Structure, function, and genomic organization of human Na+-dependent high-affinity dicarboxylate transporter

Haiping Wang1, You-Jun Fei1, Ramesh Kekuda1, Teresa L. Yang-Feng2, Lawrence D. Devoe3, Frederick H. Leibach1, Puttur D. Prasad3, and Vadivel Ganapathy1

Departments of 1 Biochemistry and Molecular Biology, and 3 Obstetrics and Gynecology, Medical College of Georgia, Augusta, Georgia 30912; and 2 Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have cloned and functionally characterized the human Na+-dependent high-affinity dicarboxylate transporter (hNaDC3) from placenta. The hNaDC3 cDNA codes for a protein of 602 amino acids with 12 transmembrane domains. When expressed in mammalian cells, the cloned transporter mediates the transport of succinate in the presence of Na+ [concentration of substrate necessary for half-maximal transport (Kt) for succinate = 20 ± 1 µM]. Dimethylsuccinate also interacts with hNaDC3. The Na+-to-succinate stoichiometry is 3:1 and concentration of Na+ necessary for half-maximal transport (KNa+0.5) is 49 ± 1 mM as determined by uptake studies with radiolabeled succinate. When expressed in Xenopus laevis oocytes, hNaDC3 induces Na+-dependent inward currents in the presence of succinate and dimethylsuccinate. At a membrane potential of -50 mV, KSuc0.5 is 102 ± 20 µM and KNa+0.5 is 22 ± 4 mM as determined by the electrophysiological approach. Simultaneous measurements of succinate-evoked charge transfer and radiolabeled succinate uptake in hNaDC3-expressing oocytes indicate a charge-to-succinate ratio of 1:1 for the transport process, suggesting a Na+-to-succinate stoichiometry of 3:1. pH titration of citrate-induced currents shows that hNaDC3 accepts preferentially the divalent anionic form of citrate as a substrate. Li+ inhibits succinate-induced currents in the presence of Na+. Functional analysis of rat-human and human-rat NaDC3 chimeric transporters indicates that the catalytic domain of the transporter lies in the carboxy-terminal half of the protein. The human NaDC3 gene is located on chromosome 20q12-13.1, as evidenced by fluorescent in situ hybridization. The gene is >80 kbp long and consists of 13 exons and 12 introns.

human placenta; electrophysiology; chromosomal localization; exon-intron organization


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MAMMALIAN SODIUM-DICARBOXYLATE cotransporters, which transport succinate and other Krebs cycle intermediates, fall into two categories based on their substrate affinity (23). The low-affinity Na+-dicarboxylate transporter is expressed in the brush-border membrane of the intestinal and renal epithelial cells and exhibits a concentration of substrate necessary for half-maximal transport (Kt) value of 0.1-0.5 mM for succinate. This transporter, designated NaDC1, has been cloned from different animal species and functionally characterized (5, 13, 21, 22, 28). The high-affinity Na+-dicarboxylate transporter is expressed in the basolateral membrane of renal proximal tubular epithelial cells, sinusoidal membrane of hepatocytes, and brain synaptosomes (23). Studies from our laboratory have shown that the maternal-facing brush-border membrane of the placental syncytiotrophoblast also expresses the high-affinity Na+-dicarboxylate transporter (8). Recently, we reported on the cloning of this high-affinity transporter, designated NaDC3, from rat placenta (12). Subsequently, Chen et al. (6) cloned the same transporter from rat kidney. When expressed in mammalian cells, the NaDC3 cloned from rat placenta induces Na+-dependent transport of succinate with a Kt value of ~2 µM. The NaDC3-specific transcript (~3.6 kb) is detectable in placenta, kidney, liver, and brain, tissues in which the high-affinity transporter has been shown to exist by functional studies.

The low-affinity as well as the high-affinity transporters interact with citrate, and pH titration studies indicate that the divalent form of citrate is preferentially recognized by the transporters (23). These two transporters, expressed in the brush-border and basolateral membranes, respectively, of the renal tubular cells play an important role in the handling of citrate by the kidneys. Cellular processes involved in the transport of citrate in the kidney are very important for a number of reasons. Citrate is a significant source of metabolic energy in this tissue (20). In addition, low urinary levels of citrate are associated with nephrolithiasis, because citrate is a potent inhibitor of calcium stone formation (25). A significant proportion of patients with renal stone diseases exhibit hypocitraturia (19). Therefore, increased absorption of citrate in the kidney with the resultant hypocitraturia is potentially linked to renal calcium stone formation. Conditions such as chronic metabolic acidosis and potassium depletion are known to enhance renal absorption of citrate (1, 11). There are also individuals who exhibit hypocitraturia with no apparent cause (idiopathic) (2). The underlying mechanism of idiopathic hypocitraturia has not been identified, but the Na+-coupled dicarboxylate transporters NaDC1 and NaDC3 are potential players in the pathogenesis of this disorder. Structural characterization of human NaDC1 (hNaDC1) and human NaDC3 (hNaDC3) and the corresponding genes will help in the screening of human patients with hypocitraturia for identification of potential genetic changes in these genes. hNaDC1 has been cloned and functionally characterized (22). The gene coding for NaDC1 maps to human chromosome 17, but the structure of the gene has not been elucidated. Here we report on the structure, function, genomic organization, and chromosomal location of the hNaDC3.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Materials. The human retinal pigment epithelial (HRPE) cell line number 165, used in expression studies, was originally provided by M. A. Del Monte (W. K. Kellogg Eye Center, Department of Ophthalmology, Ann Arbor, MI) and was routinely maintained in DMEM/F-12 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin as described before (10). Frogs (Xenopus species) were purchased from Nasco (Fort Atkinson, WI) and MEGAscript cRNA synthesis kit was obtained from Ambion (Austin, TX). SuperScript plasmid system for cDNA cloning, TRIzol reagent, oligo(dT)-cellulose, and Lipofectin were purchased from Life Technologies (Grand Island, NY). Restriction enzymes were purchased from New England Biolabs (Beverly, MA). Magna nylon transfer membranes were purchased from Micron Separations (Westboro, MA). [2,3-3H]succinic acid (sp act 37.5 Ci/mmol) was purchased from Moravek Biochemicals (Brea, CA).

