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Papers In Press, published online ahead of print August 8, 2005
Department of Physiology, University of Arizona, Tucson, AZ 85724
Corresponding Author: shwright{at}u.arizona.edu
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, CIM). We postulated that ‘homologue-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 homologue-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 CIM while retaining transport of 1-methyl-4-phenylpyridinium. In a novel homology model of the 3D structure of OCT2, E447 was found in a putative docking region within a hydrophilic cleft of the protein. In addition, 6 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 IC(sub50} values for inhibition of TEA transport by 14 different compounds and their calculated KD values for binding to the model of rbOCT2. The results suggest that homology modeling offers an opportunity to direct future site-directed studies of OCT/substrate interaction.
J. Biol. Chem, 10.1074/jbc.M506342200
Submitted on June 10, 2005
Revised on July 28, 2005
Accepted on August 8, 2005
A conserved glutamate residue in transmembrane helix 10 influences substrate specificity of rabbit OCT2 (SLC22A2)
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