Abstract

In this study, we further characterize a mutant P-glycoprotein (P-gp) that has a deletion of Phe³³⁵ and is resistant to inhibition by cyclosporins. Photoaffinity labeling with [³H]cyclosporine and [³H]azidopine revealed markedly decreased binding to the mutant P-gp compared with wild-type P-gp. Expression of the mutant P-gp in multidrug-resistant variant cell line MES-SA/DxP (DxP) cells was associated with a 2-fold higher basal ATPase activity relative to multidrug-resistant cell line MES-SA/Dx5 (Dx5) cells with wild-type P-gp. Cyclosporine inhibited ATPase activity in both cell types, whereas the cyclosporin D analog valspodar (PSC 833), vinblastine, and dactinomycin stimulated ATPase activity in Dx5 but not in mutant DxP cells. Moreover, the cell lines differed in their responses to verapamil, which produced greater stimulation of ATPase in Dx5 than DxP cells. Verapamil significantly reversed the [³H]daunorubicin accumulation defect in wild-type Dx5 cells, but it had no significant effect on [³H]daunorubicin accumulation in the mutant DxP cells. Verapamil was not transported by cells expressing either mutant or wild-type P-gp. Vanadate trapping of azido-ATP was markedly impaired in mutant P-gp. In conclusion, our data demonstrate that Phe³³⁵ of transmembrane 6 is an important amino acid residue for the formation of cyclosporine and azidopine drug-binding site(s). Phe³³⁵ also plays a role in the coupling of verapamil binding and modulation of daunorubicin intracellular accumulation in wild-type P-gp. In addition, Phe³³⁵ in transmembrane 6 may play a role in coupling drug binding to ATPase activity. The deletion of Phe³³⁵ results in a significant increase in the basal ATPase activity with a concomitant decrease in its ability to trap ATP and transport some P-gp substrates.