title: Detailed characterization of cysteine-less P-glycoprotein reveals subtle pharmacological differences in function from wild-type protein.
abstract: 1. Subtle alterations in the coupling of drug binding to nucleotide hydrolysis were observed following mutation of all seven endogenous cysteine residues to serines in the human multidrug resistance transporter, P-glycoprotein. Wild-type (wt) and the mutant (cys-less) forms of P-gp were expressed in Trichoplusia ni (High Five) cells and purified by metal affinity chromatography in order to undertake functional studies. 2. No significant differences were observed in substrate ([(3)H]-azidopine) binding to wt or cys-less P-gp. Furthermore, neither the transported substrate vinblastine, nor the modulator nicardipine, differed in their respective potencies to displace [(3)H]-azidopine from the wt or cys-less P-gp. These results suggest that respective binding sites for these drugs were unaffected by the introduced cysteine to serine substitutions. 3. The Michaelis-Menten characteristics of basal ATP hydrolysis of the two isoforms of P-gp were identical. The maximal ATPase activity in the presence of vinblastine was marginally reduced whilst the K(m) was unchanged in cys-less P-gp compared to control. However, cys-less P-gp displayed lower overall maximal ATPase activity (62%), a decreased K(m) and a lower degree of stimulation (76%) in the presence of the modulator nicardipine. 4. Therefore, the serine to cysteine mutations in P-gp may suggest that vinblastine and nicardipine transduce their effects on ATP hydrolysis through distinct conformational pathways. The wt and cys-less P-gp isoforms display similarity in their fundamental kinetic properties thereby validating the use of cys-less P-gp as a template for future cysteine-directed structure/function analysis.
authors.affiliation: Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, University of Oxford, Oxford, OX3 9DU, UK.
authors.forename: A M
authors.initials: AM
authors.lastname: Taylor
authors.forename: J
authors.initials: J
authors.lastname: Storm
authors.forename: L
authors.initials: L
authors.lastname: Soceneantu
authors.forename: K J
authors.initials: KJ
authors.lastname: Linton
authors.forename: M
authors.initials: M
authors.lastname: Gabriel
authors.forename: C
authors.initials: C
authors.lastname: Martin
authors.forename: J
authors.initials: J
authors.lastname: Woodhouse
authors.forename: E
authors.initials: E
authors.lastname: Blott
authors.forename: C F
authors.initials: CF
authors.lastname: Higgins
authors.forename: R
authors.initials: R
authors.lastname: Callaghan
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001386:Azides / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000235:genetics*; D004095:Dihydropyridines / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D018432:Drug Resistance, Multiple; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D016296:Mutagenesis; D009529:Nicardipine / Q000494:pharmacology; D019920:Photoaffinity Labels / Q000378:metabolism; D012694:Serine / Q000235:genetics; D018411:Spodoptera / Q000821:virology; D014747:Vinblastine / Q000494:pharmacology
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001386:Azides; D003432:Cross-Linking Reagents; D004095:Dihydropyridines; D019920:Photoaffinity Labels; D012694:Serine; D014747:Vinblastine; C041130:azidopine; D000255:Adenosine Triphosphate; D009529:Nicardipine; D000251:Adenosine Triphosphatases; D003545:Cysteine