Omnitemporal choreographies of all five STIM/Orai and IPRs underlie the complexity of mammalian Ca signaling.

Stromal-interaction molecules (STIM1/2) sense endoplasmic reticulum (ER) Ca depletion and activate Orai channels. However, the choreography of interactions between native STIM/Orai proteins under physiological agonist stimulation is unknown. We show that the five STIM1/2 and Orai1/2/3 proteins are non-redundant and function together to ensure the graded diversity of mammalian Ca signaling. Physiological Ca signaling requires functional interactions between STIM1/2, Orai1/2/3, and IPRs, ensuring that receptor-mediated Ca release is tailored to Ca entry and nuclear factor of activated T cells (NFAT) activation. The N-terminal Ca-binding ER-luminal domains of unactivated STIM1/2 inhibit IPR-evoked Ca release. A gradual increase in agonist intensity and STIM1/2 activation relieves IPR inhibition. Concomitantly, activated STIM1/2 C termini differentially interact with Orai1/2/3 as agonist intensity increases. Thus, coordinated and omnitemporal functions of all five STIM/Orai and IPRs translate the strength of agonist stimulation to precise levels of Ca signaling and NFAT induction, ensuring the fidelity of complex mammalian Ca signaling.