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.

The tethered peptide activation mechanism of adhesion GPCRs.

Adhesion G-protein-coupled receptors (aGPCRs) are characterized by the presence of auto-proteolysing extracellular regions that are involved in cell-cell and cell-extracellular matrix interactions1. Self cleavage within the aGPCR auto-proteolysis-inducing (GAIN) domain produces two protomers-N-terminal and C-terminal fragments-that remain non-covalently attached after receptors reach the cell surface1. Upon dissociation of the N-terminal fragment, the C-terminus of the GAIN domain acts as a tethered agonist (TA) peptide to activate the seven-transmembrane domain with a mechanism that has been poorly understood2-5. Here we provide cryo-electron microscopy snapshots of two distinct members of the aGPCR family, GPR56 (also known as ADGRG1) and latrophilin 3 (LPHN3 (also known as ADGRL3)). Low-resolution maps of the receptors in their N-terminal fragment-bound state indicate that the GAIN domain projects flexibly towards the extracellular space, keeping the encrypted TA peptide away from the seven-transmembrane domain. High-resolution structures of GPR56 and LPHN3 in their active, G-protein-coupled states, reveal that after dissociation of the extracellular region, the decrypted TA peptides engage the seven-transmembrane domain core with a notable conservation of interactions that also involve extracellular loop 2. TA binding stabilizes breaks in the middle of transmembrane helices 6 and 7 that facilitate aGPCR coupling and activation of heterotrimeric G proteins. Collectively, these results enable us to propose a general model for aGPCR activation.

The oxytocin signaling complex reveals a molecular switch for cation dependence.

Oxytocin (OT) and vasopressin (AVP) are conserved peptide signaling hormones that are critical for diverse processes including osmotic homeostasis, reproduction, lactation and social interaction. OT acts through the oxytocin receptor (OTR), a magnesium-dependent G protein-coupled receptor that is a therapeutic target for treatment of postpartum hemorrhage, dysfunctional labor and autism. However, the molecular mechanisms that underlie OTR activation by OT and the dependence on magnesium remain unknown. Here we present the wild-type active-state structure of human OTR bound to OT and miniGq/i determined by cryo-EM. The structure reveals a unique activation mechanism adopted by OTR involving both the formation of a Mg2+ coordination complex between OT and the receptor, and disruption of transmembrane helix 7 (TM7) by OT. Our functional assays demonstrate the role of TM7 disruption and provide the mechanism of full agonism by OT and partial agonism by OT analogs. Furthermore, we find that the identity of a single cation-coordinating residue across vasopressin family receptors determines whether the receptor is cation-dependent. Collectively, these results demonstrate how the Mg2+-dependent OTR is activated by OT, provide essential information for structure-based drug discovery efforts and shed light on the molecular determinants of cation dependence of vasopressin family receptors throughout the animal kingdom.

G-protein activation by a metabotropic glutamate receptor.

Family C G-protein-coupled receptors (GPCRs) operate as obligate dimers with extracellular domains that recognize small ligands, leading to G-protein activation on the transmembrane (TM) domains of these receptors by an unknown mechanism1. Here we show structures of homodimers of the family C metabotropic glutamate receptor 2 (mGlu2) in distinct functional states and in complex with heterotrimeric Gi. Upon activation of the extracellular domain, the two transmembrane domains undergo extensive rearrangement in relative orientation to establish an asymmetric TM6-TM6 interface that promotes conformational changes in the cytoplasmic domain of one protomer. Nucleotide-bound Gi can be observed pre-coupled to inactive mGlu2, but its transition to the nucleotide-free form seems to depend on establishing the active-state TM6-TM6 interface. In contrast to family A and B GPCRs, G-protein coupling does not involve the cytoplasmic opening of TM6 but is facilitated through the coordination of intracellular loops 2 and 3, as well as a critical contribution from the C terminus of the receptor. The findings highlight the synergy of global and local conformational transitions to facilitate a new mode of G-protein activation.

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.

L-type Ca2+ channel blockers promote vascular remodeling through activation of STIM proteins.

