Analysis of MDR1 P-glycoprotein conformational changes in permeabilized cells using differential immunoreactivity

The reactivity of the ATP-dependent multidrug transporter P-glycoprotein (P gp) with the conformation-sensitive monoclonal antibody UIC2 is increased i n the presence of Pgp transport substrates, ATP-depleting agents, or mutati ons that reduce the level of nucleotide binding by Pgp. We have investigate d the effects of nucleotides and vinblastine, a Pgp transport substrate, on the UIC2 reactivity of Pgp in cells permeabilized by Staphylococcus aureus alpha -toxin. ATP, ADP, and nonhydrolyzable ATP analogues decreased the UI C2 reactivity; this effect was potentiated by vanadate, a nucleotide-trappi ng agent. The Hill number for the nucleotide-induced conformational transit ion was 2 for ATP and ADP but 1 for nonhydrolyzable ATP analogues. The Hill numbers for ATP and ADP were decreased to 1 by mutations in one of the two nucleotide binding sites of Pgp, whereas mutation of both sites greatly di minished the overall effect of nucleotides. Vinblastine reversed the decrea se in the UIC2 reactivity brought about by all the nucleotides, including n onhydrolyzable analogues; this effect of vinblastine was blocked by vanadat e. These data indicate that UIC2-detectable conformational changes of Pgp a re driven by binding and debinding of nucleotides, that nucleotide hydrolys is affects the Hill number for its Pgp interactions, and that Pgp transport substrates promote nucleotide dissociation from Pgp. These findings are co nsistent with a conventional E1/E2 model that explains conformational trans itions of a transporter protein through a series of linked equilibria.