Screening of the human placental cDNA library. A placental cDNA library was constructed using poly(A)+ RNA isolated from placenta collected after uncomplicated Cesarean delivery. The SuperScript plasmid cDNA library construction system (Life Technologies) was used to clone the cDNA inserts into pSPORT vector. The screening of the cDNA library was done as described before (27, 33). The cDNA probe used for screening was an ~1.9-kbp-long EcoR I/BamH I restriction fragment of rat NaDC3 cDNA (12). The probe included the entire open reading frame, 30 nucleotides of the 5'-noncoding region, and 69 nucleotides from the 3'-noncoding region. The cDNA probe was labeled with [alpha -32P]dCTP by random priming using the ready-to-go oligolabeling beads (Amersham Pharmacia Biotech). After overnight hybridization at 65°C, the filters were washed under low-stringency conditions. Positive clones were purified by secondary screening.

DNA sequencing. Both the sense and antisense strands of the cDNA were sequenced by primer walking. Sequencing was done by Taq DyeDeoxy terminator cycle sequencing using an automated Perkin-Elmer Applied Biosystems 377 Prism DNA sequencer. The sequence was analyzed using the Baylor College of Medicine Search Launcher server at http://dot.imgen.bcm. tmc.edu:9331/ (31) and the National Center for Biotechnology Information server at http://www.ncbi.nlm.nih.gov/.

Functional expression in HRPE cells. This was done in HRPE cells using the vaccinia virus expression system as described before (12, 27, 33). Subconfluent HRPE cells grown on 24-well plates were first infected with a recombinant (VTF7-3) vaccinia virus encoding T7 RNA polymerase and then transfected with the plasmid carrying the full-length cDNA. After 10-12 h posttransfection, uptake measurements were made at room temperature with [3H]succinate. The uptake medium was 25 mM HEPES/Tris (pH 7.5), containing (in mM) 140 NaCl, 5.4 KCl, 1.8 CaCl2, 0.8 MgSO4, and 5 glucose. In experiments dealing with the anion dependence of succinate transport, the composition of the transport buffer was modified by substituting KCl and CaCl2 in the buffer with potassium gluconate and calcium gluconate, respectively. When the influence of pH on transport was investigated, transport buffers of different pH values were prepared by varying the concentrations of Tris, HEPES, and MES. When the influence of Na+ on succinate transport was investigated, the buffers containing 140 mM NaCl or 140 mM N-methyl-D-glucamine (NMDG) chloride were mixed to give uptake buffers of desired Na+ composition. In most experiments, the time of incubation was 1 min. Endogenous transport was always determined in parallel using cells transfected with pSPORT vector alone.

Functional expression in Xenopus laevis oocytes. Capped cRNA from the cloned hNaDC3-cDNA was synthesized using the MEGAscript kit (Ambion) according to manufacturer's protocol. The cRNA was dissolved in sterile water and its integrity checked on denaturing formaldehyde-agarose gel. Mature oocytes from X. laevis were isolated by treatment with collagenase A (1.6 mg/ml), manually defolliculated, and maintained at 18°C in modified Barth's medium supplemented with 10 mg/l gentamicin (26). On the following day, oocytes were injected with 50 ng cRNA. Oocytes injected with water served as control. The oocytes were used for electrophysiological studies 6 days after cRNA injection. Electrophysiological studies were done by the conventional two-microelectrode voltage-clamp method (16-18). Oocytes were superfused with a NaCl-containing transport buffer (in mM: 100 NaCl, 2 KCl, 1 MgCl2, 1 CaCl2, 3 HEPES, 3 MES, and 3 Tris, pH 7.5) followed by the same buffer containing different substrates. The membrane potential was clamped at -50 mV. Voltage pulses between +50 and -150 mV, in 20-mV increments, were applied for 100-ms durations and steady-state currents measured. The differences between the steady-state currents measured in the presence and absence of substrates were considered the substrate-induced currents. Kinetic parameters for the saturable transport of the substrates were calculated using the Michaelis-Menten equation. Data were analyzed by nonlinear regression.

When the effects of Na+ on the kinetics of succinate uptake were evaluated, the oocyte was perifused with buffer containing different concentrations of Na+ and 0.5 mM succinate. The osmolality of the buffer and the concentration of Cl- in the buffer were kept constant by substituting NaCl with NMDG chloride. The Na+-to-succinate stoichiometry (i.e., the ratio of number of Na+ cotransported per molecule of succinate) was calculated by determining the Hill coefficient for the Na+-dependent activation of succinate-induced currents as well as by simultaneous measurements of succinate-evoked charge transfer and radiolabeled succinate uptake. The electrophysiological measurements were made wherein the oocytes were incubated for 10 min with 100 µM succinate containing traces of radiolabeled succinate. After the electrophysiological measurements, the oocytes were quickly washed with ice-cold transport buffer, solubilized in 10% SDS, and the amount of radiolabeled succinate that was transported into the oocytes during the period of electrophysiological measurements was determined by liquid scintillation counting. The charge transfer that occurred during the incubation period was calculated from the electrical measurements using the Nernst equation.