Voltage-gated L-type Ca2+ channel (Cav1.2) blockers (LCCBs) are major drugs for treating hypertension, the preeminent risk factor for heart failure. Vascular smooth muscle cell (VSMC) remodeling is a pathological hallmark of chronic hypertension. VSMC remodeling is characterized by molecular rewiring of the cellular Ca2+ signaling machinery, including down-regulation of Cav1.2 channels and up-regulation of the endoplasmic reticulum (ER) stromal-interacting molecule (STIM) Ca2+ sensor proteins and the plasma membrane ORAI Ca2+ channels. STIM/ORAI proteins mediate store-operated Ca2+ entry (SOCE) and drive fibro-proliferative gene programs during cardiovascular remodeling. SOCE is activated by agonists that induce depletion of ER Ca2+, causing STIM to activate ORAI. Here, we show that the three major classes of LCCBs activate STIM/ORAI-mediated Ca2+ entry in VSMCs. LCCBs act on the STIM N terminus to cause STIM relocalization to junctions and subsequent ORAI activation in a Cav1.2-independent and store depletion-independent manner. LCCB-induced promotion of VSMC remodeling requires STIM1, which is up-regulated in VSMCs from hypertensive rats. Epidemiology showed that LCCBs are more associated with heart failure than other antihypertensive drugs in patients. Our findings unravel a mechanism of LCCBs action on Ca2+ signaling and demonstrate that LCCBs promote vascular remodeling through STIM-mediated activation of ORAI. Our data indicate caution against the use of LCCBs in elderly patients or patients with advanced hypertension and/or onset of cardiovascular remodeling, where levels of STIM and ORAI are elevated.

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.

A calcium/cAMP signaling loop at the ORAI1 mouth drives channel inactivation to shape NFAT induction.

ORAI1 constitutes the store-operated Ca2+ release-activated Ca2+ (CRAC) channel crucial for life. Whereas ORAI1 activation by Ca2+-sensing STIM proteins is known, still obscure is how ORAI1 is turned off through Ca2+-dependent inactivation (CDI), protecting against Ca2+ toxicity. Here we identify a spatially-restricted Ca2+/cAMP signaling crosstalk critical for mediating CDI. Binding of Ca2+-activated adenylyl cyclase 8 (AC8) to the N-terminus of ORAI1 positions AC8 near the mouth of ORAI1 for sensing Ca2+. Ca2+ permeating ORAI1 activates AC8 to generate cAMP and activate PKA. PKA, positioned by AKAP79 near ORAI1, phosphorylates serine-34 in ORAI1 pore extension to induce CDI whereas recruitment of the phosphatase calcineurin antagonizes the effect of PKA. Notably, CDI shapes ORAI1 cytosolic Ca2+ signature to determine the isoform and degree of NFAT activation. Thus, we uncover a mechanism of ORAI1 inactivation, and reveal a hitherto unappreciated role for inactivation in shaping cellular Ca2+ signals and NFAT activation.

Cross-talk between N-terminal and C-terminal domains in stromal interaction molecule 2 (STIM2) determines enhanced STIM2 sensitivity.

Store-operated Ca2+ entry (SOCE) is a ubiquitous pathway for Ca2+ influx across the plasma membrane (PM). SOCE is mediated by the endoplasmic reticulum (ER)-associated Ca2+-sensing proteins stromal interaction molecule 1 (STIM1) and STIM2, which transition into an active conformation in response to ER Ca2+ store depletion, thereby interacting with and gating PM-associated ORAI1 channels. Although structurally homologous, STIM1 and STIM2 generate distinct Ca2+ signatures in response to varying strengths of agonist stimulation. The physiological functions of these Ca2+ signatures, particularly under native conditions, remain unclear. To investigate the structural properties distinguishing STIM1 and STIM2 activation of ORAI1 channels under native conditions, here we used CRISPR/Cas9 to generate STIM1-/-, STIM2-/-, and STIM1/2-/- knockouts in HEK293 and colorectal HCT116 cells. We show that depending on cell type, STIM2 can significantly sustain SOCE in response to maximal store depletion. Utilizing the SOCE modifier 2-aminoethoxydiphenyl borate (2-APB), we demonstrate that 2-APB-activated store-independent Ca2+ entry is mediated exclusively by endogenous STIM2. Using variants that either stabilize or disrupt intramolecular interactions of STIM C termini, we show that the increased flexibility of the STIM2 C terminus contributes to its selective store-independent activation by 2-APB. However, STIM1 variants with enhanced flexibility in the C terminus failed to support its store-independent activation. STIM1/STIM2 chimeric constructs indicated that coordination between N-terminal sensitivity and C-terminal flexibility is required for specific store-independent STIM2 activation. Our results clarify the structural determinants underlying activation of specific STIM isoforms, insights that are potentially useful for isoform-selective drug targeting.

Identification of molecular determinants that govern distinct STIM2 activation dynamics.

The endoplasmic reticulum (ER) Ca2+ sensors stromal interaction molecule 1 (STIM1) and STIM2, which connect ER Ca2+ depletion with extracellular Ca2+ influx, are crucial for the maintenance of Ca2+ homeostasis in mammalian cells. Despite the recent progress in unraveling the role of STIM2 in Ca2+ signaling, the mechanistic underpinnings of its activation remain underexplored. We use an engineering approach to direct ER-resident STIMs to the plasma membrane (PM) while maintaining their correct membrane topology, as well as Förster resonance energy transfer (FRET) sensors that enabled in cellulo real-time monitoring of STIM activities. This allowed us to determine the calcium affinities of STIM1 and STIM2 both in cellulo and in situ, explaining the current discrepancies in the literature. We also identified the key structural determinants, especially the corresponding G residue in STIM1, which define the distinct activation dynamics of STIM2. The chimeric E470G mutation could switch STIM2 from a slow and weak Orai channel activator into a fast and potent one like STIM1 and vice versa. The systemic dissection of STIM2 activation by protein engineering sets the stage for the elucidation of the regulation and function of STIM2-mediated signaling in mammals.

