Drug binding sites on P-glycoprotein are altered by ATP binding prior to nucleotide hydrolysis.
P-glycoprotein (P-gp) confers multiple drug resistance on cancer cells by acting as a plasma membrane localized ATP-dependent drug efflux pump. Currently, there is little information on the nature of the communication between the energy-providing nucleotide binding domains (NBDs) and the drug binding sites of P-gp to generate transport of substrate. Many substrates and modulators cause alterations in ATP hydrolysis, but what effect do the various stages of the catalytic cycle have on drug interaction with P-gp? Vanadate trapping of Mg.ADP caused a reversible decrease in the binding capacity of the transported substrate [(3)H]-vinblastine and the nontransported modulator [(3)H]XR9576 to P-gp in CH(r)B30 cell membranes. The non-hydrolyzable nucleotide analogue ATP-gamma-S also caused a reduction in the binding capacity of [(3)H]-vinblastine but not for the modulator [(3)H]XR9576. This indicates that signaling to the NBDs following binding of a nontransported modulator is different to that transmitted upon interaction of a transported substrate. Second, it appears that the binding of nucleotide, rather than its hydrolysis, causes the initial conformational shift in the drug-binding site during a transport cycle.
Martin
C
C
Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, University of Oxford, Oxford, U.K.
Berridge
G
G
Mistry
P
P
Higgins
C
C
Charlton
P
P
Callaghan
R
R
D009327:4-Chloro-7-nitrobenzofurazan / Q000737:chemistry; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry; D005033:Ethylmaleimide / Q000737:chemistry; D006868:Hydrolysis; D013379:Substrate Specificity; D014638:Vanadates / Q000737:chemistry
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000817:Anilino Naphthalenesulfonates; D004791:Enzyme Inhibitors; C022571:adenosine 5'-O-(3-thiotriphosphate); D014638:Vanadates; C021310:2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D009327:4-Chloro-7-nitrobenzofurazan; D005033:Ethylmaleimide