A conserved glutamate residue in transmembrane helix 10 influences substrate specificity of rabbit OCT2 (SLC22A2)

Xiaohong Zhang, Nikhil V. Shirahatti, Daruka Mahadevan, Stephen H. Wright

Research output: Contribution to journalArticle

58 Scopus citations

Abstract

OCT1 and OCT2 are involved in renal secretion of cat ionic 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.

Original languageEnglish (US)
Pages (from-to)34813-34822
Number of pages10
JournalJournal of Biological Chemistry
Volume280
Issue number41
DOIs
StatePublished - Oct 14 2005

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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