Loss of cyclosporin and azidopine binding are associated with altered ATPase activity by a mutant p-glycoprotein with deleted Phe(335)

In this study, we further characterize a mutant P-glycoprotein (P-gp) that has a deletion of Phe(335) and is resistant to inhibition by cyclosporins. Photoaffinity labeling with [H-3] cyclosporine and [H-3] 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 ME S-SA/ DxP (DxP) cells was associated with a 2-fold higher basal ATPase acti vity 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 dac tinomycin stimulated ATPase activity in Dx5 but not in mutant DxP cells. Mo reover, the cell lines differed in their responses to verapamil, which prod uced greater stimulation of ATPase in Dx5 than DxP cells. Verapamil signifi cantly reversed the [H-3] daunorubicin accumulation defect in wild-type Dx5 cells, but it had no significant effect on [H-3] 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 marked ly impaired in mutant P-gp. In conclusion, our data demonstrate that Phe(33 5) of transmembrane 6 is an important amino acid residue for the formation of cyclosporine and azidopine drug-binding site(s). Phe(335) also plays a r ole in the coupling of verapamil binding and modulation of daunorubicin int racellular accumulation in wild-type P-gp. In addition, Phe(335) in transme mbrane 6 may play a role in coupling drug binding to ATPase activity. The d eletion of Phe(335) results in a significant increase in the basal ATPase a ctivity with a concomitant decrease in its ability to trap ATP and transpor t some P-gp substrates.