MUTATIONAL ANALYSIS OF HISTIDINE-RESIDUES IN THE RABBIT NA+ DICARBOXYLATE COTRANSPORTER NADC-1/

Succinate transport by the rabbit Na+/dicarboxylate co-transporter, Na DC-1, expressed in Xenopus oocytes was inhibited by the histidyl-selec tive reagent diethyl pyrocarbonate (DEPC). Therefore the role of histi dine residues in the function of NaDC-1 was examined by site-directed mutagenesis. All 11 histidine residues in NaDC-1 were converted to ala nine, but only mutant H106A exhibited a decrease in succinate transpor t. Additional mutations of NaDC-1 at position 106 showed that aspartic acid and asparagine, but not arginine, can substitute for histidine. Examination of succinate and citrate kinetics of H106A revealed a decr ease in V-max with no change in K-m. Cell surface biotinylation experi ments showed that the transport activity of all four mutants at positi on 106 was correlated with the amount of cell surface expression, sugg esting a role of His-106 in membrane expression rather than function. Two of the histidine mutants, H153A and H569A, exhibited insensitivity to inhibition by DEPC, indicating that these residues are involved in binding DEPC. Neither of these residues is required for transport act ivity; thus DEPC probably inhibits NaDC-1 function by hindrance of the mobility of the carrier. We conclude that histidine residues are not critical for transport function in NaDC-1, although His-106 might be i nvolved in determining protein expression or stability in the membrane .