Calcium store refilling and STIM activation in STIM- and Orai-deficient cell lines.

Mediated through the combined action of STIM proteins and Orai channels, store-operated Ca2+ entry (SOCE) functions ubiquitously among different cell types. The existence of multiple STIM and Orai genes has made it difficult to assign specific roles of each STIM and Orai homolog in mediating Ca2+ signals. Using CRISPR/Cas9 gene editing tools, we generated cells with both STIM or all three Orai homologs deleted and directly monitored store Ca2+ and Ca2+ signals. We found that unstimulated, SOCE null KO cells still retain 50~70% of ER Ca2+ stores of wildtype (wt) cells. After brief exposure to store-emptying conditions, acute refilling of ER Ca2+ stores was totally blocked in KO cells. However, after 24 h in culture, stores were eventually refilled. Thus, SOCE is critical for immediate refilling of ER Ca2+ but is dispensable for the maintenance of long-term ER Ca2+ homeostasis. Using the Orai null background triple Orai-KO cells, we examined the plasma membrane translocation properties of a series of truncated STIM1 variants. FRET analysis reveals that, even though PM tethering of STIM1 expedites the activation of STIM1 by facilitating its oligomerization, migration, and accumulation in ER-PM junctions, it is not required for the conformational switch, oligomerization, and clustering of STIM1. Even without overt puncta formation at ER-PM junctions, STIM11-491 and STIM11-666 could still rescue SOCE when expressed in STIM KO cells. Thus, ER-PM trapping and clustering of STIM molecules only facilitates the process of SOCE activation, but is not essential for the activation of Orai channels.

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.

Cross-linking of Orai1 channels by STIM proteins.

The transmembrane docking of endoplasmic reticulum (ER) Ca2+-sensing STIM proteins with plasma membrane (PM) Orai Ca2+ channels is a critical but poorly understood step in Ca2+ 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 Ca2+ oscillations and protects cells from Ca2+ overload, resulting from high agonist-induced Ca2+ 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 Ca2+ signal generation.

The STIM-Orai Pathway: Conformational Coupling Between STIM and Orai in the Activation of Store-Operated Ca2+ Entry.

Store-operated Ca2+ entry fulfills a crucial role in controlling Ca2+ signals in almost all cells. The Ca2+-sensing stromal interaction molecule (STIM) proteins in the endoplasmic reticulum (ER) undergo complex conformational changes in response to depleted ER luminal Ca2+, allowing them to unfold and become trapped in ER-plasma membrane (PM) junctions. Dimers of STIM proteins trap and gate the plasma membrane Orai Ca2+ channels within these junctions to generate discrete zones of high Ca2+ and regulate sensitive Ca2+-dependent intracellular signaling pathways. The STIM-Orai activating region (SOAR) of STIM1 becomes exposed upon store depletion and promotes trapping of Orai1 at the PM. Residue Phe-394 within SOAR forms an integral part of the high-affinity Orai1-interacting site. Our results demonstrate that only a single active site within the dimeric SOAR domain of STIM1 is required for the activation of Orai1 channel activity. This unimolecular model is strongly supported by evidence of variable STIM1:Orai1 stoichiometry reported in many studies. We hypothesize that unimolecular coupling promotes cross-linking of channels, localizing Ca2+ signals, and regulating channel activity. We have also identified a key "nexus" region in Orai1 near the C-terminal STIM1-binding site that can be mutated to constitutively activate Ca2+ entry, mimicking STIM1 activated channels. This suggests that STIM1 mediates gating of Orai1 in an allosteric manner via interaction with the Orai1 C-terminus alone. This model suggests the dual role of STIM1 in regulating both localization and gating of Orai1 channels and has important implications for the regulation of SOCE-mediated downstream signaling and the kinetics of channel activation.

The STIM-Orai coupling interface and gating of the Orai1 channel.

