P-glycoprotein substrate transport assessed by comparing cellular and vesicular ATPase activity

We compared the ATPase activity in inside-out vesicles and living cells with the aim to detect substrate transport using six substrates which differ significantly in their passive influx through the plasma membrane. In cells, the cytosolic membrane leaflet harboring the substrate binding site of P-glycoprotein has to be approached by passive diffusion through the lipid membrane, whereas in inside-out plasma membrane vesicles, it is accessible directly from the aqueous phase. Compounds exhibiting fast passive influx compared to active efflux by P-glycoprotein induced similar ATPase activity profiles in cells and inside-out plasma membrane vesicles, because the substrate concentrations in the cytosolic leaflet of the two systems were similar. Compounds exhibiting similar influx as efflux induced in contrast different ATPase activity profiles in cells and inside-out vesicles, because the substrate concentration in the cytosolic leaflet was significantly lower in cells than in inside-out vesicles because Pgp could cope with influx. Data show that Pgp transported all compounds at a rate proportional to ATP hydrolysis (i.e. all compounds were substrates). But entry into the cell was prevented only if passive influx of substrates across the lipid bilayer was in a similar range as active efflux of Pgp.