Rothnie A, Theron D, Soceneantu L, Martin C, Traikia M, Berridge G, Higgins C F, Devaux P F, Callaghan R
Nuffield Department of Clinical Laboratory Science, John Radcliffe Hospital, University of Oxford, UK.
Eur Biophys J. 2001 Oct;30(6):430-42. doi: 10.1007/s002490100156.
In tumour cell lines that display multidrug resistance, expression of P-glycoprotein (P-gp) alters many aspects of biomembrane organization in addition to its well-characterized drug transport activity. We have developed a reconstitution system to directly investigate the effect of purified P-gp on the biophysical properties of lipid bilayers. Using a mixed detergent system 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 BioBeads. P-gp was able to alter many biophysical parameters associated with lipid organization within bilayers. For example, the changes in overall fluidity and excimer formation by lipid analogues indicate modified packing organization of bilayer constituents. Surprisingly, given its role in conferring drug resistance, P-gp insertion into bilayers also caused significantly increased permeability to aqueous compounds, also reflecting a modified phospholipid environment. Translocation of various phospholipid species between leaflets of the bilayer was increased in the presence of P-gp; however, the effect was not dependent on ATP hydrolysis by the protein. Physiological 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 cholesterol in PC:PE liposomes. In addition, the degree to which the modulator verapamil was able to stimulate this basal ATPase activity was reduced by the presence of cholesterol in proteoliposomes. However, the potency of verapamil 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 organization by P-gp and moreover this led to altered protein function.
在表现出多药耐药性的肿瘤细胞系中，除了其已被充分表征的药物转运活性外，P-糖蛋白（P-gp）的表达还改变了生物膜组织的许多方面。我们开发了一种重组系统，以直接研究纯化的P-gp对脂质双层生物物理性质的影响。使用混合洗涤剂系统，通过吸附到SM-2 BioBeads上去除洗涤剂，能够以低至2.5（w/w）的脂质：蛋白质比例有效地重组P-gp。P-gp能够改变许多与双层内脂质组织相关的生物物理参数。例如，脂质类似物的整体流动性和准分子形成的变化表明双层成分的堆积组织发生了改变。令人惊讶的是，鉴于其在赋予耐药性方面的作用，P-gp插入双层也导致对水性化合物的通透性显著增加，这也反映了磷脂环境的改变。在P-gp存在的情况下，双层小叶之间各种磷脂种类的转运增加；然而，这种效应并不依赖于该蛋白的ATP水解。生理浓度的胆固醇改变了P-gp的功能及其扰乱双层组织的程度。通过在PC:PE脂质体中掺入胆固醇，P-gp的基础ATPase活性以剂量依赖的方式增加。此外，调节剂维拉帕米刺激这种基础ATPase活性的程度在蛋白脂质体中因胆固醇的存在而降低。然而，维拉帕米的效力未改变，表明这是一种特定效应，并非仅仅由药物向含胆固醇双层的较低渗透引起。总之，P-gp能够引起双层成分组织的扰动。胆固醇赋予了P-gp对双层组织这种扰动的“稳定性”，而且这导致了蛋白质功能的改变。
