P-glycoprotein does not reduce substrate concentration from the extracellular leaflet of the plasma membrane in living cells

P-glycoprotein (Pgp), a member of the ATP-binding cassette family of transp orters, is an important mediator of multidrug resistance in cancer. Pgp exh ibits a very broad specificity for substrates. These substrates share a com mon feature of being amphipathic and can orient into either ea et of the me mbrane bilayer. Current evidence suggests that Pgp recognizes and extracts substrates from the membrane bilayer, but from which leaflet is unresolved. To directly test whether Pgp can decrease substrate concentration in the e xtracellular leaflet of the plasma membrane in living cells, we used the fl uorescent lipid analogue 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-tr iene (TMA-DPH). TMA-DPH in the extracellular solution rapidly partitions in to the extracellular leaflet of the plasma membrane and exhibits slow trans bilayer flipping into the cytoplasmic leaflet. Because TMA-DPH fluorescence is confined to the extracellular leaflet in early time points after additi on but labels intracellular membranes after longer incubation, we can asses s the effect of Pgp on TMA-DPH concentration from both extracellular leafle t and intracellular membranes. Transient transfection. with a Pgp and the g reen fluorescence protein (GFP) fusion protein generated cells with heterog eneous expression levels of Pgp-GFP. Compared with nonexpressing cells, cel ls expressing Pgp-GFP showed decreased accumulation of TMA-DPH in intracell ular membranes but similar levels of accumulation in the extracellular leaf let of the plasma membrane. Additionally, in drug-selected MCF7/Adr cells, which constitutively express high levels of Pgp, inhibition of PgP by cyclo sporin A resulted in significantly increased accumulation of TMA-DPH in int racellular membranes but no difference in its accumulation in the extracell ular leaflet of the plasma membrane. These data indicate that whereas Pgp c an extract TMA-DPH from the cytoplasmic leaflet of the membrane, any activi ty Pgp may possess in the extracellular leaflet is insufficient to decrease TMA-DPH concentration there and, therefore, does not contribute to lowerin g the cellular levels. Pgp is the prototype of an increasing number of clin ically important ATP-binding cassette transporters of amphipathic. drugs an d lipids. These results may help decipher a common mechanism of these trans porters.