title: Cationic amino acids involved in dicarboxylate binding of the flounder renal organic anion transporter.
abstract: 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.
authors.affiliation: Zentrum Physiologie und Pathophysiologie, Georg-August-Universität, Göttingen, Germany.
authors.forename: Natascha A
authors.initials: NA
authors.lastname: Wolff
authors.affiliation: Zentrum Physiologie und Pathophysiologie, Georg-August-Universität, Göttingen, Germany.
authors.forename: Bettina
authors.initials: B
authors.lastname: Grünwald
authors.affiliation: Abteilung Physiologie I, Universität Tübingen, Tübingen, Germany.
authors.forename: Björn
authors.initials: B
authors.lastname: Friedrich
authors.affiliation: Abteilung Physiologie I, Universität Tübingen, Tübingen, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Lang
authors.affiliation: Zentrum Physiologie und Pathophysiologie, Georg-August-Universität, Göttingen, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Godehardt
authors.affiliation: Zentrum Physiologie und Pathophysiologie, Georg-August-Universität, Göttingen, Germany.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Burckhardt
mesh_terms: 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
chemical_list: D000596:Amino Acids; D002412:Cations; D003998:Dicarboxylic Acids; D005977:Glutarates; D027361:Organic Anion Transporters; D010130:p-Aminohippuric Acid