Construction of rat-human and human-rat NaDC3 chimeras. Rat NaDC3 (rNaDC3) cDNA and hNaDC3 cDNA have a single common site for Xmn I that lies at amino acid positions 230/231 in rNaDC3 and 232/233 in hNaDC3. Chimeric NaDC3 transporters were constructed by exchanging the two Xmn I fragments between rNaDC3 and hNaDC3 cDNAs. Because the pSPORT vector also has a site for Xmn I, the cDNAs were first linearized with a unique cutter (Hind III) present in the multiple cloning site at the 3'-end of the cDNA inserts, and the linearized plasmids were then subjected to partial digestion with Xmn I. Fragments of appropriate length were then isolated following electrophoretic separation and used for ligation. The chimera rat-human NaDC3 (rhNaDC3) contains the amino-terminal 230 amino acids from rNaDC3 and the carboxy-terminal 370 amino acids from hNaDC3. The chimera human-rat NaDC3 (hrNaDC3) contains the amino-terminal 232 amino acids from hNaDC3 and the carboxy-terminal 370 amino acids from rNaDC3.

Chromosomal localization of hNaDC3 gene. A human placental genomic library (Lambda FIX II; Stratagene, La Jolla, CA) was screened with an Aat II fragment (~0.6 kbp) of hNaDC3 cDNA, comprising the 3'-end of the hNaDC3 cDNA, as the probe. A single positive clone was identified and plaque purified. The size of the genomic insert was determined by digestion with Sal I followed by size-fractionation of the released insert on agarose gel.

The chromosomal localization of the human NaDC3 gene was done by fluorescent in situ hybridization (FISH). The entire phage DNA of the hNaDC3 genomic clone was labeled by nick translation with biotin-11-dUTP and used for hybridization to human metaphase chromosome spreads at a concentration of 50 ng/µl. FISH was carried out essentially as detailed earlier (29). The chromosomes were stained by the chromycin A3/distamycin A/4',6-diamidino-2-phenylindole technique for fluorescent microscopic analysis. A chromosome 20-specific centromere probe as well as the banding pattern were used to differentiate the p (short) and q (long) arms of chromosome 20.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Structural features of hNaDC3 cDNA. Screening of the human placental cDNA using an ~1.9-kbp fragment of the rNaDC3 cDNA as the probe led to the isolation of two positive clones. Restriction digestion using several enzymes indicated that both the clones were similar and differed only in the length of the cDNA insert. The longer of the two clones was chosen for further structural and functional characterization. The hNaDC3 cDNA is 3,481 bp long with an open reading frame of 1,809 bp (including the termination codon), encoding a protein of 602 amino acids. The open reading frame is flanked by a 5'-noncoding sequence of 19 bp long and a 1,653-bp-long 3'-noncoding sequence (GenBank accession no. AF 154121). The predicted molecular mass of the protein encoded by the open reading frame is 66.9 kDa. Hydrophobicity analysis of the predicted amino acid sequence using the algorithm of Kyte and Doolittle (15) with a transmembrane helix length between 17 and 33 amino acid residues per membrane-spanning domain indicated that the hNaDC3 protein contains 11-12 putative transmembrane domains. There are three putative N-linked glycosylation sites (Asn-312, Asn-586, and Asn-596), the first site between transmembrane domains 6 and 7 and the other two sites in the carboxy-terminal tail. When modeled such that the N-glycosylation sites face the extracellular side, both the amino terminus and the carboxy terminus are located on the extracellular side if the existence of 12 transmembrane domains is assumed. However, it has been shown in the case of the low-affinity Na+-dicarboxylate transporter NaDC1 that only the N-glycosylation site present in the carboxy terminus is actually glycosylated, even though this protein contains an additional putative glycosylation site between transmembrane domains 4 and 5 (24). Based on data obtained with epitope-specific antibodies, NaDC1 has been modeled to possess 11 transmembrane domains, with the carboxy terminus containing the actual N-glycosylation site placed on the extracellular side and the amino terminus placed on the intracellular side (24). It is likely that a similar situation exists in the case of hNaDC3. The deduced amino acid sequence also displays eight sites with consensus sequence for protein kinase C-dependent phosphorylation and one site with consensus sequence for cAMP- and cGMP-dependent protein kinase phosphorylation in putative intracellular domains. Amino acid sequence comparison of hNaDC3 against rNaDC3 shows a high degree of homology (85% identity and 91% similarity) (Fig. 1) between the two proteins, indicating that the protein is highly conserved across the species. In addition, hNaDC3 shares considerable homology (~45% identity) at the amino acid level with the previously cloned low-affinity dicarboxylate transporters. Another interesting aspect of the nucleotide sequence of hNaDC3 cDNA is that the nucleotide sequence between 744 and 2416 is 99% identical to the reverse complement of nucleotides 1-1674 of hepatocyte nuclear factor-4gamma (HNF4gamma ) cDNA (7). The significance of this identity is not known.


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Fig. 1.   Comparison of amino acid sequences of human and rat Na+-dependent high-affinity dicarboxylate transporter (hNaDC3 and rNaDC3, respectively).

To determine the tissue distribution of the hNaDC3 transcript, we performed Northern analysis using a commercially available multiple tissue RNA blot (Clontech, PaloAlto, CA). A primary transcript, ~3.6 kb in size, was detectable in placenta, brain, liver, kidney, and pancreas (data not shown). Of the tissues tested, the transcript was not detectable in heart, lung, and skeletal muscle. Among the positive tissues, the hNaDC3 transcript was especially abundant in kidney. A similar tissue distribution pattern of NaDC3 transcripts has been described in the rat (12).

Functional characterization of hNaDC3 following expression in HRPE cells. Because the cDNA was cloned in pSPORT vector with the cDNA insert directionally ligated such that the sense transcription is under the control of the T7 promoter in the plasmid, we employed the transient vaccinia virus expression system for the initial characterization of the clone. A time course study of succinate uptake in cDNA-transfected cells showed that succinate uptake was significantly higher in cells transfected with pSPORT-cDNA than in cells transfected with pSPORT alone (data not shown). The uptake was linear only within the initial 2.5 min, and consequently all the subsequent experiments were done with a 1-min incubation. At 1 min, the amount of succinate transported into cDNA-transfected cells was ~85-fold higher than the amount of succinate transported into cells transfected with vector alone. The ionic dependence of the cDNA-stimulated succinate transport was investigated by measuring succinate transport in the presence of various inorganic salts. Control transport was measured in the presence of NaCl. Replacement of Na+ in the transport buffer with other cations such as Li+, K+, and NMDG almost completely abolished the transport, indicating that Na+ is obligatory for the transport process (data not shown).

