Stimulated association of STIM1 and Orai1 is regulated by the balance of PtdIns(4,5) P 2 between distinct membrane pools

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.