Omnitemporal choreographies of all five STIM/Orai and IP₃Rs underlie the complexity of mammalian Ca²⁺ signaling

Stromal-interaction molecules (STIM1/2) sense endoplasmic reticulum (ER) Ca²⁺ 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 Ca²⁺ signaling. Physiological Ca²⁺ signaling requires functional interactions between STIM1/2, Orai1/2/3, and IP₃Rs, ensuring that receptor-mediated Ca²⁺ release is tailored to Ca²⁺ entry and nuclear factor of activated T cells (NFAT) activation. The N-terminal Ca²⁺-binding ER-luminal domains of unactivated STIM1/2 inhibit IP₃R-evoked Ca²⁺ release. A gradual increase in agonist intensity and STIM1/2 activation relieves IP₃R 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 IP₃Rs translate the strength of agonist stimulation to precise levels of Ca²⁺ signaling and NFAT induction, ensuring the fidelity of complex mammalian Ca²⁺ signaling. Ca²⁺ signals are crucial for cell function. Emrich et al. show that the five store-operated STIM/Orai proteins are non-redundant. Physiological store-operated channel activities are omnitemporal choreographies of all five STIM1/2 and Orai1/2/3 proteins and their functional interactions with IP₃Rs, ensuring the fidelity and diversity of Ca²⁺ signaling and NFAT activation.