The substrate specificity of the dicarboxylate transport system induced by the clone was evaluated by assessing the ability of various unlabeled organic anions to inhibit the cDNA-induced transport of [3H]succinate (Table 1). The most effective inhibitors were succinate, fumarate, glutarate, and alpha -ketoglutarate. It is clear from these studies that dicarboxylates of four- and five-carbon chain length are the ideal substrates of hNaDC3. Malonate and oxalate, both dicarboxylates but with shorter carbon chain lengths, were not effective inhibitors of cDNA-induced [3H]succinate transport. Similarly, the monocarboxylates lactate, pyruvate, butyrate, and valerate were also ineffective. These findings demonstrate that both the number of anionic charge as well as the length of carbon chain play a critical role in influencing the substrate recognition. Comparison of the inhibitory potency of fumarate (a trans isomer) and maleate (a cis isomer) indicates that hNaDC3 is also able to distinguish between cis and trans isomers of unsaturated dicarboxylates. Presence of a hydroxyl group, as in malate, or a keto group, as in alpha -ketoglutarate, on the alpha -carbon atom does not abolish the interaction with the transporter. Similarly, substitution at the second and third carbon atoms in the succinate molecule with a methyl group (e.g., dimethylsuccinate) does not interfere with substrate recognition by hNaDC3. Dimethylsuccinate has been shown to be a specific substrate for the high-affinity Na+-dicarboxylate transporter in isolated renal basolateral membrane vesicles (32). The present studies confirm these findings with the cloned high-affinity NaDC3. Citrate, which is predominantly trivalent at pH 7.5, also inhibited the uptake by ~40%. Acidic amino acids, aspartate and glutatmate, inhibited the uptake of succinate significantly, the inhibition by aspartate being greater (~40%) than glutamate (~20%) when the concentration of the amino acids was 2 mM.

                              
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Table 1.   Substrate specificity of hNaDC3

The kinetics of hNaDC3-induced succinate transport was determined by measuring the rate of succinate uptake at varying concentrations of succinate (2.5-80 µM) in HRPE cells expressing the cloned cDNA. The data obtained were analyzed first by nonlinear regression (Fig. 2A) and confirmed by linear regression (Fig. 2A, inset). The transport process mediated by hNaDC3 was saturable with a Kt of 20.4 ± 1.4 µM for succinate. The effect of Na+ on the transport of succinate mediated by hNaDC3 was investigated by measuring the uptake of succinate in the presence of varying concentrations of extracellular Na+ (0-140 mM) in HRPE cells expressing hNaDC3. The relationship between uptake rate and Na+ concentration was sigmoidal (Fig. 2B), suggesting the involvement of more than one Na+ per succinate molecule transported. The data were fit to the Hill equation and the Hill coefficient was calculated. These values were confirmed from the slope of the Hill plot (Fig. 2B, inset). This analysis yielded a Hill coefficient value of 2.7 ± 0.1. This suggests that three Na+-binding sites are involved in the transport process.


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Fig. 2.   A: kinetics of succinate uptake induced by hNaDC3 cDNA in human retinal pigment epithelial (HRPE) cells. Transport measurements were made in HRPE cells transfected with hNaDC3 cDNA. Uptake was measured in the presence of NaCl at pH 7.5 with 1-min incubation at room temperature. Concentration of succinate was varied over a range of 2.5-80 µM while the concentration of [3H]succinate was kept constant at 50 nM. Values represent means ± SE for 4 determinations. Inset: Eadie-Hofstee transformation of the same data. B: effect of Na+ on the uptake of succinate in HRPE cells transfected with hNaDC3 cDNA. Uptake of [3H]succinate (30 nM) was studied in HRPE cells expressing hNaDC3 cDNA with 1-min incubation in the presence of increasing concentrations of Na+ (0-140 mM). Osmolality and concentration of Cl- (140 mM) in extracellular medium were kept constant by replacing NaCl with appropriate concentrations of NMDG chloride. Values are means ± SE of 4 determinations. Inset: Hill plot of the same data. v, Uptake rate in fmol/106 cells/min; Vm, maximal uptake rate calculated from the experimental data using the Hill equation; S, substrate.

Functional characterization of hNaDC3 following expression in X. laevis oocytes. Because the hNaDC3-mediated transport of succinate is rheogenic as evidenced from the Na+-to-succinate stoichiometry of 3:1, we further characterized the transport properties of hNaDC3 by electrophysiological means using the two-microelectrode, voltage-clamp technique following the expression of the cDNA in X. laevis oocytes. Perifusion of oocytes with transport buffer containing 0.5 mM succinate in the presence of Na+ induced inward currents (~0.6-0.75 µA) (data not shown). The inward currents were absent when NaCl in the transport buffer was substituted with NMDG chloride and also in control oocytes injected with water, showing that the succinate-induced current is dependent on the presence of extracellular Na+ and the expression of hNaDC3 (data not shown). The inward currents induced by succinate were also dependent on the testing membrane potential (Fig. 3A). The magnitude of the succinate-induced currents increased as the testing membrane potential became more hyperpolarized, demonstrating that the transport activity of hNaDC3 is stimulated when the membrane potential is made more negative. The ability of a number of other Krebs cycle intermediates and their analogs to induce inward currents was also tested (Fig. 3B). The concentration of substrates in the transport buffer was 0.5 mM. At this concentration, all the compounds, which inhibited the uptake of succinate in vaccinia virus-dependent expression studies, also induced inward currents in oocytes. The induced currents were quantitatively higher in magnitude than the currents induced by succinate. The only exception was citrate. However, when the concentration of citrate was increased to 5 mM, inward currents of ~50-75 nA were induced in oocytes expressing hNaDC3 (see below). Pyruvate, lactate, oxalate, malonate, and maleate did not induce inward currents and therefore are not substrates for hNaDC3.


