Cationic amino acids involved in dicarboxylate binding of the flounder renal organic anion transporter

Three conserved cationic amino acids in predicted transmembrane domains 1, 8, and 11, respectively, of the flounder renal organic anion transporter, f ROAT, were changed by site-directed mutagenesis and the resulting mutants f unctionally characterized in Xenopus laevis oocytes. Uptake of p-aminohippu rate (PAH) in oocytes that expressed mutant H341. K394A, or R478D was marke dly reduced compared with oocytes that expressed wild-type fROAT, but was s till several-fold higher than that in water-injected control oocytes. Immun ocytochemically, no decrease in cell surface expression of the mutants coul d be detected. Only mutant R478D appeared to have a lower PAH affinity than the wild type. Similar to wild-type dependent PAH transport, uptake induce d by mutant H33I was sensitive to glutarate (GA) cis-inhibition. In contras t, mutants K394A and R478D could not be significantly affected by up to 10 mM GA, although the cRNA-dependent PAH uptake could still be almost complet ely suppressed by probenecid. Moreover, again in contrast to the wild type, neither PAH influx nor PAH efflux mediated by these two mutants could be t rans-stimulated by GA, nor did they induce GA transport. These data suggest that amino acids K394 and R478 in fROAT are required for dicarboxylate bin ding and PAH/dicarboxylate exchange.