Phosphatidylserine externalization in caveolae inhibits Ca2+ efflux through plasma membrane Ca2+-ATPase in ECV304

It has been evidenced that plasma membrane Ca2+-ATPase (PMCA) is localized at caveolae. However, the caveolar function of PMCA in living cells has never been demonstrated. In the present study, PMCA is exclusively localized at caveolae from ECV 304 cells demonstrated by sucrose gradient fractionation and the co-localization of PMCA with caveolin-1 was visualized by confocal microscopy. We found that PMCA is the main mechanism involved in Ca2+ efflux in ECV 304 cells. Treatment of cells with MβCD to disrupt caveolae significantly reduced the Ca2+ efflux, and the rate of decay is 4.45 ± 0.14 min−1 in the absence of MβCD and 1.99 ± 0.038 min−1 in the presence of MβCD. Moreover, the replenishment of cholesterol restored the reduction of the PMCA-mediated Ca2+ efflux in the presence of MβCD. Consistent with Ca2+ efflux in living cells, the activity of the reconstituted PMCA in membranes extracted from cells in vitro was decreased in the presence of MβCD. It was found that phosphatidylserine, which is normally in the inner leaflet of plasma membranes and is able to stimulate PMCA was relatively enriched in caveolae. Importantly, the treatment of cells with MβCD concomitantly increased the phosphatidylserine externalization. Taken together, our results suggest that activation of PMCA in caveolae is modulated by phosphatidylserine, and phosphatidylserine externalization induced by MβCD reduced the interaction of phosphatidylserine with PMCA, subsequently PMCA-mediated Ca2+ efflux in ECV 304 cells.