Threonine-509 is a determinant of apparent affinity for both substrate and cations in the human Na⁺/dicarboxylate cotransporter

The Na⁺/dicarboxylate cotransporter (NaDCl) is involved in the absorption of citric acid cycle intermediates from the lumen of the renal proximal tubule and small intestine. The NaDCl orthologues from human (h) and rabbit (rb) exhibit differences in citrate and cation transport properties. The citrate K_m and sodium K_Na values are much larger in human than rabbit NaDCl. Our previous study showed that transmembrane helices (TM) 7, 10, and 11 and associated loop regions contain the amino acids that are important in determining the differences in apparent citrate affinity, whereas TM10 and 11 determine differences in apparent sodium affinity. Chimera R10 (hNaDCl with a substitution of TM10 and associated loop from rbNaDCl) contains only four amino acid differences between rb and hNaDCl. This chimera has similar apparent affinity for succinate and sodium as the wild-type rbNaDCl, and an intermediate K_m for citrate. To identify individual residues in the TM10 region that determine functional differences between rb and hNaDCl, four mutants were made in which the rabbit sequence was substituted for that of the hNaDCl. Mutants with a serine or threonine at position 509 (or 512 in rbNaDCl) in TM10 have partial changes in K_m for citrate and succinate but larger changes in apparent affinity for cations and substrate specificity for four-carbon dicarboxylates. The results show that the serine or threonine at position 509 (h) or 512 (rb) is the most important determinant of functional differences in apparent affinity for substrate and cations. Furthermore, the results suggest that the cation and substrate binding sites are located in close proximity to one another in NaDCl.