Figure 5. Effect of drug substrates and inhibitors on the ATPase activity of mutant L65C before and after labelling with MTS-verapamil.

BHK cells stably expressing His-tagged mutant L65C were solubilized with n-dodecyl-β-D-maltoside and then incubated in the absence (Untreated; A) or presence (+MTS-Verapamil; B) of 0.3 mM MTS-verapamil. P-gp was then isolated by nickel-chelate chromatography, mixed with E. coli lipids and ATPase activity was measured in the presence of no drug (No Drug), 0.6 mM calcein-AM (CAM), 3 mM demecolcine (Deme), 1 mM verapamil (Ver), 0.15 mM cyclosporin A (Cyclo) or 0.6 mM trans-(E)-flupentixol (T-Flu). The fold stimulation is the ratio of the activity with drug substrate to that without drug substrate. Each value is the average of triplicate determinations.