P-glycoprotein (P-gp; ABCB1) actively transports a broad range of structurally unrelated compounds out of the cell. An important step in the transport cycle is coupling of drug binding with ATP hydrolysis. Drug substrates such as verapamil bind in a common drug-binding pocket at the interface between the TM (transmembrane) domains of P-gp and stimulate ATPase activity. In the present study, we used cysteine-scanning mutagenesis and reaction with an MTS (methanethiosulphonate) thiol-reactive analogue of verapamil (MTS-verapamil) to test whether the first TM segment [TM1 (TM segment 1)] forms part of the drug-binding pocket. One mutant, L65C, showed elevated ATPase activity (10.7-fold higher than an untreated control) after removal of unchanged MTS-verapamil. The elevated ATPase activity was due to covalent attachment of MTS-verapamil to Cys65 because treatment with dithiothreitol returned the ATPase activity to basal levels. Verapamil covalently attached to Cys65 appears to occupy the drug-binding pocket because verapamil protected mutant L65C from modification by MTS-verapamil. The ATPase activity of the MTS-verapamil-modified mutant L65C could not be further stimulated with verapamil, calcein acetoxymethyl ester or demecolcine. The ATPase activity could be inhibited by cyclosporin A but not by trans-(E)-flupentixol. These results suggest that TM1 contributes to the drug-binding pocket.
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Research Article| May 29 2006
Transmembrane segment 1 of human P-glycoprotein contributes to the drug-binding pocket
Tip W. Loo;
M. Claire Bartlett;
David M. Clarke
David M. Clarke 1
*Department of Medicine, University of Toronto, Toronto, ON, Canada M5S 1A8
†Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8
1To whom correspondence should be addressed, at Department of Medicine, University of Toronto, 1 King's College Circle, Rm. 7342, Medical Sciences Building, Toronto, ON, Canada M5S 1A8 (email firstname.lastname@example.org).
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Tip W. Loo, M. Claire Bartlett, David M. Clarke; Transmembrane segment 1 of human P-glycoprotein contributes to the drug-binding pocket. Biochem J 15 June 2006; 396 (3): 537–545. doi: https://doi.org/10.1042/BJ20060012
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