TY - JOUR
T1 - A conserved glutamate residue in transmembrane helix 10 influences substrate specificity of rabbit OCT2 (SLC22A2)
AU - Zhang, Xiaohong
AU - Shirahatti, Nikhil V.
AU - Mahadevan, Daruka
AU - Wright, Stephen H.
PY - 2005/10/14
Y1 - 2005/10/14
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.M506342200
DO - 10.1074/jbc.M506342200
M3 - Article
C2 - 16087669
AN - SCOPUS:27144555426
SN - 0021-9258
VL - 280
SP - 34813
EP - 34822
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 41
ER -