Determinants of substrate and cation affinities in the Na+/dicarboxylate cotransporter

The Na+/dicarboxylate cotransporter (NaDC-1) couples the transport of sodiu m and tricarboxylic acid cycle intermediates, such as succinate and citrate . The rabbit and human homologues (rbNaDC-1 and hNaDC-1, respectively) are 78% identical in amino acid sequence but exhibit several differences in the ir functional properties, rbNaDC-1 has a greater apparent affinity for citr ate and sodium than hNaDC-1. Furthermore, unlike hNaDC-1, rbNaDC-1 is inhib ited by low concentrations of lithium. In this study, chimeric transporters were constructed to identify the protein domains responsible for the funct ional differences between rbNaDC-1 and hNaDC-1. Individual substitutions of transmembrane domain (TMD) 7, 10 or 11 produced transporters with intermed iate properties. However, substitution of TMD 7, 10, and 11 together result ed in a transporter with the citrate K-m of the donor, suggesting that inte ractions between these domains determine the differences in apparent citrat e affinities. TMDs 10 and 11 are most important in determining the differen ces in apparent sodium affinities, and TMD 11 determines the sensitivity to lithium inhibition. We conclude that transmembrane domains 7, 10, and 11 i n NaDC-1 may contain at least one of the cation binding sites in close prox imity to the substrate binding domain.