title: The drug-binding pocket of the human multidrug resistance P-glycoprotein is accessible to the aqueous medium.
abstract: P-Glycoprotein (P-gp) is an ATP-dependent drug pump that transports a broad range of compounds out of the cell. Cross-linking studies have shown that the drug-binding pocket is at the interface between the transmembrane (TM) domains and can simultaneously bind two different drug substrates. Here, we determined whether cysteine residues within the drug-binding pocket were accessible to the aqueous medium. Cysteine mutants were tested for their reactivity with the charged thiol-reactive compounds sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) and [2-(trimethylammonium)ethyl)]methanethiosulfonate (MTSET). Residue Ile-306(TM5) is close to the verapamil-binding site. It was changed to cysteine, reacted with MTSES or MTSET, and assayed for verapamil-stimulated ATPase activity. Reaction of mutant I306C(TM5) with either compound reduced its affinity for verapamil. We confirmed that the reduced affinity for verapamil was indeed due to introduction of a charge at position 306 by demonstrating that similar effects were observed when Ile-306 was replaced with arginine or glutamic acid. Mutant I306R showed a 50-fold reduction in affinity for verapamil and very little change in the affinity for rhodamine B or colchicine. MTSES or MTSET modification also affected the cross-linking pattern between pairs of cysteines in the drug-binding pocket. For example, both MTSES and MTSET inhibited cross-linking between I306C(TM5) and I868C(TM10). Inhibition was enhanced by ATP hydrolysis. By contrast, cross-linking of cysteine residues located outside the drug-binding pocket (such as G300C(TM5)/F770C(TM8)) was not affected by MTSES or MTSET. These results indicate that the drug-binding pocket is accessible to water.
authors.affiliation: CIHR Group in Membrane Biology, Department of Medicine, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
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* / Q000235:genetics / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D001692:Biological Transport / Q000187:drug effects; D002021:Buffers; D002460:Cell Line; D003078:Colchicine / Q000494:pharmacology; D016572:Cyclosporine / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018432:Drug Resistance, Multiple; D005786:Gene Expression Regulation; D006801:Humans; D006868:Hydrolysis; D007532:Isoleucine / Q000235:genetics / Q000378:metabolism; D008698:Mesylates / Q000494:pharmacology; D009154:Mutation / Q000235:genetics; D012235:Rhodamines / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology; D014867:Water / Q000737:chemistry / Q000378:metabolism*
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002021:Buffers; D008698:Mesylates; D012235:Rhodamines; D007532:Isoleucine; D014867:Water; C109153:(2-sulfonatoethyl)methanethiosulfonate; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; D016572:Cyclosporine; D000255:Adenosine Triphosphate; D014700:Verapamil; D000251:Adenosine Triphosphatases; C029773:rhodamine B; D003545:Cysteine; D003078:Colchicine