Krebs cycle intermediates such as succinate, citrate, and alpha-ketoglutarate are transferred across plasma membranes of cells by secondary active transporters that couple the downhill movement of sodium to the concentrative uptake of substrate. Several transporters have been identified in isolated membrane vesicles and cells based on their functional properties, suggesting the existence of at least three or more Na+/dicarboxylate cotransporter proteins in a given species. Recently, several cDNAs, called NaDC-1, coding for the low-affinity Na+/dicarboxylate cotransporters have been isolated from rabbit, human, and rat kidney. The Na+/dicarboxylate cotransporters are part of a distinct gene family that includes the renal and intestinal Na+/sulfate cotransporters. Other members of this family include a Na(+)- and Li(+)-dependent dicarboxylate transporter from Xenopus intestine and a putative Na+/dicarboxylate cotransporter from rat intestine. The current model of secondary structure in NaDC-1 contains 11 transmembrane domains and an extracellular N-glycosylated carboxy terminus.

The cDNA coding for a rabbit renal Na+/dicarboxylate cotransporter, designated NaDC-1, was isolated by functional expression in Xenopus oocytes. NaDC-1 cDNA is approximately 2.3 kilobases in length and codes for a protein of 593 amino acids. NaDC-1 protein contains eight putative transmembrane domains, and the sequence and secondary structure are related to the renal Na+/sulfate transporter, NaSi-1. Northern analysis shows that the NaDC-1 message is abundant in kidney and small intestine, and related transporters may be found in liver, lung, and adrenal. The transport of succinate by NaDC-1 was sodium-dependent, sensitive to inhibition by lithium, and inhibited by a range of di- and tricarboxylic acids. This transporter also carries citrate, but it does not transport lactate. In kinetic experiments, the Km for succinate was around 0.4 mM and the Vmax was 15 nmol/oocyte/h, while the Hill coefficient of Na+ activation of succinate transport was 1.9. The transport of succinate by NaDC-1 was insensitive to changes in pH, whereas the transport of citrate increased with decreasing pH, in parallel with the concentration of divalent citrate in the medium. The results of the functional characterization indicate that NaDC-1 likely corresponds to the renal brush-border Na+/dicarboxylate cotransporter.

We have cloned a Na ؉ -dependent, high affinity dicarboxylate transporter (NaDC3) from rat placenta. NaDC3 exhibits 48% identity in amino acid sequence with rat NaDC1, a Na ؉ -dependent, low affinity dicarboxylate transporter. NaDC3-specific mRNA is detectable in kidney, brain, liver, and placenta. When expressed in mammalian cells, NaDC3 mediates Na ؉ -dependent transport of succinate with a K t of 2 M. The transport function of NaDC3 shows a sigmoidal relationship with regard to Na ؉ concentration, with a Hill coefficient of 2.7. NaDC3 accepts a number of dicarboxylates including dimethylsuccinate as substrates and excludes monocarboxylates. Li ؉ inhibits NaDC3 in the presence of Na ؉ . Transport of succinate by NaDC3 is markedly influenced by pH, the transport function gradually decreasing when pH is acidified from 8.0 to 5.5. In contrast, the influence of pH on NaDC3-mediated transport of citrate is biphasic in which a pH change from 8.0 to 6.5 stimulates the transport and any further acidification inhibits the transport. In addition, the potency of citrate to compete with NaDC3-mediated transport of succinate increases 25-fold when pH is changed from 7.5 to 5.5. These data show that NaDC3 interacts preferentially with the divalent anionic species of citrate. This represents the first report on the cloning and functional characterization of a mammalian Na ؉

Mutations in the SLC13A5 gene that codes for the Na + /citrate cotransporter, NaCT, are associated with early onset epilepsy, developmental delay and tooth dysplasia in children. In this study, we identify additional SLC13A5 mutations in nine epilepsy patients from six families. To better characterize the syndrome, families with affected children answered questions about the scope of illness and the treatment strategies. Currently, there are no effective treatments, but some antiepileptic drugs targeting the γ-aminobutyric acid system reduce seizure frequency. Acetazolamide, a carbonic anhydrase inhibitor and atypical antiseizure medication, decreases seizures in four patients. In contrast to previous reports, the ketogenic diet and fasting resulted in worsening of symptoms. The effects of the mutations on NaCT transport function and protein expression were examined by transient transfections of COS-7 cells. There was no transport activity from any of the mutant transporters, although some of the mutant transporter proteins were present on the plasma membrane. The structural model of NaCT suggests that these mutations can affect helix packing or substrate binding. We tested various treatments, including chemical chaperones and low temperatures, but none improved transport function in the NaCT mutants. Interestingly, coexpression of NaCT and the mutants results in decreased protein expression and activity of the wild-type transporter, indicating functional interaction. In conclusion, this study has identified additional SLC13A5 mutations in patients with chronic epilepsy starting in the neonatal period, with the mutations producing inactive Na + /citrate transporters.online address: http://www.molmed.org

The SLC13 family in humans and other mammals consists of sodium-coupled transporters for anionic substrates: three transporters for dicarboxylates/citrate and two transporters for sulfate. This review will focus on the di- and tricarboxylate transporters: NaDC1 (SLC13A2), NaDC3 (SLC13A3), and NaCT (SLC13A5). The substrates of these transporters are metabolic intermediates of the citric acid cycle, including citrate, succinate, and α-ketoglutarate, which can exert signaling effects through specific receptors or can affect metabolic enzymes directly. The SLC13 transporters are important for regulating plasma, urinary and tissue levels of these metabolites. NaDC1, primarily found on the apical membranes of renal proximal tubule and small intestinal cells, is involved in regulating urinary levels of citrate and plays a role in kidney stone development. NaDC3 has a wider tissue distribution and high substrate affinity compared with NaDC1. NaDC3 participates in drug and xenobiotic excretion through interactions with organic anion transporters. NaCT is primarily a citrate transporter located in the liver and brain, and its activity may regulate metabolic processes. The recent crystal structure of the Vibrio cholerae homolog, VcINDY, provides a new framework for understanding the mechanism of transport in this family. This review summarizes current knowledge of the structure, function, and regulation of the di- and tricarboxylate transporters of the SLC13 family.

The two-electrode voltage clamp was used to study the currents associated with transport of succinate by the cloned Na ؉ /dicarboxylate cotransporter, NaDC-1, expressed in Xenopus oocytes. The presence of succinate induced inward currents which were dependent on the concentrations of succinate and sodium, and on the membrane potential. At ؊50 mV, the K 0.5 succinate was 180 M and the K 0.5 Na؉ was 19 mM. The Hill coefficient was 2.3, which is consistent with a transport stoichiometry of 3 Na ؉ :1 divalent anion substrate. Currents were induced in NaDC-1 by a range of di-and tricarboxylates, including citrate, methylsuccinate, fumarate, and tricarballylate. Although Na ؉ is the preferred cation, Li ؉ was also able to support transport. The K 0.5 succinate was approximately 10-fold higher in Li ؉ compared with Na ؉ . In the presence of Na ؉ , however, Li ؉ was a potent inhibitor of transport. Millimolar concentrations of Li ؉ resulted in decreases in apparent succinate affinity and in the I max succinate . Furthermore, lithium inhibition under saturating sodium concentrations showed hyperbolic kinetics, suggesting that one of the three cation binding sites in NaDC-1 has a higher affinity for Li ؉ than Na ؉ . We conclude that NaDC-1 is an electrogenic anion transporter that accepts either Na ؉ or Li ؉ as coupling cations. However, NaDC-1 contains a single high affinity binding site for Li ؉ that, when occupied, results in transport inhibition, which may account for its potent inhibitory effects on renal dicarboxylate transport.The active transport of Krebs cycle intermediates, such as succinate and citrate, is mediated by a specific sodium-coupled transporter found on the apical membrane in epithelial cells of the kidney proximal tubule (1, 2). The Na ϩ -dicarboxylate cotransporter reabsorbs a wide range of di-and tricarboxylic acids in the form of divalent anions. This transporter is sensitive to inhibition by lithium (3), and patients receiving therapeutic doses of lithium exhibit increased renal excretion of ␣-ketoglutarate and glutarate (4). The cDNA coding for the rabbit renal Na ϩ /dicarboxylate cotransporter, NaDC-1, 1 has been cloned and sequenced (5), and the protein has been identified in renal brush border membranes (6). NaDC-1 belongs to a distinct gene family of sodium-coupled anion transporters that includes the Na ϩ /dicarboxylate cotransporters, hNaDC-1, from human kidney (7), and NaDC-2, from Xenopus intestine (8), and the renal Na ϩ /sulfate cotransporter, NaSi-1 (9). The transport mechanism of NaDC-1 is thought to involve the ordered binding of four charged substrates: 3 Na ϩ ions and 1 divalent anion substrate (10 -12), resulting in one net inward positive charge across the membrane per cycle. Experiments with rabbit renal brush border membrane vesicles support this hypothesis: sodium-dependent transport of succinate was affected by changes in membrane potential, and transport of succinate also caused a depolarization of membrane potential (12-14). However, the dependence of transport kinetics o...

