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 [(3)H]cyclosporine and [(3)H]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 [(3)H]daunorubicin accumulation defect in wild-type Dx5 cells, but it had no significant effect on [(3)H]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 Phe(335) 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 role in the coupling of verapamil binding and modulation of daunorubicin intracellular accumulation in wild-type P-gp. In addition, Phe(335) in transmembrane 6 may play a role in coupling drug binding to ATPase activity. The deletion of Phe(335) 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.
Chen
K
KG
Oncology Division, Department of Medicine, and the Cancer Biology Program, Stanford University School of Medicine, Stanford, California 94305-5115, USA.
Lacayo
N J
NJ
Durán
G E
GE
Cohen
D
D
Sikic
B I
BI
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000595:Amino Acid Sequence; D001386:Azides / Q000494:pharmacology*; D001665:Binding Sites; D002121:Calcium Channel Blockers / Q000494:pharmacology; D016572:Cyclosporine / Q000494:pharmacology*; D004095:Dihydropyridines / Q000494:pharmacology*; D004347:Drug Interactions; D004789:Enzyme Activation; D006801:Humans; D008969:Molecular Sequence Data; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D019920:Photoaffinity Labels / Q000494:pharmacology; D014316:Tritium; D014407:Tumor Cells, Cultured; D014638:Vanadates / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001386:Azides; D002121:Calcium Channel Blockers; D004095:Dihydropyridines; D019920:Photoaffinity Labels; D014316:Tritium; D014638:Vanadates; D010649:Phenylalanine; C017040:8-azidoadenosine 5'-triphosphate; C041130:azidopine; D016572:Cyclosporine; D000255:Adenosine Triphosphate; D014700:Verapamil; D000251:Adenosine Triphosphatases