The coupling mechanism of P-glycoprotein involves residue L339 in the sixth membrane spanning segment.
The transmembrane (TM) domains in P-glycoprotein (P-gp) contain the drug binding sites and undergo conformational changes driven by nucleotide catalysis to effect translocation. However, our understanding of exactly which regions are involved in such events remains unclear. A site-directed labelling approach was used to attach thiol-reactive probes to cysteines introduced into transmembrane segment 6 (TM6) in order to perturb function and infer involvement of specific residues in drug binding and/or interdomain communication. Covalent attachment of coumarin-maleimide at residue 339C within TM6 resulted in impaired ATP hydrolysis by P-gp. The nature of the effect was to reduce the characteristic modulation of basal activity caused by transported substrates, modulators and the potent inhibitor XR9576. Photoaffinity labelling of P-gp with [(3)H]-azidopine indicated that residue 339C does not alter drug binding per se. However, covalent modification of this residue appears to prevent conformational changes that lead to drug stimulation of ATP hydrolysis.
Rothnie
Alice
A
Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, UK.
Storm
Janet
J
McMahon
Roisin
R
Taylor
Andrew
A
Kerr
Ian D
ID
Callaghan
Richard
R
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003374:Coumarins / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D006868:Hydrolysis; D007313:Insecta; D007700:Kinetics; D016014:Linear Models; D016297:Mutagenesis, Site-Directed; D009529:Nicardipine / Q000494:pharmacology; D017239:Paclitaxel / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011804:Quinolines / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013997:Time Factors; D014747:Vinblastine / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D003374:Coumarins; D020033:Protein Isoforms; D011804:Quinolines; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D014747:Vinblastine; D000255:Adenosine Triphosphate; C030123:coumarin; D009529:Nicardipine; D000251:Adenosine Triphosphatases; C402343:tariquidar; D003545:Cysteine; D017239:Paclitaxel