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J Biol Chem, Vol. 274, Issue 50, 35388-35392, December 10, 1999
From the Medical Research Council Group in Membrane Biology,
Department of Medicine and Department of Biochemistry, University of
Toronto, Toronto, Ontario M5S 1A8, Canada
The drug-binding domain of the human multidrug
resistance P-glycoprotein (P-gp) probably consists of residues from
multiple transmembrane (TM) segments. 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 the presence of
verapamil, vinblastine, or colchicine. Mutants G939V, F942A, T945A,
Q946A, A947L, Y953A, A954L, and G955V had altered drug-stimulated ATPase activities. Direct evidence for binding of drug substrate was
then determined by cysteine-scanning mutagenesis of the residues in
TM11 and inhibition 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
Phe942 and Thr945 in TM11, together with
residues previously identified in TM6 (Leu339 and
Ala342) and TM12 (Leu975, Val982,
and Ala985) (Loo, T. W., and Clarke, D. M. (1997)
J. Biol. Chem. 272, 31945-31948) form part of the
drug-binding domain of P-gp.
Identification of Residues in the Drug-binding Domain of Human
P-glycoprotein
ANALYSIS OF TRANSMEMBRANE SEGMENT 11 BY CYSTEINE-SCANNING
MUTAGENESIS AND INHIBITION BY DIBROMOBIMANE*
*
This work was supported by National Institutes of Health
Grant RO1 CA80900, the Medical Research Council of Canada, and the Canadian Cystic Fibrosis Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Scientist of the Medical Research Council of Canada and the
Zellers Senior Scientist of the Canadian Cystic Fibrosis Foundation. To
whom all correspondence should be addressed: Dept. of Medicine, University of Toronto, Rm. 7342, Medical Sciences Bldg., 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada. Tel./Fax:
416-978-1105.
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