We have shown that ABCA1 mediates unfolding of the apoA1 N-terminal helical hairpin during apoA1 lipidation. Others have shown that an acidic pH exposes the hydrophobic surface of apoA1. We postulated that the vacuolar ATPase (V-ATPase) proton pump facilitates apoA1 unfolding and promotes ABCA1 mediated cholesterol efflux.
We found that V-ATPase inhibitors dose dependently decreased ABCA1 mediated cholesterol efflux to apoA1 in baby hamster kidney (BHK) cells and RAW264.7 cells; and similarly, siRNA knockdown of ATP6V 0C inhibited ABCA1 mediated cholesterol efflux to apoA1 in RAW264.7 cells. Although ABCA1 expression did not alter total cellular levels of V-ATPase, ABCA1 increased the cell surface levels of the V 0A1 and V 1E1 subunits of V-ATPase. We generated a FITC/Alexa647 double-labeled fluorescent ratiometric apoA1 pH indicator whose FITC/Alexa647 emission ratio decreased as the pH drops. We found that ABCA1 induction in BHK cells led to acidification of the cell associated apoA1 pH indicator, compared to control cells without ABCA1 expression. The V-ATPase inhibitor bafilomycin A1, dose dependently inhibited the apoA1 pH shift in ABCA1 expressing cells, without affecting the levels of cell associated apoA1. However, we were not able to detect ABCA1 mediated extracellular proton release. We showed that acidic pH facilitated apoA1 unfolding, apoA1 solubilization of phosphatidycholine:phosphatidyserine liposomes, and increased lipid fluidity of these liposomes.