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Fig. 3.   A: current-voltage (I-V) relationship for succinate-evoked inward currents. HNaDC3 was expressed in X. laevis oocytes by injection of hNaDC3 cRNA. Currents were measured at different holding membrane potentials in NaCl-containing buffer in the presence of 0.5 mM succinate. B: substrate specificity of hNaDC3 as determined by substrate-induced current in hNaDC3-expressing oocytes. Membrane potential was clamped at -50 mV and oocytes were perifused with potential substrates (0.5 mM) of hNaDC3. Substrate-induced currents were measured and expressed as percentage of inward currents evoked by succinate. DM-succinate, dimethylsuccinate; alpha -KG, alpha -ketoglutarate.

Figure 4 details the kinetic analyses of hNaDC3-induced transport of succinate and dimethylsuccinate. Substrate-generated inward currents were measured at different testing membrane potentials with increasing concentrations of succinate and dimethylsuccinate. The current increased in magnitude with increasing hyperpolarization of the membrane potential and also with increasing concentrations of succinate (Fig. 4A) and dimethylsuccinate (Fig. 4C). The substrate-induced currents showed saturation kinetics with respect to substrate concentration at different testing membrane potentials. The kinetic constants concentration of substrate necessary for half-maximal current (K0.5) and maximal current (Imax) for the transport of the substrates at different testing membrane potentials were individually calculated by nonlinear regression analyses. The kinetic constant K0.5 decreased gradually as the testing membrane potential became more hyperpolarized from 10 to -150 mV (Fig. 4, B and D). At a testing membrane potential of 10 mV, the K0.5 for succinate was 196 ± 25 µM, which decreased to 102 ± 20 µM at -50 mV. The corresponding values for dimethylsuccinate are 246 ± 38 µM at 10 mV and 112 ± 23 µM at -50 mV. This indicates that the affinity of the carrier for its substrates increased with increasing hyperpolarization of the membrane potential. Further hyperpolarization beyond -50 mV, however, did not influence the substrate affinity to any significant extent. The kinetic constant Imax, on the other hand, increased continuously as the testing membrane potential became more hyperpolarized (data not shown). For succinate, the Imax value increased almost twofold from 405 ± 14 to 750 ± 33 nA when the membrane potential was hyperpolarized from -50 to -150 mV, indicating that hNaDC3 is stimulated by an inside-negative membrane potential. Under similar conditions, the Imax value for dimethylsuccinate increased from 318 ± 19 to 508 ± 28 nA.


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Fig. 4.   Kinetic analyses of succinate- and dimethylsuccinate (DMS)-evoked inward currents in X. laevis oocytes expressing hNaDC3. Oocytes, injected with hNaDC3 cRNA, were perifused with increasing concentrations of succinate or DMS in NaCl-containing buffer. Membrane potential was clamped at -50 mV and substrate-induced currents were measured at different testing membrane potentials. A: influence of succinate concentration on succinate-induced currents at various testing membrane potentials. Concentration of succinate was varied from 5 to 500 µM. B: influence of testing membrane potential on KSuc0.5. Testing membrane potential was varied from -150 to + 30 mV with 20-mV increments. The KSuc0.5 values were calculated by fitting the data from substrate saturation studies to a Michaelis-Menten equation describing a single saturable system. C: influence of DMS concentration on DMS-induced currents at various testing membrane potentials. Concentration of DMS was varied from 5 to 200 µM. D: influence of testing membrane potential on KDMS0.5. The KDMS0.5 values were calculated as described previously for succinate.

The relationship between extracellular Na+ concentration and substrate-evoked currents was determined at various testing membrane potentials. The concentration of Na+ in the transport buffer was varied from 0 to 60 mM, and the inward currents induced by succinate (0.5 mM) were measured. The testing membrane potential was changed from -10 to -150 mV in steps of 20 mV. The data obtained indicate that the relationship between the succinate-induced currents and extracellular Na+ concentration is sigmoidal at all testing membrane potentials (Fig. 5A). Also, the substrate-induced currents increased with increasing hyperpolarization. The data were fit into the Hill equation and the value of the Hill coefficient (nH), the number of binding sites for Na+, was calculated. This value was >2 (~2.4 at -50 mV) at all testing membrane potentials (Fig. 5B), indicating participation of more than two Na+-binding sites in the Na+-dependent activation of succinate transport. We then determined the charge-to-uptake ratio by simultaneously measuring the substrate-dependent charge transfer and the amount of substrate transported for a specific length of time (8-10 min) (Fig. 5, C and D). The amount of substrate transported during this period was determined by including radiolabeled substrate of known specific activity during the incubation. Data obtained indicate that the charge-to-substrate ratio for succinate is 1.1, indicating that, for every succinate molecule transported, there is a net transfer of one positive charge into the cells. Because succinate is divalent at pH 7.5, transport of every succinate molecule should therefore be accompanied with the cotransport of 3 Na+. Thus, even though the Hill coefficient calculated from the Na+ activation kinetics refers to the number of Na+ binding sites that may or may not be involved in actual translocation of Na+, the data on charge-to-substrate ratio for succinate indicate that each of the Na+ binding sites predicted from the Hill coefficient is indeed associated with Na+ transport.


