FRET-based sensor analysis reveals caveolae are spatially distinct Ca2+ stores in endothelial cells

Ca2+-regulating and Ca2+-dependent molecules enriched in caveolae are typically shaped as plasmalemmal invaginations or vesicles. Caveolae structure and subcellular distribution are critical for Ca2+ release from endoplasmic reticulum Ca2+ stores and for Ca2+ influx from the extracellular space into the cell. However, Ca2+ dynamics inside caveolae have never been directly measured and remain uncharacterized. To target the fluorescence resonance energy transfer (FRET)-based Ca2+ sensing protein D1, a mutant of cameleon, to the intra-caveolar space, we made a cDNA construct encoding a chimeric protein of lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) and D1 (LOXD1). Immunofluorescence and immunoelectron microscopy confirmed that a significant portion of LOXD1 was localized with caveolin-1 at morphologically apparent caveolar vesicles in endothelial cells. LOXD1 detected ATP-induced transient Ca2+ decreases by confocal FRET imaging in the presence or absence of extracellular Ca2+. This ATP-induced Ca2+ decrease was abolished following knockdown of caveoin-1, suggesting an association with caveolae. The X-ray spectra obtained by the spot analysis of electron-opaque pyroantimonate precipitates further confirmed that ATP-induced calcium decreases in intra-caveolar vesicles. In conclusion, subplasmalemmal caveolae function as Ca2+-releasable Ca2+ stores in response to ATP. This intracellular local Ca2+ delivery system may contribute to the complex spatiotemporal organization of Ca2+ signaling.