title: FRET-based sensor analysis reveals caveolae are spatially distinct Ca2+ stores in endothelial cells.
abstract: 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.
authors.affiliation: Department of Molecular Vascular Endocrinology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan. Electronic address: isshiki-tky@umin.ac.jp.
authors.forename: Masashi
authors.initials: M
authors.lastname: Isshiki
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Nishimoto
authors.forename: Risuke
authors.initials: R
authors.lastname: Mizuno
authors.forename: Toshiro
authors.initials: T
authors.lastname: Fujita
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology; D002352:Carrier Proteins / Q000378:metabolism; D002417:Cattle; D021941:Caveolae / Q000378:metabolism; D051242:Caveolin 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D007477:Ions / Q000737:chemistry; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D051242:Caveolin 1; D007477:Ions; C543905:LOXHD1 protein, mouse; D034741:RNA, Small Interfering; D000255:Adenosine Triphosphate; D002118:Calcium
keywords: Ca(2+) regulation; Cameleon; ER; Endothelial cells; FRET; IP(3); LOX-1; Vascular endothelial function; endoplasmic reticulum; inositol 1,4,5-trisphosphate; lectin-like oxidized low-density lipoprotein receptor 1