The chloride dependence of the human organic anion transporter 1 (hOAT1) is blunted by mutation of a single amino acid.
Organic anion transporter 1 (OAT1) is key for the secretion of organic anions in renal proximal tubules. These organic anions comprise endogenous as well as exogenous compounds including frequently used drugs of various chemical structures. The molecular basis for the polyspecificity of OAT1 is not known. Here we mutated a conserved positively charged arginine residue (Arg(466)) in the 11(th) transmembrane helix of human OAT1. The replacement by the positively charged lysine (R466K) did not impair expression of hOAT1 at the plasma membrane of Xenopus laevis oocytes but decreased the transport of p-aminohippurate (PAH) considerably. Extracellular glutarate inhibited and intracellular glutarate trans-stimulated wild type and mutated OAT1, suggesting for the mutant R466K an unimpaired interaction with dicarboxylates. However, when Arg(466) was replaced by the negatively charged aspartate (R466D), glutarate no longer interacted with the mutant. PAH uptake by wild type hOAT1 was stimulated in the presence of chloride, whereas the R466K mutant was chloride-insensitive. Likewise, the uptake of labeled glutarate or ochratoxin A was chloride-dependent in the wild type but not in R466K. Kinetic experiments revealed that chloride did not alter the apparent K(m) for PAH but influenced V(max) in wild type OAT1-expressing oocytes. In R466K mutants the apparent K(m) for PAH was similar to that of the wild type, but V(max) was not changed by chloride removal. We conclude that Arg(466) influences the binding of glutarate, but not interaction with PAH, and interacts with chloride, which is a major determinant in substrate translocation.
Rizwan
Ahsan N
AN
Abteilung Vegetative Physiologie und Pathophysiologie, Zentrum Physiologie und Pathophysiologie, Georg-August-Universität Göttingen, Humboldtallee 23, 37073 Göttingen, Germany.
Krick
Wolfgang
W
Burckhardt
Gerhard
G
D019943:Amino Acid Substitution; D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002712:Chlorides / Q000378:metabolism*; D015870:Gene Expression; D005977:Glutarates / Q000378:metabolism*; D006801:Humans; D017136:Ion Transport / Q000187:drug effects / Q000235:genetics; D007700:Kinetics; D020125:Mutation, Missense; D009793:Ochratoxins / Q000494:pharmacology; D009865:Oocytes / Q000378:metabolism; D027362:Organic Anion Transport Protein 1 / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary / Q000235:genetics; D014982:Xenopus laevis
D002121:Calcium Channel Blockers; D002712:Chlorides; D005977:Glutarates; D009793:Ochratoxins; D027362:Organic Anion Transport Protein 1; C025589:ochratoxin A