The importance of cholesterol in maintenance of P-glycoprotein activity and its membrane perturbing influence

In tumour cell lines that display multidrug resistance, expression of P-gly coprotein (P-gp) alters many aspects of biomembrane organization in additio n to its well-characterized drug transport activity. We have developed a re constitution system to directly investigate the effect of purified P-gp on the biophysical properties of lipid bilayers. Using a mixed detergent syste m it was possible to efficiently reconstitute P-gp at lipid:protein ratios as low as, 2.5 (w/w) by removal of detergent using adsorption to SM-2 BioBe ads. R-gp was, able to alter many biophysical parameters, associated with l ipid organization within bilayers. For example, the changes in overall flui dity and excimer formation by lipid analogues indicate modified packing org anization of bilayer constituents. Surprisingly, given its role in conferri ng drug resistance, P-gp insertion into bilayers also caused significantly, increased permeability to aqueous compounds, also reflecting a modified ph ospholipid environment. Translocation of various, phospholipid species betw een, leaflets of the bilayer was increased in the presence of P-gp; however , the effect was not dependent on ATP hydrolysis by the protein. Physiologi cal concentrations of cholesterol modified P-gp function and the degree to which it perturbed bilayer organization. The basal ATPase activity, of P-gp was increased in a dose-dependent fashion by the incorporation of choleste rol in PC:PE liposomes. In addition, the degree to, which the modulator ver apamil was able to stimulate this basal ATPase activity was reduced by the presence of cholesterol in proteoliposomes. However, the potency of verapam il was unaltered, suggesting a specific effect, not simply caused by lower drug penetration into the cholesterol containing bilayers. In summary, P-gp is able to cause perturbation in the organization of bilayer constituents. Cholesterol imparted "stability" to this perturbation of bilayer organizat ion by P-gp and moreover this led to altered protein function.