title: Cholesterol interaction with the daunorubicin binding site of P-glycoprotein.
abstract: The inherent complexities of cholesterol disposition and metabolism preclude a single transmembrane active transport avenue for this steroid-precursor, cell-membrane constituent. Yet the ABC (ATP binding cassette) transporters are inextricably linked to elements of cholesterol disposition. Recent observations have suggested that, under certain settings, the ABC transporter P-glycoprotein (P-gp) performs a direct role in cholesterol disposition. The gene product of MDR1 (multidrug resistance transporter), P-glycoprotein also confers protection against xenobiotics. Using a whole cell assay in which the retention of a marker substrate is evaluated and quantified, we studied the ability of cholesterol to inhibit directly the function of this transporter. In a NIH-G185 cell line presenting an overexpressed amount of the human transporter P-gp, cholesterol caused dramatic inhibition of daunorubicin transport with an IC(50) of about 8 microM yet had no effect on the parent cell line nor rhodamine 123 transport. Additionally, using the ATP-hydrolysis assay, we showed that cholesterol increases P-gp-mediated ATP hydrolysis by approximately 1.6-fold with a K(s) of 5 microM. Suggesting that cholesterol directly interacts with the substrate binding site of P-gp, these results are consistent with cholesterol being transported by MDR1 P-gp.
authors.affiliation: Drug Metabolism and Pharmacokinetics, Schering-Plough Research Institute, Lafayette, New Jersey, 07848, USA.
authors.forename: E
authors.initials: E
authors.lastname: Wang
authors.forename: C N
authors.initials: CN
authors.lastname: Casciano
authors.forename: R P
authors.initials: RP
authors.lastname: Clement
authors.forename: W W
authors.initials: WW
authors.lastname: Johnson
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D001693:Biological Transport, Active / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival; D002784:Cholesterol / Q000378:metabolism* / Q000494:pharmacology; D003630:Daunorubicin / Q000378:metabolism*; D005434:Flow Cytometry; D005453:Fluorescence; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism; D011485:Protein Binding; D020112:Rhodamine 123 / Q000378:metabolism
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D020112:Rhodamine 123; D000255:Adenosine Triphosphate; D002784:Cholesterol; D003630:Daunorubicin