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J. Biol. Chem., Vol. 276, Issue 18, 14972-14979, May 4, 2001
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From the Canadian Institutes for Health Research Group in Membrane
Biology, Department of Medicine and Department of Biochemistry,
University of Toronto, Toronto, Ontario M5S 1A8, Canada
Defining the residues involved in the
binding of a substrate provides insight into how the human multidrug
resistance P-glycoprotein (P-gp) can transport a wide range of
structurally diverse compounds out of the cell. Because verapamil is
the most potent stimulator of P-gp ATPase activity, we synthesized a
thiol-reactive analog of verapamil (MTS-verapamil) and used it with
cysteine-scanning mutagenesis to identify the reactive residues within
the drug-binding domain of P-gp. MTS-verapamil stimulated the ATPase
activity of Cys-less P-gp and had a Km value (25 µM) that was similar to that of verapamil. 252 P-gp
mutants containing a single cysteine within the predicted transmembrane
(TM) segments were expressed in HEK 293 cells and purified by
nickel-chelate chromatography and assayed for inhibition by
MTS-verapamil. The activities of 15 mutants, Y118C (TM2), V125C (TM2),
S222C (TM4), L339C (TM6), A342C (TM6), A729C (TM7), A841C (TM9),
N842C (TM9), I868C (TM10), A871C (TM10), F942C (TM11), T945C (TM11),
V982C (TM12), G984C (TM12), and A985C (TM12), were inhibited by
MTS-verapamil. Four mutants, S222C (TM4), L339C (TM6), A342C (TM6), and
G984C (TM12), were significantly protected from inhibition by
MTS-verapamil by pretreatment with verapamil. Less protection was
observed in mutants I868C (TM10), F942C (TM11) and T945C (TM11). These
results indicate that residues in TMs 4, 6, 10, 11, and 12 must
contribute to the binding of verapamil.
Defining the Drug-binding Site in the Human Multidrug Resistance
P-glycoprotein Using a Methanethiosulfonate Analog of Verapamil,
MTS-verapamil*
*
This work was supported in part by National Institutes of
Health Grant RO1 CA80900 and by grants from the Canadian Institutes for
Health Research (CIHR) 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.
An Investigator of the CIHR. To whom 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; E-mail:
david.clarke@utoronto.ca.
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