Assaraf Y G, Borgnia M J
Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel.
Eur J Biochem. 1994 Jun 15;222(3):813-24. doi: 10.1111/j.1432-1033.1994.tb18928.x.
It has been proposed that the multidrug resistance (MDR) transporter, P-glycoprotein (P-170), may be physiologically involved in the transport of polypeptides. As a step towards understanding the interaction of P-170 with polypeptides, we isolated various gramicidin-D-resistant mammalian cell lines. Gramicidin D is a hydrophobic pentadecapeptide ionophore that forms proton and alkali metal cation-permeable channels in lipid bilayers. Gramicidin-D-resistant cells displayed a prominent MDR gene amplification, P-170 overexpression, reduced drug accumulation, and consequent resistance to MDR-type cytotoxic agents. Modulators of the MDR phenotype, including verapamil, reserpine and quinidine, rendered these cells sensitive to gramicidin D. Using these cell lines, we established an assay that probes for the intra-membranal interaction between P-170 and gramicidin D. Gramicidin-D channel formation was followed by cellular accumulation of 86Rb+. Ionophore-resistant cells, and other MDR cells, did not show an appreciable increase in 86Rb+ influx rates, in the presence of increasing gramicidin-D concentrations. In contrast, parental cells displayed a dose-dependent increase in the 86Rb+ influx rates. Interestingly, in the absence of serum, gramicidin-D-resistant cells resumed the wild-type, ionophore-dose-dependent increase in 86Rb+ influx rates. MDR modulators caused a resumption of channel formation in ionophore-resistant cells. We conclude that acquisition of the MDR phenotype is an efficient means of cellular protection against gramicidin D. Hence, a new approach is offered in which P-170 interaction with gramicidin D is quantitatively followed by a rapid assessment of the biological activity (i.e. channel formation) of the substrate itself. Possible mechanisms of P-170 interaction with free ionophore monomers, and membrane-associated gramicidin D are discussed.
有人提出，多药耐药（MDR）转运蛋白P-糖蛋白（P-170）可能在多肽转运中发挥生理作用。作为了解P-170与多肽相互作用的第一步，我们分离出了多种对短杆菌肽D具有抗性的哺乳动物细胞系。短杆菌肽D是一种疏水性十五肽离子载体，可在脂质双层中形成质子和碱金属阳离子可渗透的通道。对短杆菌肽D具有抗性的细胞表现出显著的MDR基因扩增、P-170过表达、药物积累减少以及对MDR型细胞毒性药物的抗性。包括维拉帕米、利血平和奎尼丁在内的MDR表型调节剂使这些细胞对短杆菌肽D敏感。利用这些细胞系，我们建立了一种检测方法，用于探究P-170与短杆菌肽D在膜内的相互作用。短杆菌肽D通道形成后，细胞会积累86Rb+。在短杆菌肽D浓度增加的情况下，对离子载体具有抗性的细胞和其他MDR细胞的86Rb+流入速率没有明显增加。相比之下，亲代细胞的86Rb+流入速率呈剂量依赖性增加。有趣的是，在无血清的情况下，对短杆菌肽D具有抗性的细胞恢复了野生型，86Rb+流入速率呈离子载体剂量依赖性增加。MDR调节剂导致对离子载体具有抗性的细胞恢复通道形成。我们得出结论，获得MDR表型是细胞抵御短杆菌肽D的一种有效保护方式。因此，我们提供了一种新方法，通过快速评估底物本身的生物活性（即通道形成）来定量跟踪P-170与短杆菌肽D的相互作用。文中还讨论了P-170与游离离子载体单体以及膜相关短杆菌肽D相互作用的可能机制。