In virtually all cells, store-operated Ca2+ entry signals are vital in controlling a spectrum of functions. The signals are mediated by STIM proteins in the ER and Orai channels in the PM which undergo a dynamic coupling process within discrete ER-PM junctional regions. This coupling is initiated by depletion of ER stored Ca2+ triggering STIM proteins to undergo an intricate activation process. Thereafter, STIM proteins become trapped in the ER-PM junctions where they tether and gate PM Orai Ca2+ channels. STIM1 exists as a dimer, with a single STIM-Orai activating region (SOAR) buried in the resting protein that becomes exposed upon activation. An exposed region on SOAR including the Phe-394 residue forms a critical Orai1 interacting site. Using dimeric SOAR concatemers, we reveal only one of the two sites in the SOAR dimer is needed for Orai1 activation. This unimolecular interaction of SOAR with Orai1 suggests STIM1 can cross-link Orai channels with important significance for Ca2+ signaling. A critical "nexus" region in Orai1 close to the STIM1-binding site can be mutated to constitutively activate the channel mimicking the gating action of STIM1. This indicates STIM1 remotely controls Orai1 channel gating through an allosteric switch triggered by STIM1 binding only to the exposed C-terminal tail of the Orai1 channel.

The STIM1-binding site nexus remotely controls Orai1 channel gating.

The ubiquitously expressed Orai Ca2+ channels are gated through a unique process of intermembrane coupling with the Ca2+-sensing STIM proteins. Despite the significance of Orai1-mediated Ca2+ signals, how gating of Orai1 is triggered by STIM1 remains unknown. A widely held gating model invokes STIM1 binding directly to Orai1 pore-forming helix. Here we report that an Orai1 C-terminal STIM1-binding site, situated far from the N-terminal pore helix, alone provides the trigger that is necessary and sufficient for channel gating. We identify a critical 'nexus' within Orai1 connecting the peripheral C-terminal STIM1-binding site to the Orai1 core helices. Mutation of the nexus transforms Orai1 into a persistently open state exactly mimicking the action of STIM1. We suggest that the Orai1 nexus transduces the STIM1-binding signal through a conformational change in the inner core helices, and that STIM1 remotely gates the Orai1 channel without the necessity for direct STIM1 contact with the pore-forming helix.

The Orai1 Store-operated Calcium Channel Functions as a Hexamer.

Orai channels mediate store-operated Ca2+ signals crucial in regulating transcription in many cell types, and implicated in numerous immunological and inflammatory disorders. Despite their central importance, controversy surrounds the basic subunit structure of Orai channels, with several biochemical and biophysical studies suggesting a tetrameric structure yet crystallographic evidence indicating a hexamer. We systematically investigated the subunit configuration of the functional Orai1 channel, generating a series of tdTomato-tagged concatenated Orai1 channel constructs (dimers to hexamers) expressed in CRISPR-derived ORAI1 knock-out HEK cells, stably expressing STIM1-YFP. Surface biotinylation demonstrated that the full-length concatemers were surface membrane-expressed. Unexpectedly, Orai1 dimers, trimers, tetramers, pentamers, and hexamers all mediated similar and substantial store-operated Ca2+ entry. Moreover, each Orai1 concatemer mediated Ca2+ currents with inward rectification and reversal potentials almost identical to those observed with expressed Orai1 monomer. In Orai1 tetramers, subunit-specific replacement with Orai1 E106A "pore-inactive" subunits revealed that functional channels utilize only the N-terminal dimer from the tetramer. In contrast, Orai1 E106A replacement in Orai1 hexamers established that all the subunits can contribute to channel formation, indicating a hexameric channel configuration. The critical Ca2+ selectivity filter-forming Glu-106 residue may mediate Orai1 channel assembly around a central Ca2+ ion within the pore. Thus, multiple E106A substitutions in the Orai1 hexamer may promote an alternative "trimer-of-dimers" channel configuration in which the C-terminal E106A subunits are excluded from the hexameric core. Our results argue strongly against a tetrameric configuration for Orai1 channels and indicate that the Orai1 channel functions as a hexamer.

STIM1 dimers undergo unimolecular coupling to activate Orai1 channels.

The endoplasmic reticulum (ER) Ca(2+) sensor, STIM1, becomes activated when ER-stored Ca(2+) is depleted and translocates into ER-plasma membrane junctions where it tethers and activates Orai1 Ca(2+) entry channels. The dimeric STIM1 protein contains a small STIM-Orai-activating region (SOAR)--the minimal sequence sufficient to activate Orai1 channels. Since SOAR itself is a dimer, we constructed SOAR concatemer-dimers and introduced mutations at F394, which is critical for Orai1 coupling and activation. The F394H mutation in both SOAR monomers completely blocks dimer function, but F394H introduced in only one of the dimeric SOAR monomers has no effect on Orai1 binding or activation. This reveals an unexpected unimolecular coupling between STIM1 and Orai1 and argues against recent evidence suggesting dimeric interaction between STIM1 and two adjacent Orai1 channel subunits. The model predicts that STIM1 dimers may be involved in crosslinking between Orai1 channels with implications for the kinetics and localization of Orai1 channel opening.