The channel Orai1 requires Ca²⁺ store depletion in the endoplasmic reticulum and an interaction with the Ca²⁺ sensor STIM1 to mediate Ca²⁺ signaling. Alterations in Orai1-mediated Ca²⁺ influx have been linked to several pathological conditions including immunodeficiency, tubular myopathy, and cancer. We screened large-scale cancer genomics data sets for dysfunctional Orai1 mutants. Five of the identified Orai1 mutations resulted in constitutively active gating and transcriptional activation. Our analysis showed that certain Orai1 mutations were clustered in the transmembrane 2 helix surrounding the pore, which is a trigger site for Orai1 channel gating. Analysis of the constitutively open Orai1 mutant channels revealed two fundamental gates that enabled Ca²⁺ influx: Arginine side chains were displaced so they no longer blocked the pore, and a chain of water molecules formed in the hydrophobic pore region. Together, these results enabled us to identify a cluster of Orai1 mutations that trigger Ca²⁺ permeation associated with gene transcription and provide a gating mechanism for Orai1.

Disease-associated mutations in the Ca ²⁺ sensor STIM1 alter its conformation and disrupt Ca ²⁺ ion binding.

The family of stromal interaction molecules (STIM) includes two widely expressed single‐pass endoplasmic reticulum (ER) transmembrane proteins and additional splice variants that act as precise ER‐luminal Ca²⁺ sensors. STIM proteins mainly function as one of the two essential components of the so‐called Ca²⁺ release‐activated Ca²⁺ (CRAC) channel. The second CRAC channel component is constituted by pore‐forming Orai proteins in the plasma membrane. STIM and Orai physically interact with each other to enable CRAC channel opening, which is a critical prerequisite for various downstream signalling pathways such as gene transcription or proliferation. Their activation commonly requires the emptying of the intracellular ER Ca²⁺ store. Using their Ca²⁺ sensing capabilities, STIM proteins confer this Ca²⁺ content‐dependent signal to Orai, thereby linking Ca²⁺ store depletion to CRAC channel opening. Here we review the conformational dynamics occurring along the entire STIM protein upon store depletion, involving the transition from the quiescent, compactly folded structure into an active, extended state, modulation by a variety of accessory components in the cell as well as the impairment of individual steps of the STIM activation cascade associated with disease. image

The initial activation step in the gating of ubiquitously expressed Orai1 calcium (Ca²⁺) ion channels represents the activation of the Ca²⁺-sensor protein STIM1 upon Ca²⁺ store depletion of the endoplasmic reticulum. Previous studies using constitutively active Orai1 mutants gave rise to, but did not directly test, the hypothesis that STIM1-mediated Orai1 pore opening is accompanied by a global conformational change of all Orai transmembrane domain (TM) helices within the channel complex. We prove that a local conformational change spreads omnidirectionally within the Orai1 complex. Our results demonstrate that these locally induced global, opening-permissive TM motions are indispensable for pore opening and require clearance of a series of Orai1 gating checkpoints. We discovered these gating checkpoints in the middle and cytosolic extended TM domain regions. Our findings are based on a library of double point mutants that contain each one loss-of-function with one gain-of-function point mutation in a series of possible combinations. We demonstrated that an array of loss-of-function mutations are dominant over most gain-of-function mutations within the same as well as of an adjacent Orai subunit. We further identified inter- and intramolecular salt-bridge interactions of Orai subunits as a core element of an opening-permissive Orai channel architecture. Collectively, clearance and synergistic action of all these gating checkpoints are required to allow STIM1 coupling and Orai1 pore opening. Our results unravel novel insights in the preconditions of the unique fingerprint of CRAC channel activation, provide a valuable source for future structural resolutions, and help to understand the molecular basis of disease-causing mutations.

The single-pass transmembrane protein Stromal Interaction Molecule 1 (STIM1), located in the endoplasmic reticulum (ER) membrane, possesses two main functions: It senses the ER-Ca ²⁺ concentration and directly binds to the store-operated Ca ²⁺ channel Orai1 for its activation when Ca ²⁺ recedes. At high resting ER-Ca ²⁺ concentration, the ER-luminal STIM1 domain is kept monomeric but undergoes di/multimerization once stores are depleted. Luminal STIM1 multimerization is essential to unleash the STIM C-terminal binding site for Orai1 channels. However, structural basis of the luminal association sites has so far been elusive. Here, we employed molecular dynamics (MD) simulations and identified two essential di/multimerization segments, the α7 and the adjacent region near the α9-helix in the sterile alpha motif (SAM) domain. Based on MD results, we targeted the two STIM1 SAM domains by engineering point mutations. These mutations interfered with higher-order multimerization of ER-luminal fragments in biochemical assays and puncta formation in live-cell experiments upon Ca ²⁺ store depletion. The STIM1 multimerization impeded mutants significantly reduced Ca ²⁺ entry via Orai1, decreasing the Ca ²⁺ oscillation frequency as well as store-operated Ca ²⁺ entry. Combination of the ER-luminal STIM1 multimerization mutations with gain of function mutations and coexpression of Orai1 partially ameliorated functional defects. Our data point to a hydrophobicity-driven binding within the ER-luminal STIM1 multimer that needs to switch between resting monomeric and activated multimeric state. Altogether, these data reveal that interactions between SAM domains of STIM1 monomers are critical for multimerization and activation of the protein.

The activation of the Ca²⁺-channel Orai1 via the physiological activator stromal interaction molecule 1 (STIM1) requires structural rearrangements within the entire channel complex involving a series of gating checkpoints. Focusing on the gating mechanism operating along the peripheral transmembrane domain (TM) 3/TM4-interface, we report here that some charged substitutions close to the center of TM3 or TM4 lead to constitutively active Orai1 variants triggering nuclear factor of activated T-cell (NFAT) translocation into the nucleus. Molecular dynamics simulations unveil that this gain-of-function correlates with enhanced hydration at peripheral TM-interfaces, leading to increased local structural flexibility of the channel periphery and global conformational changes permitting pore opening. Our findings indicate that efficient dehydration of the peripheral TM-interfaces driven by the hydrophobic effect is critical for maintaining the closed state of Orai1. We conclude that a charge close to the center of TM3 or TM4 facilitates concomitant hydration and widening of peripheral TM interfaces to trigger constitutive Orai1 pore opening to a level comparable to or exceeding that of native activated Orai1.