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