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J. Biol. Chem., Vol. 281, Issue 49, 38071-38079, December 8, 2006

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A Three-dimensional Model of Human Organic Anion Transporter 1

AROMATIC AMINO ACIDS REQUIRED FOR SUBSTRATE TRANSPORT^*

From the Laboratory of Pharmacology and Chemistry, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709

Organic anion transporters (OATs) play a critical role in the handling of endogenous and exogenous organic anions by excretory and barrier tissues. Little is known about the OAT three-dimensional structure or substrate/protein interactions involved in transport. In this investigation, a theoretical three-dimensional model was generated for human OAT1 (hOAT1) based on fold recognition to the crystal structure of the glycerol 3-phosphate transporter (GlpT) from Escherichia coli. GlpT and hOAT1 share several sequence motifs as major facilitator superfamily members. The structural hOAT1 model shows that helices 5, 7, 8, 10, and 11 surround an electronegative putative active site (830ų). The site opens to the cytoplasm and is surrounded by three residues not previously examined for function (Tyr²³⁰ (domain 5) and Lys⁴³¹ and Phe⁴³⁸ (domain 10)). Effects of these residues on p-aminohippurate (PAH) and cidofovir transport were assessed by point mutations in a Xenopus oocyte expression system. Membrane protein expression was severely limited for the Y230A mutant. For the K431A and F438A mutants, [³H]PAH uptake was less than 30% of wild-type hOAT1 uptake after protein expression correction. Reduced V_max values for the F438A mutant confirmed lower protein expression. In addition, the F438A mutant exhibited an increased affinity for cidofovir but was not significantly different for PAH. Differences in handling of PAH and cidofovir were also observed for the Y230F mutant. Little uptake was determined for cidofovir, whereas PAH uptake was similar to wild-type hOAT1. Therefore, the hOAT1 structural model has identified two new residues, Tyr²³⁰ and Phe⁴³⁸, which are important for substrate/protein interactions.

Received for publication, September 13, 2006

^* This work was supported by the Intramural Research Program of the National Institutes of Health, NIEHS. 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: P. O. Box 12233, F1-03 Research Triangle Park, NC 27709. Tel.: 919-541-4054; Fax: 919-541-5737; E-mail: pritcha3{at}niehs.nih.gov.

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