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 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.