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
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ABSTRACT |
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SLC13 gene family; citric acid cycle intermediate; lithium
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.
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METHODS |
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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. 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.
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RESULTS |
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DISCUSSION |
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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 215 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
-ketoglutarate or adipate (19). The xNaDC-3 resembles other NaDC-3 orthologs because it is strongly inhibited by 2,3-dimethylsuccinate,
-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 1520 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]-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.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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