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. 2013 Jun 3;288(28):20326–20333. doi: 10.1074/jbc.M113.484550

FIGURE 6.

FIGURE 6.

Replacement of Ala-266 with aromatic residues restores F1086A activity. A, wild-type P-gp or A266X mutants containing the F1086A mutation were expressed in the absence of drug substrates. B, mutant A266D/F1086A was expressed in the presence (+) or absence (−) of cyclosporine A (Cyclo). Samples from A or B were subjected to immunoblot analysis. The positions of mature (170 kDa) and immature (150 kDa) P-gps are indicated. C, ATPase activities of wild-type P-gp and F1086A mutants containing replacements to Ala-266 were measured in the presence of verapamil. Mutant A266D was first expressed in the presence of cyclosporine A before isolation of the protein by nickel-chelate chromatography. Each value is the mean ± S.D. (n = 3). D, membranes prepared from cells expressing mutants T333C/L975C (None), T333C/L975C/F1086A, or T333C/L975C/F1086A/A266F were treated with BMOE in the presence (+) or absence (−) of ATP. Samples were subjected to immunoblot analysis. The positions of cross-linked (X-link) and mature (170 kDa) P-gps are indicated.