FIGURE 3.

Cross-linking of ICL1 and ICL3 with M4M highly stimulates ATPase activity. A, model showing predicted orientations of Leu¹⁷⁵, Asp¹⁷⁷, and Asn⁸²⁰ in the closed conformation. B, membranes prepared from HEK 293 cells expressing the ICL1/ICL3 mutants D177C/N820C or L175C/N820C were treated with cross-linkers M1M, M4M, or M17M or no cross-linker (Control). The reactions were stopped by addition of SDS sample buffer containing no thiol-reducing agent and samples subjected to immunoblot analysis. The positions of the cross-linked (X-link) and mature (170 kDa) P-gps are indicated. C, membranes expressing mutants D177C/N820C or L175C/N820C were treated with or without (None) cross-linker and histidine-tagged P-gp isolated by nickel-chelate chromatography. The isolated P-gps were mixed with lipid and ATPase activities were measured in the absence (−) or presence (+) of 0.3 mm verapamil. In some cases the samples were treated with 10 mm dithiothreitol (+DTT) before assay. Each value is the mean ± S.D. (n = 3).