FIGURE 4.
Cross-linking of A259C (IH2) to W803C (IH3) inhibits activity. A, membranes prepared from cells expressing histidine-tagged mutant A259C/W803C were treated without (−) or with (+) 0.5 mm CuCl2 for 10 min at 0 °C in the absence (None) or presence of 5 mm ATP, adenosine 5′-(β,γ-imino)triphosphate (AMP.PNP), or ADP. The reactions were stopped by addition of SDS sample buffer containing no reducing agent, and samples were then subjected to immunoblot analysis. The positions of the cross-linked (X-link) and mature (170-kDa) P-gps are indicated. B, the amount of cross-linked protein relative to total P-gp (170-kDa plus 150-kDa P-gp) was quantitated from three different transfections + S.D. C, the histidine-tagged mutant A259C/W803C was isolated by nickel-chelate chromatography, reconstituted with lipid, and treated without (−) or with (+) 0.2 mm CuCl2 for 10 min at 20 °C. The reaction was stopped with 1 mm EDTA. One sample was treated with 10 mm dithiothreitol (+DTT) after incubation with CuCl2. Samples were then assayed for verapamil-stimulated ATPase activity. Each value is the mean ± S.D. (n = 3).