KINETIC EVIDENCE SUGGESTING THAT THE MULTIDRUG TRANSPORTER DIFFERENTIALLY HANDLES INFLUX AND EFFLUX OF ITS SUBSTRATES

A kinetic approach was used to analyze the mechanism by which a substi tution of valine for glycine at position 185 in the multidrug transpor ter alters its substrate specificity so that colchicine and etoposide transport is increased, daunorubicin transport is unchanged, and vinbl astine transport is decreased. Time courses for uptake and efflux of c olchicine, vinblastine, etoposide, and daunorubicin for NIH/3T3 mouse cells transfected with wild-type (MDR1-G185) and mutant (MDR1-V185) st rains of the human mdr1 gene were determined at room temperature in th e presence and absence of an energy supply. The initial rate of vinbla stine uptake was reduced approximately 5-fold by glucose feeding of AT P-depleted wild-type (MDR1-G185) cells but was only halved in MDR1-V18 5 transfectants. In contrast, glucose feeding decreased the initial ra te of colchicine uptake approximately 4-fold in the MDR1-V185 (mutant) transfectant but not in the MDR1-G185 (wild-type) transfectant. Efflu x of colchicine was accelerated >5-fold in both the MDR1-V185 (mutant) and MDR1-G185 (wild-type) transfectants when glucose was given to rai se ATP levels. The effects on initial rates of colchicine uptake accou nted semiquantitatively for the increased colchicine resistance of MDR 1-V185 (mutant) transfectants. Similar effects were found for etoposid e in the MDR-V185 transfectants. Quinidine in the external medium grea tly inhibited drug entry rates but had little effect on efflux, wherea s verapamil inhibited both uptake and efflux. A possible interpretatio n 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.