Function of MRP1/ABCC1 is not dependent on cholesterol or cholesterol-stabilized lipid rafts

Abstract

MRP1 (multidrug-resistance-related protein 1)/ABCC1 (ATP-binding cassette transporter C1) has been localized in cholesterol-enriched lipid rafts, which suggests a role for these lipid rafts and/or cholesterol in MRP1 function. In the present study, we have shown for the first time that nearly complete oxidation of free cholesterol in the plasma membrane of BHK-MRP1 (MRP1-expressing baby hamster kidney) cells did not affect MRP1 localization in lipid rafts or its efflux function, using 5-carboxyfluorescein diacetate as a substrate. Inhibition of cholesterol biosynthesis, using lovastatin in combination with RO 48-8071, an inhibitor of oxidosqualene cyclase, resulted in a shift of MRP1 out of lipid raft fractions, but did not affect MRP1-mediated efflux in Neuro-2a (neuroblastoma) cells. Short-term methyl-β-cyclodextrin treatment was equally effective in removing free cholesterol from Neuro-2a and BHK-MRP1 cells, but affected MRP1 function only in the latter. The kinetics of loss of both MRP1 efflux function and lipid raft association during long-term methyl-β-cyclodextrin treatment did not match the kinetics of free cholesterol removal in both cell lines. Moreover, MRP1 activity was measured in vesicles consisting of membranes isolated from BHK-MRP1 cells using the substrate cysteinyl leukotriene C4 and was not changed when the free cholesterol level of these membranes was either decreased or increased. In conclusion, MRP1 activity is not correlated with the level of free cholesterol or with localization in cholesterol-dependent lipid rafts.