Transport of LDS-751 from the cytoplasmic leaflet of the plasma membrane by the rhodamine-123-selective site of P-glycoprotein

P-glycoprotein is an ATP-dependent transporter of an extremely wide variety of lipophilic compounds. We showed previously [Shapiro, A. B. & Ling, V. (1997a) Eur: J. Biochem. 250, 130-137] that P-glycoprotein contains two drug transporting sites, dubbed H (for Hoechst 33342-selective) and R (for rhodamine-123-selective), that interact with positive cooperativity. The H site transports 2-[2-(4-ethoxyphenyl)-6-benzimidazolyl]-6-(1-methyl-4-piperazyl)benzimidazole (Hoechst 33342) from the cytoplasmic leaflet of the plasma membrane to the aqueous extracellular medium [Shapiro, A. B. gr Ling, V. (1997b) Eur J. Biochem. 250, 122-129]. The environment from which the R site transports its substrates is unknown. In this paper, we used the fluorescent DNA dye 2-{4-[4-(dimethylamino)phenyl]-1,3-butadienyl}-3-ethylbenzothiazolium perchlorate (LDS-751), a substrate of the R site, to address this issue. LDS-751 which, like Hoechst 33342, exhibits Lipid-dependent fluorescence and slow transleaflet diffusion, allowed us to use the same methodology that we used for the H site to study the location of the R site. As with Hoechst 33342, the specific initial rate of LDS-751 transport by P-glycoprotein-rich, isolated plasma membrane vesicles from CH(R)B30 cells was directly proportional to the amount of membrane-bound LDS-751 and inversely proportional to the concentration of free, aqueous LDS-751. This result demonstrates that the R site of P-glycoprotein transports LDS-751 out of the lipid membrane. The slight decrease, instead of an increase, in the initial rate of active transport of LDS-751 with the amount of time elapsed for slow diffusion of LDS-751 from the cytoplasmic leaflet to the extracellular leaflet indicates that the R site of P-glycoprotein removes LDS-751 from the cytoplasmic leaflet of the plasma membrane. Thus, both known drug-transporting sites of P-glycoprotein remove their substrates from the cytoplasmic leaflet. Since all of the P-glycoprotein substrates we have examined so far are recognized by one or both of the two known drug-transporting sites, these two sites in the cytoplasmic leaflet of the plasma membrane may be able to account for all substrate transport by P-glycoprotein.