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J. Biol. Chem., Vol. 282, Issue 18, 13402-13409, May 4, 2007

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The Chloride Dependence of the Human Organic Anion Transporter 1 (hOAT1) Is Blunted by Mutation of a Single Amino Acid^*

From the Abteilung Vegetative Physiologie und Pathophysiologie, Zentrum Physiologie und Pathophysiologie, Georg-August-Universität Göttingen, Humboldtallee 23, 37073 Göttingen, Germany

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 (Arg⁴⁶⁶) 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 Arg⁴⁶⁶ 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 Arg⁴⁶⁶ influences the binding of glutarate, but not interaction with PAH, and interacts with chloride, which is a major determinant in substrate translocation.

Received for publication, October 19, 2006 , and in revised form, February 22, 2007.

^* This work was supported by Deutsche Forschungsgemeinschaft Grants Bu 571/7-5 and GRK335. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 49-551-395881; Fax: 49-551-395883; E-mail: gburckh{at}gwdg.de.

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