Orai channel C-terminal peptides are key modulators of STlM-Orai coupling and calcium signal generation

Junctional coupling between endoplasmic reticulum (ER) Ca 2+ -sensor STIM proteins and plasma membrane (PM) Orai channels mediates Ca 2+ signals in most cells. We reveal that PM-tethered, fluorescently tagged C-terminal M4x (fourth transmembrane helix contains a cytoplasmic C-terminal extension) peptides from Orai channels undergo a Leu-specific signature of direct interaction with the STIM1 Orai-activating region (SOAR), exactly mimicking STIM1 binding to gate Orai channels. The 20-amino-acid Orai3-M4x peptide associates avidly with STIM1 within ER-PM junctions, functions to competitively block native Ca 2+ signals, and mediates a key modification of STIM-Orai coupling induced by 2-aminoethoxydiphenyl borate. By blocking STIM-Orai coupling, the Orai3-M4x peptide reveals the critical role of Orai channels in driving Ca 2+ oscillatory signals and transcriptional control through NFAT. The M4x peptides interact independently with SOAR dimers consistent with unimolecular coupling between Orai subunits and STIM1 dimers. We reveal the critical role of M4x helices in defining the coupling interface between STIM and Orai proteins to mediate store-operated Ca 2+ signals. Baraniak et al. use plasma-membrane-tethered Orai channel C-terminal peptides to reveal a Leu signature of interaction with the active site of STIM1 exactly mimicking the STIM-Orai interaction. The Orai peptides block store-operated Ca 2+ entry and reveal the critical role of Orai C-terminal helices in defining the STIM-Orai coupling interface.