Identification of residues in the drug-binding domain of human P-glycoprotein - Analysis of transmembrane segment 11 by cysteine-scanning mutagenesisand inhibition by dibromobimane
Tw. Loo et Dm. Clarke, Identification of residues in the drug-binding domain of human P-glycoprotein - Analysis of transmembrane segment 11 by cysteine-scanning mutagenesisand inhibition by dibromobimane, J BIOL CHEM, 274(50), 1999, pp. 35388-35392
The drug-binding domain of the human multidrug resistance P-glycoprotein (P
-gp) probably consists of residues from multiple transmembrane (TM) segment
s. In this study, we tested whether the amino acids in TM11 participate in
binding drug substrates, Each residue in TM11 was initially altered by site
-directed mutagenesis and assayed for drug-stimulated ATPase activity in th
e presence of verapamil, vinblastine, or colchicine, Mutants G939V, F942A,
T945A, Q946A, A947L, Y953A, A954L, and G955V had altered drug-stimulated AT
Pase activities. Direct evidence for binding of drug substrate was then det
ermined by cysteine-scanning mutagenesis of the residues in TM11 and inhibi
tion of drug-stimulated ATPase activity by dibromobimane, a thiol-reactive
substrate. Dibromobimane inhibited the drug-stimulated ATPase activities of
two mutants, F942C and T945C, by more than 75%, These results suggest that
residues Phe(942) and Thr(945) in TM11, together with residues previously
identified in TM6 (Leu(339) and Ala(342)) and TM12 (Leu(975), Val(982), and
Ala(985)) (Loo, T. W., and Clarke, D. M. (1997) J. Biol. Chem. 272, 31945-
31948) form part of the drug-binding domain of P-gp.