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, fROAT, were changed by site-directed mutagenesis and the resulting mutants functionally characterized in Xenopus laevis oocytes. Uptake of p-aminohippurate (PAH) in oocytes that expressed mutant H34I, K394A, or R478D was markedly reduced compared with oocytes that expressed wild-type fROAT, but was still several-fold higher than that in water-injected control oocytes. Immunocytochemically, no decrease in cell surface expression of the mutants could be detected. Only mutant R478D appeared to have a lower PAH affinity than the wild type. Similar to wild-type-dependent PAH transport, uptake induced by mutant H34I was sensitive to glutarate (GA) cis-inhibition. In contrast, 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 completely suppressed by probenecid. Moreover, again in contrast to the wild type, neither PAH influx nor PAH efflux mediated by these two mutants could be trans-stimulated by GA, nor did they induce GA transport. These data suggest that amino acids K394 and R478 in fROAT are required for dicarboxylate binding and PAH/dicarboxylate exchange.
Wolff
Natascha A
NA
Zentrum Physiologie und Pathophysiologie, Georg-August-Universität, Göttingen, Germany.
Grünwald
Bettina
B
Zentrum Physiologie und Pathophysiologie, Georg-August-Universität, Göttingen, Germany.
Friedrich
Björn
B
Abteilung Physiologie I, Universität Tübingen, Tübingen, Germany.
Lang
Florian
F
Abteilung Physiologie I, Universität Tübingen, Tübingen, Germany.
Godehardt
Stefan
S
Zentrum Physiologie und Pathophysiologie, Georg-August-Universität, Göttingen, Germany.
Burckhardt
Gerhard
G
Zentrum Physiologie und Pathophysiologie, Georg-August-Universität, Göttingen, Germany.
D000596:Amino Acids / Q000502:physiology*; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002412:Cations / Q000378:metabolism*; D003998:Dicarboxylic Acids / Q000378:metabolism*; D005260:Female; D005432:Flounder / Q000378:metabolism*; D005977:Glutarates / Q000493:pharmacokinetics / Q000494:pharmacology; D007668:Kidney / Q000378:metabolism*; D009154:Mutation / Q000502:physiology; D009865:Oocytes; D027361:Organic Anion Transporters / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D014982:Xenopus laevis; D010130:p-Aminohippuric Acid / Q000037:antagonists & inhibitors / Q000378:metabolism
D000596:Amino Acids; D002412:Cations; D003998:Dicarboxylic Acids; D005977:Glutarates; D027361:Organic Anion Transporters; D010130:p-Aminohippuric Acid