The SLC13 gene family consists of five members in humans, with corresponding orthologs from different vertebrate species. All five genes code for sodium-coupled transporters that are found on the plasma membrane. Two of the transporters, NaS1 and NaS2, carry substrates such as sulfate, selenate and thiosulfate. The other members of the family (NaDC1, NaDC3, and NaCT) are transporters for di- and tri-carboxylates including succinate, citrate and alpha-ketoglutarate. The SLC13 transporters from vertebrates are electrogenic and they produce inward currents in the presence of sodium and substrate. Substrate-independent leak currents have also been described. Structure-function studies have identified the carboxy terminal half of these proteins as the most important for determining function. Transmembrane helices 9 and 10 may form part of the substrate permeation pathway and participate in conformational changes during the transport cycle. This review also discusses new members of the SLC13 superfamily that exhibit both sodium-dependent and sodium-independent transport mechanisms. The Indy protein from Drosophila, involved in determining lifespan, and the plant vacuolar malate transporter are both sodium-independent dicarboxylate transporters, possibly acting as exchangers. The purpose of this review is to provide an update on new advances in this gene family, particularly on structure-function studies and new members of the family.

The rabbit and human Na(+)-dicarboxylate cotransporters, NaDC-1 and hNaDC-1, were expressed in Xenopus oocytes, and the transport of succinate, citrate, and glutarate was compared. Both transporters had similar affinities for succinate and glutarate, with Michaelis-Menten constant (K(m)) values of approximately 0.5- 0.8 mM (succinate) and 6-7 mM (glutarate), verifying that they are low-affinity sodium-dependent dicarboxylate transporters. The two transporters differed in their handling of citrate. At pH 7.5, the K(m) value for citrate was 0.9 mM in the rabbit NaDC-1 and 7 mM in the human hNaDC-1. However, the human transporter was more sensitive to pH than the rabbit. At pH 5.5, the K(m) value for citrate decreased to 1.2 mM in hNaDC-1 and decreased to 0.3 mM in the rabbit transporter. Both transporters had Hill coefficients between 1.6 and 2.1, suggesting that multiple sodium ions are coupled to the transport of divalent anions. However, the human transporter, hNaDC-1, had a lower apparent affinity for sodium (KNa, 78 mM) than the rabbit transporter (KNa, 41 mM). In addition, the human hNaDC-1 was relatively insensitive to inhibition by lithium, furosemide, and flufenamate compared with the rabbit NaDC-1. The differences between the human and rabbit transporters may account for observed differences in renal handling of citrate between species.