Stein W D, Cardarelli C, Pastan I, Gottesman M M
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Mol Pharmacol. 1994 Apr;45(4):763-72.
A kinetic approach was used to analyze the mechanism by which a substitution of valine for glycine at position 185 in the multidrug transporter alters its substrate specificity so that colchicine and etoposide transport is increased, daunorubicin transport is unchanged, and vinblastine transport is decreased. Time courses for uptake and efflux of colchicine, vinblastine, etoposide, and daunorubicin for NIH/3T3 mouse cells transfected with wild-type (MDR1-G185) and mutant (MDR1-V185) strains of the human mdr1 gene were determined at room temperature in the presence and absence of an energy supply. The initial rate of vinblastine uptake was reduced approximately 5-fold by glucose feeding of ATP-depleted wild-type (MDR1-G185) cells but was only halved in MDR1-V185 transfectants. In contrast, glucose feeding decreased the initial rate of colchicine uptake approximately 4-fold in the MDR1-V185 (mutant) transfectant but not in the MDR1-G185 (wild-type) transfectant. Efflux of colchicine was accelerated > 5-fold in both the MDR1-V185 (mutant) and MDR1-G185 (wild-type) transfectants when glucose was given to raise ATP levels. The effects on initial rates of colchicine uptake accounted semiquantitatively for the increased colchicine resistance of MDR1-V185 (mutant) transfectants. Similar effects were found for etoposide in the MDR-V185 transfectants. Quinidine in the external medium greatly inhibited drug entry rates but had little effect on efflux, whereas verapamil inhibited both uptake and efflux. A possible interpretation of these data is that the multidrug transporter extracts drugs from the external and internal halves of the membrane bilayer by different paths, which are distinguishable by mutation and inhibitors.
采用动力学方法分析多药转运蛋白中第185位的甘氨酸被缬氨酸取代后改变其底物特异性的机制，结果显示秋水仙碱和依托泊苷的转运增加，柔红霉素的转运不变，长春碱的转运减少。在有或无能量供应的情况下，于室温测定转染了人mdr1基因野生型（MDR1-G185）和突变型（MDR1-V185）菌株的NIH/3T3小鼠细胞对秋水仙碱、长春碱、依托泊苷和柔红霉素的摄取及流出的时间进程。用葡萄糖处理耗尽ATP的野生型（MDR1-G185）细胞后，长春碱的初始摄取速率降低约5倍，但在转染MDR1-V185的细胞中仅减半。相反，在转染MDR1-V185（突变型）的细胞中，葡萄糖处理使秋水仙碱的初始摄取速率降低约4倍，而在转染MDR1-G185（野生型）的细胞中则无此现象。当给予葡萄糖以提高ATP水平时，秋水仙碱在转染MDR1-V185（突变型）和MDR1-G185（野生型）的细胞中的流出均加速>5倍。对秋水仙碱初始摄取速率的影响在半定量上解释了转染MDR1-V185（突变型）细胞对秋水仙碱抗性增加的原因。在转染MDR-V185的细胞中，依托泊苷也有类似的效应。细胞外培养基中的奎尼丁极大地抑制了药物进入速率，但对流出影响很小，而维拉帕米则同时抑制摄取和流出。这些数据的一种可能解释是，多药转运蛋白通过不同途径从膜双层的外部和内部提取药物，这些途径可通过突变和抑制剂来区分。
