V-ATPase (Vacuolar ATPase) Activity Required for ABCA1 (ATP-Binding Cassette Protein A1)-Mediated Cholesterol Efflux

Abstract

Objective 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. Approach and Results 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 ATP6V0C 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 V0A1 and V1E1 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. Conclusions Our results support a model that ABCA1 recruits V-ATPase to the plasma membrane where V-ATPase mediates apoA1 acidification and membrane remodeling that promote apoA1 unfolding and ABCA1 mediated HDL biogenesis and lipid efflux.

Cationic amphiphilic antihistamines inhibit STAT3 via Ca2+-dependent lysosomal H+ efflux

“…Next, we tested whether CADs disrupted the H + gradient across the lysosomal membrane. Employing pH-sensitive (fluorescein isothiocyanate [FITC]) and pH-insensitive (tetramethylrhodamine [TMR]) dextrans as a pair of ratiometric sensors that reflect the relative lysosomal pH, 39 we demonstrated that terfenadine and ebastine significantly increased the lysosomal pH within 1 h ( Figure 2 E). Consistently, the fluorescence intensity of LysoTracker green that accumulates in lysosomes and other cytoplasmic acidic organelles was reduced significantly upon 1 h treatment with CADs ( Figure 2 F).…”

Hyperlipidaemia and IFNgamma/TNFalpha Synergism are associated with cholesterol crystal formation in Endothelial cells partly through modulation of Lysosomal pH and Cholesterol homeostasis

“…Future work should determine the impact of inflammation on regulators of lysosomal function and lipid homeostasis under normo- or hyperlipidaemic conditions. These processes should be investigated in parallel as we found evidence that inflammatory induced lysosomal dysfunction and intracellular lipid homeostasis seem to be tightly connected especially since a recent study demonstrated vATPases to be of crucial importance for ABCA1-mediated HDL efflux [70] , a process known to be impaired in psoriasis patients as well. [12] Future therapeutic strategies could address lysosomal re-acidification as a mechanism by which to ultimately decrease CVD risk in chronically inflamed patients.…”

PHY34 inhibits autophagy through V-ATPase V0A2 subunit inhibition and CAS/CSE1L nuclear cargo trafficking in high grade serous ovarian cancer

“…Additionally, RNA-Seq analysis found ABCA1 as one of the most highly downregulated transcripts (log fold 2.95 (OVCAR3) and log fold 2.1 (OVCAR8)). ABCA1 is a target known to be directly affected by V-ATPase inhibition [ 50 ].

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