Abstract 9121: Intra-endoplasmic Reticulum Ca2+ Flow Recharges Preferentially to Subcompartments of ER at Caveolin-rich Cell Edges in Endothelial Cells

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Subcellular distribution of caveolin-1, a marker protein of caveolae, is polarized especially in migrating or subconfluent endothelial cells ( Fig. 1 ). In response to ATP, IP 3 production and Ca 2+ release originated at caveolin-1-rich cell edges (CRCE) and then propagated as spatiotemporally organized Ca 2+ waves ( Fig. 2 ). Here in this study, we investigated subcellular Ca 2+ dynamics in the endoplasmic reticulum (ER) Ca 2+ stores by using confocal microscopy and an ER-targeted version of cameleon (D1ER), a GFP-based Ca 2+ -sensing protein. As expected, FRET imaging using an ER-targeted Ca 2+ sensor revealed that the ATP-mediated decrease in local intra-ER Ca 2+ concentration ([Ca 2+ ] er ) at CRCE was more rapid and larger than in other subdomains ( Figs. 2, 3 ). Interestingly, the decreased [Ca 2+ ] er at such CRCE more rapidly returned to the baseline in the presence of normal extracellular Ca 2+ . Even in the absence of extracellular Ca 2+ , when maximal ATP stimulation continued to decrease global [Ca 2+ ] er , [Ca 2+ ] er at CRCE exhibited the opposite incremental recharging. Removal of extracellular Ca 2+ or thapsigargin treatment did not affect such local recharging of Ca 2+ , suggesting no major roles of store-operated Ca 2+ entry (SOCE) or reuptake of released Ca 2+ into the ER by a Ca 2+ -ATPase. Thus, we conclude that the local ER Ca 2+ store comprises functional subcompartments at CRCE and that an ATP-induced [Ca 2+ ] er decrease at the caveolin-1-rich region is preferentially recharged by intra-ER Ca 2+ flow from the other ER subcompartments ( Fig. 4 ).