Stimulated association of STIM1 and Orai1 is regulated by the balance of PtdIns(4,5)P2 between distinct membrane pools

We have previously shown that PIP5KI beta and PIP5KI gamma generate functionally distinct pools of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P-2] 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 PIP5KI beta and inhibited by overexpression of PIP5KI gamma. These different PIP5KI isoforms cause differential enhancement of PtdIns(4,5)P-2 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)P-2 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)P-2 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)P-2 in the distinctive membrane domains. Our results are consistent with a model of PtdIns(4,5)P-2 balance, in which store-depletion-stimulated STIM1-Orai1 association is positively regulated by the ordered lipid pool of PtdIns(4,5)P-2 and negatively regulated by PtdIns(4,5)P-2 in disordered lipid domains.