title: Transmembrane segment 7 of human P-glycoprotein forms part of the drug-binding pocket.
abstract: P-gp (P-glycoprotein; ABCB1) protects us by transporting a broad range of structurally unrelated compounds out of the cell. Identifying the regions of P-gp that make up the drug-binding pocket is important for understanding the mechanism of transport. The common drug-binding pocket is at the interface between the transmembrane domains of the two homologous halves of P-gp. It has been shown in a previous study [Loo, Bartlett and Clarke (2006) Biochem. J. 396, 537-545] that the first transmembrane segment (TM1) contributed to the drug-binding pocket. In the present study, we used cysteine-scanning mutagenesis, reaction with an MTS (methanethiosulfonate) thiol-reactive analogue of verapamil (termed MTS-verapamil) and cross-linking analysis to test whether the equivalent transmembrane segment (TM7) in the C-terminal-half of P-gp also contributed to drug binding. Mutation of Phe728 to cysteine caused a 4-fold decrease in apparent affinity for the drug substrate verapamil. Mutant F728C also showed elevated ATPase activity (11.5-fold higher than untreated controls) after covalent modification with MTS-verapamil. The activity returned to basal levels after treatment with dithiothreitol. The substrates, verapamil and cyclosporin A, protected the mutant from labelling with MTS-verapamil. Mutant F728C could be cross-linked with a homobifunctional thiol-reactive cross-linker to cysteines I306C(TM5) and F343C(TM6) that are predicted to line the drug-binding pocket. Disulfide cross-linking was inhibited by some drug substrates such as Rhodamine B, calcein acetoxymethyl ester, cyclosporin, verapamil and vinblastine or by vanadate trapping of nucleotides. These results indicate that TM7 forms part of the drug-binding pocket of P-gp.
authors.affiliation: Department of Medicine, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
authors.forename: Tip W
authors.initials: TW
authors.lastname: Loo
authors.forename: M Claire
authors.initials: MC
authors.lastname: Bartlett
authors.forename: David M
authors.initials: DM
authors.lastname: Clarke
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D008698:Mesylates / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D017433:Protein Structure, Secondary; D013379:Substrate Specificity; D014638:Vanadates / Q000378:metabolism; D014700:Verapamil / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D003432:Cross-Linking Reagents; D004220:Disulfides; D008698:Mesylates; D014638:Vanadates; C074502:methanethiosulfonate; D014700:Verapamil; D000251:Adenosine Triphosphatases; D003545:Cysteine