SUMMARY Stromal-interaction molecules (STIM1/2) sense endoplasmic reticulum (ER) Ca 2+ 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 2+ signaling. Physiological Ca 2+ signaling requires functional interactions between STIM1/2, Orai1/2/3, and IP 3 Rs, ensuring that receptor-mediated Ca 2+ release is tailored to Ca 2+ entry and nuclear factor of activated T cells (NFAT) activation. The N-terminal Ca 2+ -binding ER-luminal domains of unactivated STIM1/2 inhibit IP 3 R-evoked Ca 2+ release. A gradual increase in agonist intensity and STIM1/2 activation relieves IP 3 R 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 IP 3 Rs translate the strength of agonist stimulation to precise levels of Ca 2+ signaling and NFAT induction, ensuring the fidelity of complex mammalian Ca 2+ signaling.

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Two-pore channels are endo-lysosomal cation channels with malleable selectivity filters that drive endocytic ion flux and membrane traffic. Here we show that TPC2 can differentially regulate its cation permeability when co-activated by its endogenous ligands, NAADP and PI(3,5)P2. Whereas NAADP rendered the channel Ca2+-permeable and PI(3,5)P2 rendered the channel Na+-selective, a combination of the two increased Ca2+ but not Na+ flux. Mechanistically, this was due to an increase in Ca2+ permeability independent of changes in ion selectivity. Functionally, we show that cell permeable NAADP and PI(3,5)P2 mimetics synergistically activate native TPC2 channels in live cells, globalizing cytosolic Ca2+ signals and regulating lysosomal pH and motility. Our data reveal that flux of different ions through the same pore can be independently controlled and identify TPC2 as a likely coincidence detector that optimizes lysosomal Ca2+ signaling.

SummaryInvertebrates express one endoplasmic reticulum (ER)-resident Ca2+-sensing stromal-interaction molecule (Stim) and one Orai plasma membrane channel protein. Stim conveys store depletion to Orai, mediating the evolutionarily conserved Ca2+ release-activated Ca2+ (CRAC) current. The crucial role of their vertebrate homologues, STIM1 and Orai1 in mediating CRAC activity in mammals is well-established. However, mammals possess two STIM and three Orai isoforms and the choreography of their interactions under physiological receptor activation is unknown. We show that the five mammalian STIM1/2 and Orai1/2/3 isoforms have non-redundant functions. Yet, all five isoforms are always required together to ensure the graded diversity of mammalian Ca2+ signaling events in response to the full spectrum of agonist strengths. Receptor-activated Ca2+ signaling across the range of stimulus intensities requires functional interactions between not only STIM1/2 and Orai1/2/3, but also IP3R, ensuring that receptor-mediated Ca2+ release is precisely tailored to Ca2+ entry and activation of nuclear factor of activated T-cells (NFAT). This is orchestrated by two interdependent and counterbalancing paradigms: the N-termini Ca2+-binding ER-luminal domains of unactivated STIM1/2 inhibit IP3R-evoked Ca2+ release. Gradual increase in agonist intensity leads to gradual STIM1/2 activation and relief of IP3R inhibition. Concomitantly, the cytosolic C-termini of activated STIM1/2 differentially interact with Orai1/2/3 proteins as agonist intensity increases. Thus, coordinated and omnitemporal functions of all five STIM/Orai proteins and IP3Rs at the ER-lumen and cytosol translate the strength of agonist stimulation to precise levels of Ca2+ release, Ca2+ entry and NFAT induction, ensuring the diversity and fidelity of complex mammalian Ca2+ signaling.HighlightsAll five STIM/Orai and IP3R are always required together in mammalian Ca2+ signallingUnactivated STIM1/2 inhibit IP3R and activated STIM1/2 cooperatively activate Orai1/2/3STIM1 contribution increases and that of STIM2 decreases as agonist intensifiesGraded IP3R disinhibition and Orai activation tailor receptor activity to NFAT induction

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