Cholesterol interaction with the daunorubicin binding site of P-glycoprotein

The inherent complexities of cholesterol disposition and metabolism preclud e a single transmembrane active transport avenue for this steroid-precursor , cell-membrane constituent. Yet the ABC (ATP binding cassette) transporter s are inextricably linked to elements of cholesterol disposition. Recent ob servations have suggested that, under certain settings, the ABC transporter P-glycoprotein (P-gp) performs a direct role in cholesterol disposition. T he gene product of MDR1 (multidrug resistance transporter), P-glycoprotein also confers protection against xenobiotics. Using a whole cell assay in wh ich the retention of a marker substrate is evaluated and quantified, we stu died the ability of cholesterol to inhibit directly the function of this tr ansporter. In a NIH-G185 cell line presenting an overexpressed amount of th e human transporter P-gp, cholesterol caused dramatic inhibition of daunoru bicin transport with an IC50 of about 8 mu M yet had no effect on the paren t cell line nor rhodamine 123 transport. Additionally, using the ATP-hydrol ysis assay, we showed that cholesterol increases P-gp-mediated ATP hydrolys is by approximately 1.6-fold with a K-s of 5 mu M. Suggesting that choleste rol directly interacts with the substrate binding site of P-gp, these resul ts are consistent with cholesterol being transported by MDR1 P-gp. (C) 2000 Academic Press.