The Orai Channel C-Terminal Peptide Defines the STIM-Orai Coupling Interface

The junctional coupling between endoplasmic reticulum (ER) Ca2+-sensor STIM proteins and plasma membrane (PM) Orai channels is a universal Ca2+ signaling pathway.  Despite its importance, the crucial STIM-Orai coupling interface mediating channel gating remains unclear.  We reveal PM-tethered, fluorescently-tagged C-terminal M4x peptides from mammalian Orai channels undergo a leucine-specific signature of direct interaction with the STIM1 Orai-activating region (SOAR), exactly mimicking STIM1-mediated binding to gate intact Orai channels. The 20-amino acid Orai3 M4x peptide associates avidly with STIM1 within ER-PM junctions, functions as a competitive blocker of native Ca2+ signals, and mediates a key modification of STIM-Orai coupling induced by 2-aminoethoxydiphenyl borate.  The M4x peptides do not co-associate but interact independently with SOAR dimers consistent with unimolecular coupling between Orai channel subunits and STIM1 dimers.  These studies reveal the critical role of the M4x helices in defining the coupling interface between STIM and Orai proteins to mediate store-operated Ca2+ signals.