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

Ca 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 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 interactions. 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 surface. 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 understood.

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.

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 mechanism. Here we show structures of homodimers of the family C metabotropic glutamate receptor 2 (mGlu2) in distinct functional states and in complex with heterotrimeric G. 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.

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

Junctional coupling between endoplasmic reticulum (ER) Ca-sensor STIM proteins and plasma membrane (PM) Orai channels mediates Ca 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 signals, and mediates a key modification of STIM-Orai coupling induced by 2-aminoethoxydiphenyl borate.

The Intricate Coupling Between STIM Proteins and Orai Channels.

Store-operated Ca entry signals are critical for cellular regulation. This intricate signaling pathway involves coupling of proteins in two different membranes: the ER-resident Ca-sensing STIM proteins and the highly Ca-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.

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

Voltage-gated L-type Ca channel (Ca1.2) 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.

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.

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

ORAI1 constitutes the store-operated Ca release-activated Ca (CRAC) channel crucial for life. Whereas ORAI1 activation by Ca-sensing STIM proteins is known, still obscure is how ORAI1 is turned off through Ca-dependent inactivation (CDI), protecting against Ca toxicity. Here we identify a spatially-restricted Ca/cAMP signaling crosstalk critical for mediating CDI.

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

Store-operated Ca entry (SOCE) is a ubiquitous pathway for Ca influx across the plasma membrane (PM). SOCE is mediated by the endoplasmic reticulum (ER)-associated Ca-sensing proteins stromal interaction molecule 1 (STIM1) and STIM2, which transition into an active conformation in response to ER Ca store depletion, thereby interacting with and gating PM-associated ORAI1 channels. Although structurally homologous, STIM1 and STIM2 generate distinct Ca signatures in response to varying strengths of agonist stimulation.

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.

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

Store-operated Ca entry signals are mediated by plasma membrane Orai channels activated through intermembrane coupling with Ca-sensing STIM proteins in the endoplasmic reticulum (ER). The nature of this elaborate Orai-gating mechanism has remained enigmatic. Based on the Orai structure, mammalian Orai1 channels are hexamers comprising three dimeric subunit pairs.

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 Ca 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 Ca signals. Using CRISPR/Cas9 gene editing tools, we generated cells with both STIM or all three Orai homologs deleted and directly monitored store Ca and Ca signals.

Cross-linking of Orai1 channels by STIM proteins.

The transmembrane docking of endoplasmic reticulum (ER) Ca-sensing STIM proteins with plasma membrane (PM) Orai Ca channels is a critical but poorly understood step in Ca 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.

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

Store-operated Ca entry fulfills a crucial role in controlling Ca signals in almost all cells. The Ca-sensing stromal interaction molecule (STIM) proteins in the endoplasmic reticulum (ER) undergo complex conformational changes in response to depleted ER luminal Ca, allowing them to unfold and become trapped in ER-plasma membrane (PM) junctions. Dimers of STIM proteins trap and gate the plasma membrane Orai Ca channels within these junctions to generate discrete zones of high Ca and regulate sensitive Ca-dependent intracellular signaling pathways.

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

In virtually all cells, store-operated Ca 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 Ca triggering STIM proteins to undergo an intricate activation process.

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

The ubiquitously expressed Orai Ca channels are gated through a unique process of intermembrane coupling with the Ca-sensing STIM proteins. Despite the significance of Orai1-mediated Ca 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.

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

Orai channels mediate store-operated Ca 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.

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.