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Fig. 5.   Influence of Na+ on succinate-induced currents in hNaDC3-expressing oocytes and determination of charge-to-succinate stoichiometry using simultaneous measurements of succinate-induced currents and succinate uptake. A: influence of Na+ concentration on succinate-induced currents at different testing membrane potentials. Succinate-induced currents were measured at different concentrations of extracellular Na+ (1.25-60 mM) and at varying extent of membrane polarization (-10 to -150 mV with 20-mV increments). Concentration of succinate used was 0.5 mM. B: influence of testing membrane potential (Vtest) on Hill coefficient nH. The nH values were calculated by fitting the data from the Na+ activation kinetics to a Hill equation. Inset: Hill plot at -50 mV testing membrane potential. I, inward current; Imax, maximal inward current calculated by fitting the data to Hill equation. C: representative tracing of succinate-induced current used in determination of charge-to-substrate stoichiometry. The oocyte was perifused with succinate (100 µM) plus traces of radiolabeled succinate, and inward currents were measured over a period of 8-10 min. At the end of the experiment, the amount of succinate transported into the oocyte was calculated by measuring the radioactivity associated with the oocyte. The area within the curve was integrated to calculate the charge transferred during incubation with succinate. D: comparison of succinate transfer and charge transfer. Data are from 6 different oocytes.

The effect of pH on substrate-induced currents was investigated using succinate and citrate as substrates in voltage-clamped, hNaDC3-expressing X. laevis oocytes. Succinate, with its pKa2 of 5.6, exists predominantly as a divalent anion when the pH of the solution is in the range of 6.5-7.5. In contrast, citrate is predominantly trivalent at pH 7.5, but the divalent species increases considerably as the pH is lowered to 6.5 (pKa3 for citrate = 6.4). As seen in Fig. 6A, the succinate (0.5 mM)-induced current decreased significantly when the pH was changed from 7.5 to 6.5 (~300 vs. ~175 nA). Because the concentration of the divalent form of succinate changes only minimally under these conditions, the data suggest that hNaDC3 is inhibited by lower pH. However, when citrate (5 mM) was used as the substrate, the substrate-induced current increased severalfold when the pH was changed from 7.5 to 6.5 (~40 vs. ~180 nA; Fig. 6B). This increase in citrate-induced current is likely to be predominantly due to the increased concentration of the divalent form of citrate. These data suggest that the divalent forms of succinate and citrate are the preferred substrates of hNaDC3.


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Fig. 6.   Effects of pH on succinate- and citrate-induced currents in hNaDC3-expressing oocytes. Oocytes, injected with hNaDC3 cRNA, were perifused with either 0.5 mM succinate (A) or 5.0 mM citrate (B) in Na+-containing buffer at pH 7.5 or 6.5. Membrane potential was clamped at -50 mV. Representative chart recording obtained with a single oocyte is shown.

With rNaDC3, it has been observed that, in the absence of Na+, Li+ is able to substitute for Na+ to a limited extent and that, in the presence of Na+, Li+ is able to inhibit dicarboxylate transport at very low concentrations. The inhibition reached a plateau when the concentration of Li+ was 2.5 mM. Increasing the concentration of Li+ beyond 2.5 mM up to 40 mM had no significant effect on the transport. In hNaDC3-expressing oocytes, Li+ induced detectable inward currents when succinate was added to the transport buffer in the absence of Na+ (data not shown). This indicates that, as in the case of rNaDC3, Li+ can substitute for Na+ to a limited extent in the case of hNaDC3. However, in the presence of saturating concentrations of Na+ (70 mM), Li+ inhibited succinate-induced currents in hNaDC3-expressing oocytes. The inhibition increased with increasing concentrations of Li+ and was evident at all testing membrane potentials (Fig. 7A). The inhibition of the substrate-induced current was saturable with respect to Li+ and followed Michalis-Menten kinetics (Fig. 7B). The apparent inhibition constant (Ki) for Li+ was also membrane potential dependent, the value of which increased with increasing hyperpolarization of the membrane potential. At a membrane potential of -50 mV, the Ki value was 2.6 ± 0.1 mM. The value increased to 5.0 ± 0.9 at -150 mV, indicating that the inhibition of substrate-induced currents by Li+ decreases with increasing hyperpolarization of the membrane potential. The inhibition data obtained with Li+ were also fit into the Hill equation, and the Hill coefficient for the inhibition of succinate-induced currents by Li+ was calculated. The value obtained was ~1, suggesting that Li+ interacts with one of the three Na+ binding sites to inhibit the transport of dicarboxylates by hNaDC3.


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Fig. 7.   Effect of Li+ on succinate-evoked currents in the presence of Na+. Succinate-induced currents were measured in X. laevis oocytes expressing hNaDC3 in the presence of different concentrations of Li+ (0-30 mM). Concentrations of succinate and Na+ were kept constant at 500 µM and 70 mM, respectively. Osmolarity of the buffer was kept constant by substituting LiCl with choline chloride. A: I-V relationship for succinate-induced currents at varying concentrations of Li+. B: inhibition of succinate-induced currents at varying concentrations of Li+ at -50, -90, and -130 mV testing membrane potentials.

