Inhibition of oxidative cross-linking between engineered cysteine residues at positions 332 in predicted transmembrane segments (TM) 6 and 975 in predicted TM12 of human P-glycoprotein by drug substrates.
Each homologous half of P-glycoprotein consists of a transmembrane domain with six potential transmembrane segments and an ATP-binding domain. Labeling studies with photoactive drug analogs show that labeling occurs within or close to predicted transmembrane segments (TM) 6 (residues 331-351) and TM12 (residues 974-994). To test if these segments are in near-proximity we generated 42 different P-glycoprotein mutants in which we re-introduced a pair of cysteine residues into a Cys-less P-glycoprotein, one within TM6 (residues 332-338) and one within TM12 (residues 975-980) and assayed for cross-linking between the cysteines. All the mutants retained verapamil-stimulated ATPase activity. We found that only the mutant containing Cys-332 and Cys-975 was cross-linked in the presence of oxidant as judged by its decreased mobility on SDS gels. Similar results were obtained when the same mutations were introduced into Cys-less NH2-terminal and COOH-terminal half-molecules of P-glycoprotein followed by coexpression and treatment with oxidant. Cross-linking between Cys-332 and Cys-975, however, was inhibited by verapamil or vinblastine but not by colchicine. These results suggest that residues Cys-332 and Cys-975, which occupy equivalent positions when TM6 and TM12 are aligned, are close to each other in the tertiary structure of P-glycoprotein.
Loo
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Medical Research Council Group in Membrane Biology, Department of Medicine and Department of Biochemistry, University of Toronto, Ontario M5S 1A8, Canada.
Clarke
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D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D003078:Colchicine / Q000494:pharmacology; D003432:Cross-Linking Reagents; D003545:Cysteine; D018076:DNA, Complementary; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016877:Oxidants / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry*; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D014700:Verapamil / Q000494:pharmacology; D014747:Vinblastine / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D016877:Oxidants; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014747:Vinblastine; D014700:Verapamil; D000251:Adenosine Triphosphatases; D003545:Cysteine; D003078:Colchicine