Functional reconstitution of P-glycoprotein reveals an apparent near stoichiometric drug transport to ATP hydrolysis.

We have recently described an ATP-driven, valinomycin-dependent 86Rb+uptake into proteoliposomes reconstituted with mammalian P-glycoprotein (Eytan, G. D., Borgnia, M. J., Regev, R., and Assaraf, Y. G. (1994) J. Biol. Chem. 269, 26058-26065). P-glycoprotein mediated the ATP-dependent uptake of 86Rb+-ionophore complex into the proteoliposomes, where the radioactive cation was accumulated, thus, circumventing the obstacle posed by the hydrophobicity of P-glycoprotein substrates in transport studies. Taking advantage of this assay and of the high levels of P-glycoprotein expression in multidrug-resistant Chinese hamster ovary cells, we measured simultaneously both the ATPase and transport activities of P-glycoprotein under identical conditions and observed 0.5-0.8 ionophore molecules transported/ATP molecule hydrolyzed. The amount of 86Rb+ ions transported within 1 min via the ATP- and valinomycin-dependent P-glycoprotein was equivalent to an intravesicular cation concentration of 8 mM. Thus, this stoichiometry and transport capacity of P-glycoprotein resemble various ion-translocating ATPases, that handle millimolar substrate concentrations. This constitutes the first demonstration of comparable rates of P-glycoprotein-catalyzed substrate transport and ATP hydrolysis.