FRET-based sensor analysis reveals caveolae are spatially distinct Ca stores in endothelial cells |
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Authors: | Masashi Isshiki Mitsuhiro Nishimoto Risuke Mizuno Toshiro Fujita |
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Affiliation: | 1. Department of Molecular Vascular Endocrinology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan;2. Division of Clinical Epigenetics, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo 113-8655, Japan |
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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. |
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Keywords: | LOX-1, lectin-like oxidized low-density lipoprotein receptor 1 ER, endoplasmic reticulum IP3, inositol 1,4,5-trisphosphate |
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