Reconstitution of drug transport by purified P-glycoprotein.
P-glycoprotein confers multidrug resistance upon cells in which it is highly expressed, reducing the effectiveness of numerous cytotoxic drugs, including many of those used for chemotherapy of cancer. Although P-glycoprotein is widely believed to function as an ATP-dependent drug efflux pump, the unusually broad substrate specificity of P-glycoprotein has engendered the proposal of other, less direct mechanisms. None of the hypothetical mechanisms has been definitively tested, however, in a purified system where other cellular components and processes are absent. We have used a fluorescent substrate of P-glycoprotein, Hoechst 33342, to measure transport activity in real-time of highly purified P-glycoprotein in a reconstituted liposome system in which the P-glycoprotein has a uniformly inside-out orientation. Using this system, we demonstrated MgATP-dependent, chemosensitizer-inhibitable transport of Hoechst 33342. Transport was prevented by omission of Mg2+, by substitution of nonhydrolyzable adenylyl-beta,gamma-imidodiphosphate for ATP, by inhibition of the ATPase activity of P-glycoprotein with vanadate and N-ethylmaleimide, and by the chemosensitizers verapamil and amiodarone. Measurements of intraliposomal pH during Hoechst 33342 transport detected no large pH changes in P-glycoprotein-containing liposomes. These results are inconsistent with a mechanism in which P-glycoprotein affects drug accumulation by directly altering intracellular pH. The Hoechst 33342 transport assay results are consistent with mechanisms in which P-glycoprotein alone is sufficient to transport drugs out of the membrane bilayer.
Shapiro
A B
AB
Division of Molecular and Structural Biology, Ontario Cancer Institute, Canada.
Ling
V
V
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000302:isolation & purification / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000032:analysis; D000638:Amiodarone / Q000494:pharmacology; D000818:Animals; D001562:Benzimidazoles / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D002474:Cell-Free System; D006224:Cricetinae; D004247:DNA / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism*; D005960:Glucosides / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008055:Lipids / Q000494:pharmacology; D008081:Liposomes / Q000378:metabolism; D010316:Particle Size; D011487:Protein Conformation; D014700:Verapamil / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001562:Benzimidazoles; D005456:Fluorescent Dyes; D005960:Glucosides; D008055:Lipids; D008081:Liposomes; C018619:octyl-beta-D-glucoside; D004247:DNA; D014700:Verapamil; D000251:Adenosine Triphosphatases; D000638:Amiodarone; C017807:bisbenzimide ethoxide trihydrochloride