We have previously shown that PIP5KIβ and PIP5KIγ generate functionally distinct pools of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] important for antigen-stimulated Ca2+ entry in mast cells. In the present study, we find that association of the endoplasmic reticulum (ER) Ca2+ sensor, STIM1, and the store-operated Ca2+ channel, Orai1, stimulated by thapsigargin-mediated ER store depletion, is enhanced by overexpression of PIP5KIβ and inhibited by overexpression of PIP5KIγ. These different PIP5KI isoforms cause differential enhancement of PtdIns(4,5)P2 in detergent-resistant membrane (DRM) fractions, which comprise ordered lipid regions, and detergent-solubilized membrane (DSM) fractions, which comprise disordered lipid regions. Consistent with these results, the inositol 5-phosphatase L10-Inp54p, which is targeted to ordered lipids, decreases PtdIns(4,5)P2 in the DRM fraction and inhibits thapsigargin-stimulated STIM1–Orai1 association and store-operated Ca2+ entry, whereas the inositol 5-phosphatase S15-Inp54p, which is targeted to disordered lipids, decreases PtdIns(4,5)P2 in the DSM fraction and enhances STIM1–Orai1 association. Removal of either the STIM1 C-terminal polylysine sequence (amino acids 677–685) or an N-terminal polyarginine sequence in Orai1 (amino acids 28–33) eliminates this differential sensitivity of STIM1–Orai1 association to PtdIns(4,5)P2 in the distinctive membrane domains. Our results are consistent with a model of PtdIns(4,5)P2 balance, in which store-depletion-stimulated STIM1–Orai1 association is positively regulated by the ordered lipid pool of PtdIns(4,5)P2 and negatively regulated by PtdIns(4,5)P2 in disordered lipid domains.

SummaryMast cell activation initiated by antigen-mediated crosslinking of IgE receptors results in stimulated exocytosis of secretory lysosomes in the process known as degranulation. Much has been learned about the molecular mechanisms important for this process, including the crucial role of Ca 2+ mobilization, but spatio-temporal relationships between stimulated Ca 2+ mobilization and granule exocytosis are incompletely understood. Here we use a novel imaging-based method that uses fluorescein isothiocyanate (FITC)-dextran as a reporter for granule exocytosis in RBL mast cells and takes advantage of the pH sensitivity of FITC. We demonstrate the selectivity of FITCdextran, accumulated by fluid-phase uptake, as a marker for secretory lysosomes, and we characterize its capacity to delineate different exocytotic events, including full fusion, kiss-and-run transient fusion and compound exocytosis. Using this method, we find strong dependence of degranulation kinetics on the duration of cell to substrate attachment. We combine imaging of degranulation and Ca 2+ dynamics to demonstrate a spatial relationship between the sites of Ca 2+ wave initiation in extended cell protrusions and exocytosis under conditions of limited antigen stimulation. In addition, we find that the spatially proximal Ca 2+ signaling and secretory events correlate with participation of TRPC1 channels in Ca 2+ mobilization.