Cholesterol Interaction with the Daunorubicin Binding Site of P-Glycoprotein

• Published in: Biochemical and biophysical research communications 2000-10, Vol.276 (3), p.909-916
• Main Authors: Wang, Er-jia, Casciano, Christopher N., Clement, Robert P., Johnson, William W.
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; ATP-Binding Cassette, Sub-Family B, Member 1 - antagonists & inhibitors; ATP-Binding Cassette, Sub-Family B, Member 1 - metabolism; Binding Sites; Biological Transport, Active - drug effects; Cell Line; Cell Survival; cholesterol; Cholesterol - metabolism; Cholesterol - pharmacology; Daunorubicin - metabolism; Flow Cytometry; Fluorescence; Humans; Hydrolysis - drug effects; Inhibitory Concentration 50; Kinetics; MDR; Microsomes - drug effects; Microsomes - metabolism; P-glycoprotein; Protein Binding; Rhodamine 123 - metabolism; transport
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1006/bbrc.2000.3554

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 IC50 of about 8 μM 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 Ks of 5 μM. 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.