Structure-function studies using human and rat NaDC3 chimeras. Although both rNaDC3 and hNaDC3 are high-affinity dicarboxylate transporters, the affinity of the two proteins for succinate differs by an order of magnitude when measured under identical experimental conditions. While rNaDC3 has an affinity of ~2 µM for succinate when analyzed in the mammalian cell expression system with HRPE cells, the corresponding value for hNaDC3 is ~20 µM under similar conditions. Comparison of the amino acid sequences of rNaDC3 and hNaDC3 shows that the majority of the differences between these two sequences are clustered in two groups, one between amino acids 164-220 and another between amino acids 300-330 (Fig. 1). Therefore, the region of the transporter protein involved in substrate recognition should be located in one of these two stretches of amino acids. To determine which of these two regions is actually involved in substrate recognition, we constructed chimeric proteins in which these two regions of hNaDC3 and rNaDC3 were exchanged and determined the affinity for succinate of the resulting chimeric proteins. One of the chimeras, called hrNaDC3, has the amino-terminal 232 amino acids from hNaDC3 and the carboxy-terminal 370 amino acids from rNaDC3. The second chimera, called rhNaDC3, has the amino-terminal 230 amino acids from rNaDC3 and the carboxy-terminal 370 amino acids from hNaDC3. Kinetic analysis was performed with [3H]succinate as the substrate following the transient expression of the cDNAs in HRPE cells (Fig. 8). There were significant differences in the expression levels of different cDNAs and therefore the maximum velocity values were normalized in kinetic analysis. The Kt value obtained for the rhNaDC3 chimera (19 ± 1 µM) was comparable with the Kt value obtained for hNaDC3 (14 ± 1 µM). Similarly, the Kt value of the hrNaDC3 chimera (2.2 ± 0.2 µM) was similar to the Kt value of rNaDC3 (3.0 ± 0.3 µM). Therefore, the carboxy-terminal half of the protein determines the substrate affinity, indicating that the substrate binding site is located in this region.


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Fig. 8.   Kinetic analyses of human-rat and rat-human NaDC3 chimeras (hrNaDC3 and rhNaDC3, respectively) in HRPE cells. hNaDC3, rNaDC3, hrNaDC3, and rhNaDC3 cDNAs were expressed in HRPE cells by vaccinia virus-dependent expression system. Uptake of succinate was measured in the presence of NaCl at pH 7.5 with 1-min incubation at room temperature. Concentration of succinate was varied over a range of 5-80 µM for hNaDC3 (black-down-triangle ) and rhNaDC3 (down-triangle) cDNAs and over 1-15 µM for rNaDC3 () and hrNaDC3 (open circle ) cDNAs. Concentration of [3H]succinate was kept constant at 50 nM. Eadie-Hofstee plots are shown with Vmax values normalized to 100% for each cDNA to adjust for variations in expression levels.

Chromosomal localization of hNaDC3. An hNaDC3-specific genomic clone was first isolated from a human placental genomic library. Digestion of the positive clone with Sal I released the full-length insert of ~15 kbp. Regional localization of the hNaDC3 gene was carried out by FISH. The full-length genomic clone labeled with biotin was used as the probe to hybridize human metaphase chromosomes. The hybridization signal was found to be localized on chromosome 20, band q12-13.1 (data not shown). A chromosome 20-specific centromere probe was used together with the hNaDC3-specific genomic probe to establish the identity of the chromosome. A blast search of the High Throughput Genomic Sequences database using the nucleotide sequence of hNaDC3 revealed that the human gene for hNaDC3 has already been cloned (accession no. AL034424). The chromosomal location of this gene corroborated the results obtained by FISH in the present study. A comparison of the nucleotide sequences of the genomic sequence entry and hNaDC3 cDNA indicated that the human NaDC3 gene is >80 kbp long and that the genomic sequence entry is incomplete with the first 129 bases at the 5' end of the cDNA not being represented in the genomic clone. The sequence comparison has enabled us to deduce the exon-intron organization of the gene (Fig. 9). The gene consists of 13 exons and 12 introns. We have confirmed by PCR using human genomic DNA as the template that the 129 bp at the 5' end of the cDNA constitute the first exon. In the PCR analysis, the upper primer constituted the first 21 bases of the 5' end of the cDNA and the lower primer constituted the reverse complement of the last 19 nucleotides of the first exon. On PCR, a 129-bp fragment was amplified, the identity of which was confirmed by sequencing.


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Fig. 9.   Exon-intron organization of the human NaDC3 gene and its organizational relationship to hNaDC3 cDNA. Black boxes in the gene represent the protein coding regions of the exons and shaded boxes represent 5' noncoding region in exon 1, and 3' noncoding region in exon 13. Numbers in cDNA represent the position of the first nucleotide of each exon.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have reported here on the cloning and functional characterization of the human high-affinity Na+-dependent dicarboxylate transporter. Functional characterization of hNaDC3 was done in this study using two different expression systems, a vaccinia virus-dependent mammalian expression system as well as the X. laevis oocyte expression system. The affinity of the transporter for succinate, as determined using the two expression systems, varied significantly. The Kt value was ~20 µM in the mammalian cell expression system. The corresponding value was about fivefold higher (~ 100 µM at -50 mV) in the X. laevis oocyte expression system. This difference is most likely related to the differences in the experimental conditions employed in these two techniques. For example, the membrane potential is not clamped at any particular value in mammalian cells during uptake measurements, and, in addition, the membrane potential is not expected to stay constant in this experimental system during the time of uptake measurement due to transport-associated depolarization of the membrane potential. In contrast, the membrane potential is clamped at one particular value (-50 mV), which stays constant during the entire period of transport measurement in the X. laevis oocyte expression system. The time period used in uptake measurements may also be a contributing factor. The electrophysiological measurements using the X. laevis expression system use considerably shorter time periods than the 1-min period used in the mammalian cell expression system. The rat high-affinity Na+-dicarboxylate transporter behaves in a similar way. The Kt for succinate for this transporter is ~2 µM in the mammalian expression system (12) and ~15 µM in the X. laevis oocyte expression system (6). Even though the hNaDC3 exhibits a Kt for succinate of 20-100 µM depending on the experimental system, it is still a high-affinity transport system compared with hNaDC1. The X. laevis oocyte expression system has yielded a Kt value of ~400 µM for hNaDC1 (22).

