FIGURE 4.

Point mutations at the NBD2-TMD contact point have a greater impact on P-gp maturation and activity than those at the NBD1-TMD site. A, HEK 293 cells were transiently transfected with A52-tagged wild-type P-gp (WT) or mutants with changes to residues in NBD1 that is adjacent to IH4 (Leu-443, Tyr-444) or to homologous residues in NBD2 (Phe-1086, Tyr-1087). Whole cell SDS extracts were subjected to immunoblot analysis and the amount of mature P-gp relative to total was determined (Percent Mature). Each value is the mean ± S.D. (n = 3). B, histidine-tagged mutants were expressed in the presence of cyclosporine A to promote maturation. P-gps were isolated, mixed with lipid and ATPase activity determined in the presence of verapamil. The results are derived from three different transfections + S.D. (n = 3 different transfections).