A calcium/cAMP signaling loop at the ORAI1 mouth drives channel inactivation to shape NFAT induction

ORAI1 constitutes the store-operated Ca²⁺ release-activated Ca²⁺ (CRAC) channel crucial for life. Whereas ORAI1 activation by Ca²⁺-sensing STIM proteins is known, still obscure is how ORAI1 is turned off through Ca²⁺-dependent inactivation (CDI), protecting against Ca²⁺ toxicity. Here we identify a spatially-restricted Ca²⁺/cAMP signaling crosstalk critical for mediating CDI. Binding of Ca²⁺-activated adenylyl cyclase 8 (AC8) to the N-terminus of ORAI1 positions AC8 near the mouth of ORAI1 for sensing Ca²⁺. Ca²⁺ permeating ORAI1 activates AC8 to generate cAMP and activate PKA. PKA, positioned by AKAP79 near ORAI1, phosphorylates serine-34 in ORAI1 pore extension to induce CDI whereas recruitment of the phosphatase calcineurin antagonizes the effect of PKA. Notably, CDI shapes ORAI1 cytosolic Ca²⁺ signature to determine the isoform and degree of NFAT activation. Thus, we uncover a mechanism of ORAI1 inactivation, and reveal a hitherto unappreciated role for inactivation in shaping cellular Ca²⁺ signals and NFAT activation.