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 a cting as a plasma membrane localized ATP-dependent drug efflux pump. Curren tly, there is little information on the nature of the communication between the energy-providing nucleotide binding domains (NBDs) and the drug bindin g sites of P-gp to generate transport of substrate. Many substrates and mod ulators 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 [H-3]-vinblastine and the nontransported modulat or [H-3]XR9576 to P-gp in CH(r)B30 cell membranes. The non-hydrolyzable nuc leotide analogue ATP-gamma-S also caused a reduction in the binding capacit y of [H-3]-vinblastine but not for the modulator [H-3]XR9576, This indicate s that signaling to the NBDs following binding of a nontransported modulato r is different to that transmitted upon interaction of a transported substr ate. Second, it appears that the binding of nucleotide, rather than its hyd rolysis, causes the initial conformational shift in the drug-binding site d uring a transport cycle.