}-ketoglutarate or adipate (19). The xNaDC-3 resembles other NaDC-3 orthologs because it is strongly inhibited by 2,3-dimethylsuccinate, {alpha}-ketoglutarate, and adipate, and it does not handle citrate well. There was measurable transport of citrate in cells expressing xNaDC-3 but only about twofold above background, compared with more than sixtyfold for other substrates, such as succinate and glutarate. For comparison, the NaDC-1 transporters have low affinities for succinate, with Km ~0.5 mM, and they prefer four carbon dicarboxylates without substitutions at the third carbon (27, 28).

The xNaDC-3 is a sodium-dependent dicarboxylate transporter with a likely transport stoichiometry of 3 Na+:1 succinate2–. Sodium activation experiments show a sigmoid relationship between sodium concentrations and transport of succinate, similar to the other members of the SLC13 family. There was transport of succinate when the sodium was replaced by lithium, ~25% of that in sodium, yet lithium did not inhibit the uptake of succinate. The lithium activation experiment also suggests that multiple lithium ions may be involved in transport since the activation curve appeared sigmoidal, although it could not be fitted because of lack of saturation. The transporter may have a low affinity for lithium. In some members of the SLC13 family, lithium acts as a high-affinity inhibitor of one of the sodium binding sites (20). The cation selectivity of xNaDC-3 resembles that of the other NaDC-3 orthologs (19) rather than the Xenopus NaDC-2 (1). The Xenopus NaDC-2 is a low affinity Na+/dicarboxylate cotransporter from the intestine that transports succinate equally well in sodium and lithium.

Despite the fact that the transport properties of xNaDC-3 are very similar to those of other NaDC-3 orthologs, the electrophysiological properties of xNaDC-3 are quite different. The presence of sodium or lithium activates large leak currents in xNaDC-3 that appear to be inhibited by substrate. The leak currents are not carried by sodium or lithium because their reversal potential is independent of the cation concentration. The results of the anion replacement experiments are consistent with at least part of the current being carried by anions, such as Cl. The general anion permeability sequence was SCN > NO3 = I > Br > Cl > Gluconate. Unlike the activation of substrate-dependent transport, which involves multiple cations, the activation of the leak currents likely involves binding of sodium or lithium to a single site, which has a higher affinity than the coupled-transporter cation binding sites. The Km for Na+ or Li+ for activation of the leak was ~15–20 mM, compared with ~50 mM for uptake of succinate.

The leak currents in xNaDC-3 very different from those seen in many other Na+-coupled transporters. One common type of leak current represents an uncoupled transport of sodium, and is often inhibited by substrate. As examples, the Na+/dicarboxylate cotransporter, hNaDC-1, the Na+/glucose, Na+/iodide, and Na+/phosphate cotransporters exhibit this type of leak current with sodium (3, 5, 7, 22, 28). Some of the GABA transporters, such as GAT1 and GAT3, exhibit leak currents in lithium, which also represent partial reactions of the transporter, uncoupled to substrate (8, 14, 15). A different type of current is seen in the Na+/glutamate transporters, such as EAAT1, which exhibit chloride channel activity in the presence of substrate (25). The leak currents in xNaDC-3 do not appear to be carried by sodium or lithium, and they are not activated by substrate, which makes them very different from previously reported leak currents in other transporters. The currents measured in xNaDC-3 in the presence of substrate and cation are consistent with a mixture of activation of cation/substrate cotransport current and inhibition of the cation-dependent leak current. In lithium, the leak current is predominant. In sodium, the Na+/substrate cotransport current is larger than in lithium and the leak current is smaller; therefore, currents in the presence of substrate appear to be inward but independent of voltage. When choline is used to estimate the substrate-dependent current, however, the voltage dependence resembles that of other NaDC transporters.

This study represents the first report of a Na+/dicarboxylate transporter found in heart. The expression of NaDC-3 in Xenopus heart may be specific for amphibians, however, because NaDC-3 does not appear to be expressed in mammalian heart (4, 12, 19). Furthermore, none of the other NaDC isoforms, such as NaDC-1 and NaCT, has been found in mammalian heart (11, 17). Interestingly, there is a partial sequence in the EST database suggesting that hNaDC-3 is expressed in human aorta (Genbank C15976), but this result needs to be verified experimentally. Although there have been no studies to characterize the transport pathways for citric acid cycle intermediates across the plasma membrane in cardiomyocytes in mammals, there is evidence for both uptake and efflux of these metabolites. For example, rat hearts perfused with 13C-labeled precursors release [13C]citrate to the perfusate, which suggests the existence of a plasma membrane transport pathway for the efflux of citrate (24). Succinate is also released from cardiac myocytes, particularly during hypoxia (10). There is evidence of uptake pathways in heart because rat hearts take up [13C]fumarate, [14C]malate, and [14C]{alpha}-ketoglutarate from perfusion solutions (10, 13). These transport pathways for uptake and efflux of citric acid cycle intermediates are not likely to represent NaDC-3 transporters, and could possibly be mediated by anion exchangers. Frog hearts may use NaDC-3 for uptake of dicarboxylates and would need an additional transporter for efflux, unless the sodium-independent transport by NaDC-3 is sufficient.

In conclusion, this study has identified an unknown cDNA from the EST database as the high-affinity Na+/dicarboxylate cotransporter from Xenopus laevis. The message for this transporter is found in the kidney, heart, liver, and intestine. Transport by xNaDC-3 exhibits a high affinity for succinate, and its substrate specificity is similar to that of other NaDC-3 orthologs from mammals and fish. The xNaDC-3 has some transport activity in the absence of sodium, with some transport of radiotracer succinate in both choline and lithium buffer. The electrophysiological properties of xNaDC-3 are unique and exhibit large leak currents that are activated by sodium and lithium. The results suggest that xNaDC-3 may carry anions independently of substrate.


    GRANTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This research was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-46269.


    ACKNOWLEDGMENTS
 
Dr. Aileen Ritchie provided invaluable discussions and insight into the interpretation of the electrophysiological measurements. We also thank Kate Randolph for help in preparing the reagents used in this study.


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. M. Pajor, Dept. of Human Biological Chemistry and Genetics, Univ. of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0645 (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. Section 1734 solely to indicate this fact.


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