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J. Biol. Chem., Vol. 280, Issue 41, 34813-34822, October 14, 2005
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1
From the
Departments of Physiology and Medicine, University of Arizona, Tucson, Arizona 85724
OCT1 and OCT2 are involved in renal secretion of cationic drugs. Although they have similar selectivity for some substrates (e.g. tetraethylammonium (TEA)), they have distinct selectivities for others (e.g. cimetidine). We postulated that "homolog-specific residues," i.e. the 24 residues that are conserved in OCT1 orthologs as one amino acid and in OCT2 as a different one, influence homolog-specific selectivity and examined the influence on substrate binding of three of these conserved residues that are found in the C-terminal half of the rabbit orthologs of OCT1/2. The N353L and R403I substitutions (OCT2 to OCT1) did not significantly change the properties of OCT2. However, the E447Q replacement shifted substrate selectivity toward an OCT1-like phenotype. Substitution of glutamate with cationic amino acids (E447K and E447R) abolished transport activity, and the E447L mutant displayed markedly reduced transport of TEA and cimetidine while retaining transport of 1-methyl-4-phenylpyridinium. In a novel homology model of the three-dimensional structure of OCT2, Glu447 was found in a putative docking region within a hydrophilic cleft of the protein. In addition, six residues identified in separate studies as exerting significant effects on OCT binding were also found within the putative cleft region. There was a significant correlation (r2 = 0.82) between the IC50 values for inhibition of TEA transport by 14 different compounds and their calculated KD values for binding to the model of rabbit OCT2. The results suggest that homology modeling offers an opportunity to direct future site-directed studies of OCT/substrate interaction.
Received for publication, June 10, 2005 , and in revised form, July 28, 2005.
* This work was supported in part by National Institutes of Health Grants DK58251, ES06694, and HL07249. 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.
The on-line version of this article (available at http://www.jbc.org) contains supplemental Table A and Fig. A.
1 To whom correspondence should be addressed: Dept. of Physiology, College of Medicine, University of Arizona, Tucson, AZ 85724. Tel.: 520-626-4253; Fax: 520-626-2383; E-mail: shwright{at}u.arziona.edu.
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