Role of cationic amino acids in the Na+/dicarboxylate co-transporter NaDC-1

The role of cationic amino acids in the Na+/dicarboxylate cotransporter NaD C-1 was investigated by site-directed mutagenesis and subsequent expression of mutant transporters in Xenopus oocytes. Of the ten residues chosen for mutagenesis, eight (Lys-34, Lys-107, Arg-108, Lys-333, Lys-390, Arg;-368, L ys-414 and Arg-541) were found to be non-essential for function or targetin g. Only two conserved residues, Lys-84 (at the cytoplasmic end of helix 3) and Arg-349 (at the extracellular end of helix 7), were found to be importa nt for transport. Both mutant transporters were expressed at the plasma mem brane. The mutation of Lys-84 to Ala resulted in an increased K-m for succi nate of 1.8 mM, compared with 0.3 mM in the wild-type NaDC-1. The R349A mut ant had Na+ and citrate kinetics that were similar to those of the wild typ e. However, succinate handling in the R349A mutant was altered, with eviden ce of inhibition at high succinate concentrations. In conclusion, charge ne utralization of Lys-84 and Arg-349 in NaDC-1 affects succinate handling, su ggesting that these residues might have roles in substrate binding.