Identification of residues within the drug-binding domain of the human multidrug resistance P-glycoprotein by cysteine-scanning mutagenesis and reaction with dibromobimane

P-glycoprotein (P-gp) can transport a wide variety of cytotoxic compounds t hat have diverse structures. Therefore, the drug-binding domain of the huma n multidrug resistance P-gp likely consists of residues from multiple trans membrane (TM) segments. In this study, we completed cysteine-scanning mutag enesis of all the predicted TRI segments of P-gp (TMs 1-5 and 7-10) and tes ted for inhibition by a thiol-reactive substrate (dibromobimane) to identif y residues within the drug-binding domain. The activities of 189 mutants we re analyzed. Verapamil-stimulated ATPase activities of seven mutants (Y118C and V125C (TM2), S222C (TM4), I306C (TM5), S766C (TM9), and I868C and G872 C (TM10)) were inhibited by more than 50% by dibromobimane. The activities of mutants S222C (TM4), I306C (TM5), I868C (TM10), and G872C (TM10), but no t that of mutants Y118C (TM2), V125C (TM2), and S776C (TM9), were protected from inhibition by dibromobimane by pretreatment with verapamil, vinblasti ne, or colchicine. These results and those from previous studies (Loo, T. W , and Clarke, D. M. (1997) J. BioL Chem 272, 31945-31948; Loo, T. W. and Cl arke, D. M. (1999) J. Biol Chem 274, 35388-35392) indicate that the drug-bi nding domain of P-gp consists of residues in TMs 4 5, 6, 10, 11, and 12.