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 Co predicted transmembrane segments (TM) 6 (residues 331-351) and TM12 (residues 974-994), To test if these segments ape 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 an 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 TMS and TM12 are aligned, are close to each other in the tertiary structure of P-glycoprotein.