Heterologous expression studies carried out suggest that the Na+-to-succinate stoichiometry of hNaDC3 is 3:1, indicating that the transport process is electrogenic and that the transport process is energized by the transmembrane Na+ gradient as well as the potential difference across the membrane. Electrophysiological studies involving the measurement of substrate-evoked inward currents conclusively demonstrate that the transport process is indeed electrogenic. Similar to rNaDC3, hNaDC3 has a higher affinity for dicarboxylates of 4- and 5-carbon chain length and accepts dimethylsuccinate as a substrate. The effect of pH on citrate transport under voltage-clamped conditions clearly demonstrates that dianionic citrate is the transportable form of citrate. There is very little citrate-induced current observed at pH 7.5, the pH at which most of the citrate is in the trianionic form. At this pH, the succinate-induced current is severalfold higher. When the pH is decreased to 6.5, succinate-induced current decreases significantly, but citrate-induced current increases, since the amount of dianionic form of citrate is greater than the trianionic form at this pH. hNaDC3 behaves very similar to rNaDC3 with respect to the influence of Li+ on succinate transport. In the absence of Na+, Li+ induces inward currents at very low levels in the presence of succinate. In the presence of Na+, Li+ inhibits succinate-induced currents, indicating that Li+ interacts with one of the Na+ binding sites to bring about the inhibition of succinate transport. It is of interest to note the significant difference in the potency of Li+ inhibition between hNaDC1 and hNaDC3. hNaDC1, the low-affinity Na+-dicarboxylate transporter, is not very sensitive to inhibition by Li+, with a Ki value of ~10 mM (22). In contrast, the human high-affinity Na+-dicarboxylate transporter NaDC3 is relatively much more sensitive to inhibition by Li+, with a Ki value of ~2.5 mM. Human patients who are treated with Li+ for therapeutic reasons excrete Krebs cycle intermediates in urine at increased levels compared with controls (3). It is possible that the inhibition of the basolateral high-affinity transporter rather than the inhibition of the brush-border low-affinity transporter is responsible for the increased urinary excretion of Krebs cycle intermediates in these patients.

The affinity of hNaDC3 for succinate is ~20 µM, which is closer to rNaDC1 (~25 µM), the low-affinity dicarboxylate transporter (5) than rNaDC3 (~2 µM), the high-affinity dicarboxylate transporter (6, 12). At the amino acid level, hNaDC3 has a higher homology to rNaDC3 than rNaDC1. Kinetic analysis of human and rat NaDC3 chimeras clearly indicates that the region of transporter protein involved in substrate recognition is located in the carboxy-terminal part of the protein. The hrNaDC3 chimera with amino-terminal 232 amino acids from hNaDC3 and carboxy-terminal 370 amino acids from rNaDC3 has an affinity for succinate that is comparable with that of rNaDC3. Similarly, the rhNaDC3 chimera with amino-terminal 230 amino acids from rNaDC3 and 370 carboxy-terminal amino acids from hNaDC3 has an affinity for succinate comparable with that of hNaDC3.

An interesting aspect of the hNaDC3 cDNA is that the nucleotide sequence between 744 and 2416 is 99% identical to the reverse complement of nucleotides 1-1674 of HNF4gamma cDNA (7). HNF4gamma is an orphan member of the nuclear receptor superfamily (30) and is believed to play an important role in early embryogenesis (4). The transcripts of HNF4gamma (5.5 and 4.1 kb in size) are detected in kidney, pancreas, small intestine, colon, and testis. Because the nucleotide sequence of hNaDC3 and HNF4gamma are reverse complementary to each other, one would expect that the two proteins are coded by the two opposing strands of DNA. However, this is not the case. The gene for HNF4gamma has been localized to human chromosome 8, whereas the gene for NaDC3 is located on chromosome 20. Thus hNaDC3 and HNF4gamma are products of genes present on different chromosomes but have considerable stretch of their nucleotide sequence reverse complementary to each other. This would have potentially very interesting consequences with respect to when and where these two genes are expressed. Because hNaDC3 mRNA would be the antisense mRNA of HNF4gamma mRNA to a considerable extent, coexpression of both mRNAs in the same tissue at the same time may have potentially serious consequences. Interestingly, three other known isoforms of HNF4 have been localized to chromosome 20 (14); however, none of them show any sequence similarity at the nucleotide level either to hNaDC3 cDNA or the human NaDC3 gene.

The structural and functional characteristics of hNaDC3 and the organization of this transporter gene reported here may be of clinical relevance. Renal handling of citrate is an important factor associated with nephrolithiasis. Precipitation of calcium salts in the tubular lumen is effectively prevented by maintaining high levels of citrate in the luminal fluid. It has been shown that kidney tubular cells absorb citrate and dicarboxylates across the brush-border membrane from the filtrate as well as across the basolateral membrane from the blood (9). The low-affinity Na+-dicarboxylate transporter NaDC1 and the high-affinity Na+-dicarboxylate transporter NaDC3 are responsible for the citrate transport process in the brush-border membrane and basolateral membrane, respectively. Even though NaDC1 is expected to play a critical role in determining urinary levels of citrate due to its brush-border membrane localization, the basolateral citrate/dicarboxylate transport via NaDC3 also has the potential to modulate the luminal absorption of citrate. Systemic acid/base balance is an important regulatory factor in urinary citrate excretion. Changes in basolateral absorption of citrate and dicarboxylates via NaDC3 may affect cellular metabolism in tubular cells and consequently affect intracellular pH. Such changes in acid/base balance will have profound influence on luminal citrate absorption and hence on urinary citrate levels. Therefore, genetic alterations in NaDC3 function may be of potential relevance to the molecular basis of pathogenesis of idiopathic nephrolithiasis. The structure of the NaDC3 gene, mRNA, and protein reported here will be useful in future studies involving screening of patients with idiopathic nephrolithiasis for genetic changes in NaDC3 function.


    ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grants HD-33347 and DA-10045.


    FOOTNOTES

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.

Address for reprint requests and other correspondence: V. Ganapathy, Dept. of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912-2100 (E-mail: vganapat{at}mail.mcg.edu).

Received 11 November 1999; accepted in final form 17 December 1999.


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