Cross-linking of Orai1 channels by STIM proteins

The transmembrane docking of endoplasmic reticulum (ER) Ca 2+ -sensing STIM proteins with plasma membrane (PM) Orai Ca 2+ channels is a critical but poorly understood step in Ca 2+ signal generation. STIM1 protein dimers unfold to expose a discrete STIM–Orai activating region (SOAR1) that tethers and activates Orai1 channels within discrete ER–PM junctions. We reveal that each monomer within the SOAR dimer interacts independently with single Orai1 subunits to mediate cross-linking between Orai1 channels. Superresolution imaging and mobility measured by fluorescence recovery after photobleaching reveal that SOAR dimer cross-linking leads to substantial Orai1 channel clustering, resulting in increased efficacy and cooperativity of Orai1 channel function. A concatenated SOAR1 heterodimer containing one monomer point mutated at its critical Orai1 binding residue (F394H), although fully activating Orai channels, is completely defective in cross-linking Orai1 channels. Importantly, the naturally occurring STIM2 variant, STIM2.1, has an eight-amino acid insert in its SOAR unit that renders it functionally identical to the F394H mutant in SOAR1. Contrary to earlier predictions, the SOAR1–SOAR2.1 heterodimer fully activates Orai1 channels but prevents cross-linking and clustering of channels. Interestingly, combined expression of full-length STIM1 with STIM2.1 in a 5:1 ratio causes suppression of sustained agonist-induced Ca 2+ oscillations and protects cells from Ca 2+ overload, resulting from high agonist-induced Ca 2+ release. Thus, STIM2.1 exerts a powerful regulatory effect on signal generation likely through preventing Orai1 channel cross-linking. Overall, STIM-mediated cross-linking of Orai1 channels is a hitherto unrecognized functional paradigm that likely provides an organizational microenvironment within ER–PM junctions with important functional impact on Ca 2+ signal generation.