Abstract 9781: A FRET-based Sensor Detects Caveolae as Spatially Distinct Ca2+ Stores in Endothelial Cells
Objective: Endothelial function is tightly regulated by spatiotemporally organized intracellular Ca2+ signaling. A variety of Ca2+-regulating and Ca2+-dependent molecules, such as IP3 receptor-like protein, Ca2+-ATPase and endothelial nitric oxide synthase, are enriched in caveolae, which are mobile and typically shaped as plasmalemmal invaginations or vesicles. As previously reported by us and others, the structure and subcellular distribution of caveolae are critical for Ca2+ release from endoplasmic reticulum (ER) Ca2+ stores and for Ca2+ influx from the extracellular space into the cell. However, the Ca2+ dynamics inside caveolae have not previously been detected and remain unclear. The aim of this study was to detect and analyze the Ca2+ changes in subplasmalemmal caveolar vesicles.
Methods: To target the FRET-based Ca2+-sensing protein D1, a mutant of cameleon, to the intracaveolar space, we created a cDNA construct (LOXD1) encoding a chimeric protein with lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) and D1 at its N-terminus and C-terminus, respectively. Localization analysis of LOXD1 and FRET imaging were performed by confocal microscopy. We also analyzed the LOXD1 localization by electron microscopy and the intracaveolar Ca2+ contents by a pyroantimonate precipitation method.
Results: First, immunofluorescence and immunoelectron microscopy confirmed that LOXD1 was mainly expressed outside the plasma membrane, with a significant portion inside the subplasmalemmal caveolae. Second, FRET imaging with LOXD1 detected ATP-induced transient Ca2+ decreases in the presence or absence of extracellular Ca2+. Third, X-ray spectra obtained by spot analyses of electron-opaque pyroantimonate precipitates detected ATP-induced calcium decreases in intracaveolar vesicles.
Conclusions:Subplasmalemmal caveolae function as mobile, Ca2+-releasable Ca2+ stores in response to IP3. This intracellular local Ca2+ delivery system may contribute to the highly complex spatiotemporal organization of Ca2+ signaling, and to the versatility of endothelial functions with less Ca2+ depletion in ER Ca2+ stores.
- © 2012 by American Heart Association, Inc.