title: P-glycoprotein substrate transport assessed by comparing cellular and vesicular ATPase activity.
abstract: We compared the P-glycoprotein ATPase activity in inside-out plasma membrane vesicles and living NIH-MDR1-G185 cells with the aim to detect substrate transport. To this purpose we used 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 their concentrations in the cytosolic leaflets were similar. Compounds exhibiting similar influx as efflux induced in contrast different ATPase activity profiles in cells and inside-out vesicles. Their concentration was significantly lower in the cytosolic leaflet of cells than in the cytosolic leaflet of inside-out membrane vesicles, indicating that P-glycoprotein could cope with passive influx. P-glycoprotein thus transported all compounds at a rate proportional to ATP hydrolysis (i.e. all compounds were substrates). However, it prevented substrate entry into the cytosol only if passive influx of substrates across the lipid bilayer was in a similar range as active efflux.
authors.affiliation: Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, Switzerland.
authors.forename: Pierluigi
authors.initials: P
authors.lastname: Nervi
authors.forename: Xiaochun
authors.initials: X
authors.lastname: Li-Blatter
authors.forename: Päivi
authors.initials: P
authors.lastname: Aänismaa
authors.forename: Anna
authors.initials: A
authors.lastname: Seelig
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000143:Acids; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000388:Air; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D016572:Cyclosporine / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D005110:Extracellular Space / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D051379:Mice; D008823:Micelles; D041681:NIH 3T3 Cells; D010539:Permeability / Q000187:drug effects; D013379:Substrate Specificity / Q000187:drug effects; D013997:Time Factors; D014162:Transfection; D022161:Transport Vesicles / Q000201:enzymology*; D014867:Water
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000143:Acids; D003902:Detergents; D008823:Micelles; D014867:Water; D016572:Cyclosporine; D000251:Adenosine Triphosphatases