ABCA1-mediated nascent HDL formation is precisely regulated by the plasma membrane cholesterol

Intracellular cholesterol transport is essential for maintaining cellular cholesterol homeostasis. ATP-binding cassette A1 (ABCA1) continuously moves cholesterol from the inner leaflet to the outer leaflet of the plasma membrane (PM) to maintain low inner leaflet cholesterol levels. When PM inner leaflet cholesterol levels exceed ER cholesterol levels, which are maintained at approximately 5 mol% by the complex of sterol regulatory element-binding protein (SREBP) and SREBP cleavage-activating protein (SCAP), Aster-A/GramD1a transports the excess cholesterol to the ER. Furthermore, ABCA1 removes excess PM cholesterol by promoting its efflux as nascent high-density lipoprotein (HDL) particles. Thus, cellular cholesterol homeostasis is maintained by the coordinated action of SCAP-SREBP, Aster-A/GramD1a, and ABCA1. While the regulation of SCAP-SREBP and Aster-A/GramD1a is well-understood, the mechanism governing ABCA1 activity remains less understood. In this study, we investigated the impact of PM cholesterol levels on ABCA1-mediated cholesterol and phosphatidylcholine (PC) efflux. Cells were treated with various concentrations of methyl-β-cyclodextrin (MβCD) or MβCD-cholesterol for 30 min to modulate PM cholesterol levels. We found that the initial velocities of both cholesterol and PC efflux were dependent solely on PM cholesterol levels, despite both being substrates for ABCA1. Intriguingly, when PM cholesterol levels dropped below 70% of the level observed in cells cultured in the presence of 10% FBS, both cholesterol and PC efflux ceased, even in the presence of abundant PC in the PM. Our findings suggest that ABCA1-mediated nascent HDL formation is precisely regulated to maintain optimal PM cholesterol levels.