An apical Phe-His pair defines the Orai1-coupling site and its occlusion within STIM1.

Ca2+ signal-generation through inter-membrane junctional coupling between endoplasmic reticulum (ER) STIM proteins and plasma membrane (PM) Orai channels, remains a vital but undefined mechanism. We identify two unusual overlapping Phe-His aromatic pairs within the STIM1 apical helix, one of which (F394-H398) mediates important control over Orai1-STIM1 coupling. In resting STIM1, this locus is deeply clamped within the folded STIM1-CC1 helices, likely near to the ER surface. The clamped environment in holo-STIM1 is critical-positive charge replacing Phe-394 constitutively unclamps STIM1, mimicking store-depletion, negative charge irreversibly locks the clamped-state. In store-activated, unclamped STIM1, Phe-394 mediates binding to the Orai1 channel, but His-398 is indispensable for transducing STIM1-binding into Orai1 channel-gating, and is spatially aligned with Phe-394 in the exposed Sα2 helical apex. Thus, the Phe-His locus traverses between ER and PM surfaces and is decisive in the two critical STIM1 functions-unclamping to activate STIM1, and conformational-coupling to gate the Orai1 channel.

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

Junctional coupling between endoplasmic reticulum (ER) Ca2+-sensor STIM proteins and plasma membrane (PM) Orai channels mediates Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ signals.

The Intricate Coupling Between STIM Proteins and Orai Channels.

Store-operated Ca2+ entry signals are critical for cellular regulation. This intricate signaling pathway involves coupling of proteins in two different membranes: the ER-resident Ca2+-sensing STIM proteins and the highly Ca2+-selective PM Orai channels. The molecular nature of the STIM-Orai coupling interface in ER-PM junctions and consequent Orai channel gating, are processes under intense study. We describe recent developments in determining the mechanism of Orai activation through the key STIM-Orai Activating Region (SOAR) of STIM1. We describe the unexpected unimolecular coupling of STIM with Orai and explain the observed variable stoichiometry of STIM-Orai interactions. We also define the discrete C-terminal regions in Orai channels that initially latch onto STIM proteins and mediate allosteric activation of the channel. A critical "nexus" region closely associated with the STIM-activated C-terminus of Orai1, propagates the STIM-binding signal through the four tightly-associated transmembrane helices of Orai1, finally to modify the pore-forming helices and effect channel opening.

The remote allosteric control of Orai channel gating.

Calcium signals drive an endless array of cellular responses including secretion, contraction, transcription, cell division, and growth. The ubiquitously expressed Orai family of plasma membrane (PM) ion channels mediate Ca2+ entry signals triggered by the Ca2+ sensor Stromal Interaction Molecule (STIM) proteins of the endoplasmic reticulum (ER). The 2 proteins interact within curiously obscure ER-PM junctions, driving an allosteric gating mechanism for the Orai channel. Although key to Ca2+ signal generation, molecular understanding of this activation process remain obscure. Crystallographic structural analyses reveal much about the exquisite hexameric core structure of Orai channels. But how STIM proteins bind to the channel periphery and remotely control opening of the central pore, has eluded such analysis. Recent studies apply both crystallography and single-particle cryogenic electron microscopy (cryo-EM) analyses to probe the structure of Orai mutants that mimic activation by STIM. The results provide new understanding on the open state of the channel and how STIM proteins may exert remote allosteric control of channel gating.

Pore properties of Orai1 calcium channel dimers and their activation by the STIM1 ER calcium sensor.

Store-operated Ca2+ entry signals are mediated by plasma membrane Orai channels activated through intermembrane coupling with Ca2+-sensing STIM proteins in the endoplasmic reticulum (ER). The nature of this elaborate Orai-gating mechanism has remained enigmatic. Based on the Drosophila Orai structure, mammalian Orai1 channels are hexamers comprising three dimeric subunit pairs. We utilized concatenated Orai1 dimers to probe the function of key domains within the channel pore and gating regions. The Orai1-E106Q selectivity-filter mutant, widely considered a dominant pore blocker, was surprisingly nondominant within concatenated heterodimers with Orai1-WT. The Orai1-E106Q/WT heterodimer formed STIM1-activated nonselective cation channels with significantly enlarged apparent pore diameter. Other Glu-106 substitutions entirely blocked the function of heterodimers with Orai1-WT. The hydrophobic pore-lining mutation V102C, which constitutively opens channels, was suppressed by Orai1-WT in the heterodimer. In contrast, the naturally occurring R91W pore-lining mutation associated with human immunodeficiency was completely dominant-negative over Orai-WT in heterodimers. Heterodimers containing the inhibitory K85E mutation extending outward from the pore helix gave an interesting partial effect on both channel activation and STIM1 binding, indicating an important allosteric link between the cytosolic Orai1 domains. The Orai1 C-terminal STIM1-binding domain mutation L273D powerfully blocked STIM1-induced channel activation. The Orai1-L273D/WT heterodimer had drastically impaired STIM1-induced channel gating but, unexpectedly, retained full STIM1 binding. This reveals the critical role of Leu-273 in transducing the STIM1-binding signal into the allosteric conformational change that initiates channel gating. Overall, our results provide important new insights into the role of key functional domains that mediate STIM1-induced gating of the Orai1 channel.