Pumping of drugs by P-glycoprotein: a two-step process?

The apparent inhibition constant, Kapp, for the blockade of P-glycoprotein (P-gp) by four drugs, verapamil, cyclosporin A, XR9576 (tariquidar), and vinblastine, was measured by studying their ability to inhibit daunorubicin and calcein-AM efflux from four strains of Ehrlich cells with different levels of drug resistance and P-gp content. For daunorubicin as a transport substrate, Kapp was independent of [P-gp] for verapamil but increased strictly linearly with [P-gp] for vinblastine, cyclosporin A, and XR9576. A theoretical analysis of the kinetics of drug pumping and its reversal shows that Kapp for inhibition should increase linearly with the amount of pumps present in the membrane for a reverser that inhibits pumping from the cytoplasmic face. In contrast, if the reverser acts by blocking transport from the outer face, i.e., preemptively, Kapp should be independent of the number of pumps present. The experimental data suggest that verapamil blocks pumping at the extracellular face of the membrane, whereas the other three blockers act on pumping from the cytoplasmic phase. The maximum degree of inhibition was the same for all four blockers; thus, they do not act in parallel but rather, in serial, i.e., a drug that is pumped from the cytoplasmic phase has to pass the preemptive route upon leaving the cell. Our results are consistent with the Sauna-Ambudkar two-step model for pumping by P-gp. We suggest that the vinblastine/cyclosporin A/XR9576-binding site accepts daunorubicin at the cytoplasmic face and transfers it to the verapamil-binding site, from where daunorubicin is emptied at the extracellular surface.