Multidrug resistance protein 1 regulates lipid asymmetry in erythrocyte membranes

The role of multidrug resistance protein 1 (MRF1) in the maintenance of transbilayer lipid asymmetry in the erythrocyte membrane was investigated. The transbilayer distribution of endogenous phospholipids and [(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl (NBD)-labelled lipid analogues was compared in the absence and the presence of inhibitors of MRF1. At equilibrium the transbilayer distribution of the NBD analogues (in the absence of MRP1 inhibitors) was very similar to that of the endogenous lipids. Inhibition of MRP1 by verapamil or indomethacin resulted in a shift in the amount of probe that was internalized: approx. 50% of NBD-labelled phosphatidylcholine (PtdCho) and 9% of NBD-sphingomyelin (NBD-Spm) were no longer extractable by BSA in cells treated with inhibitor, in comparison with 25%, and 3% for control cells respectively. To verify whether inhibition of MRP1 also affected the distribution of the endogenous phospholipids, phospholipase A2 and sphingomyelinase were used to assess the amount of each of the various lipid classes present in the membrane outer leaflet. No shift in phospholipid distribution was observed after 5 h of incubation with verapamil or indomethacin. However, after 48 h of incubation with these inhibitors, significantly smaller amounts of PtdCho and Spm were present in the outer membrane leaflet. No appreciable change was observed in the distribution of phosphatidylethanolamine or phosphatidylserine, Decreased hydrolysis of PtdCho and Spm was not due to endovesicle formation, as revealed by electron microscopy. This is the first report to show that MRP1 has a role in the maintenance of the outwards orientation of endogenous choline-containing phospholipids in the erythrocyte membrane.