P-glycoprotein in proteoliposomes with low residual detergent: the effects of cholesterol.
OBJECTIVE
There is evidence that cholesterol affects the ATPase and transport functions of P-glycoprotein (P-gp). To study the influence of cholesterol on P-gp in a well defined lipid environment, we reconstituted P-gp in egg phosphatidylcholine (PhC) and PhC/cholesterol proteoliposomes with negligible residual amounts of detergents.


METHODS
P-gp proteoliposomes were prepared by continuous dialysis from micelles consisting of P-gp, lipids, sodium dodecyl sulfate and cholate. Basal and modulator-induced ATPase activities were studied in an established enzyme assay. Modulator affinities to P-gp and to the lipid bilayers were determined by equilibrium dialysis.


RESULTS
In the absence of cholesterol the basal ATPase activity was six fold lower than in the presence of 20 or 40% cholesterol, and no P-gp binding and ATPase induction was detected for the tested modulators verapamil and progesterone. In proteoliposomes containing 20 and 40% cholesterol, respectively, the modulators showed significant P-gp binding and ATPase activation. The concentration of the modulators for half maximal activation of the ATPase was higher with 40% than with 20% cholesterol.


CONCLUSIONS
Cholesterol influences P-gp in three ways: (a) it enhances its basal ATPase activity, (b) it renders P-gp sensitive towards the modulators verapamil and progesterone and (c) it affects the modulator concentration at half maximal ATPase activation.
Bucher
Karsten
K
Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zurich, Wolfgang-Pauli-Strasse 10, 8093, Zurich, Switzerland.
Belli
Sara
S
Wunderli-Allenspach
Heidi
H
Krämer
Stefanie D
SD
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D002784:Cholesterol / Q000494:pharmacology*; D002793:Cholic Acids / Q000494:pharmacology; D007941:Leukemia P388; D008051:Lipid Bilayers / Q000378:metabolism; D051379:Mice; D011374:Progesterone / Q000378:metabolism; D011510:Proteolipids / Q000378:metabolism*; D014700:Verapamil / Q000378:metabolism
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002793:Cholic Acids; D008051:Lipid Bilayers; D011510:Proteolipids; C020485:proteoliposomes; D011374:Progesterone; D002784:Cholesterol; D014700:Verapamil; D000251:Adenosine Triphosphatases; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate