Characterization of a Rat Na+-Dicarboxylate Cotransporter

Abstract

The metabolism of Krebs cycle intermediates is of fundamental importance for eukaryotic cells. In the kidney, these intermediates are transported actively into epithelial cells. Because citrate is a potent inhibitor for calcium stone formation, excessive uptake results in nephrolithiasis due to hypocitraturia. We report the cloning and characterization of a rat kidney dicarboxylate transporter (SDCT1). In situ hybridization revealed that SDCT1 mRNA is localized in S3 segments of kidney proximal tubules and in enterocytes lining the intestinal villi. Signals were also detected in lung bronchioli, the epididymis, and liver. When expressed in Xenopus oocytes, SDCT1 mediated electrogenic, sodium-dependent transport of most Krebs cycle intermediates (Km = 20-60 microM), including citrate, succinate, alpha-ketoglutarate, and oxaloacetate. Of note, the acidic amino acids L- and D-glutamate and aspartate were also transported, although with lower affinity (Km = 2-18 mM). Transport of citrate was pH-sensitive. At pH 7.5, the Km for citrate was high (0.64 mM), whereas at pH 5.5, the Km was low (57 microM). This is consistent with the concept that the -2 form of citrate is the transported species. In addition, maximal currents at pH 5.5 were 70% higher than those at pH 7.5, and our data show that the -3 form acts as a competitive inhibitor. Simultaneous measurements of substrate-evoked currents and tracer uptakes under voltage-clamp condition, as well as a thermodynamic approach, gave a Na+ to citrate or a Na+ to succinate stoichiometry of 3 to 1. SDCT1-mediated currents were inhibited by phloretin. This plant glycoside also inhibited the SDCT1-specific sodium leak in the absence of substrate, indicating that at least one Na+ binds to the transporter before the substrate. The data presented provide new insights into the biophysical characteristics and physiological implications of a cloned dicarboxylate transporter.