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.

• Keywords
• AND-gate, CRAC channel, Electrophysiology, Gating, Gating checkpoints, Opening-permissive conformation, Orai1, STIM1, Signal propagation,
• MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Phosphatidylcholines chemistry   metabolism   MeSH
• Ion Channel Gating genetics   MeSH
• Genetic Vectors chemistry   metabolism   MeSH
• HEK293 Cells MeSH
• Protein Interaction Domains and Motifs MeSH
• Protein Conformation, alpha-Helical MeSH
• Protein Conformation, beta-Strand MeSH
• Humans MeSH
• Liposomes chemistry   metabolism   MeSH
• Luminescent Proteins genetics   metabolism   MeSH
• Patch-Clamp Techniques MeSH
• Mutation MeSH
• Neoplasm Proteins chemistry   genetics   metabolism   MeSH
• ORAI1 Protein chemistry   genetics   metabolism   MeSH
• Stromal Interaction Molecule 1 chemistry   genetics   metabolism   MeSH
• Gene Expression Regulation MeSH
• Recombinant Proteins chemistry   genetics   metabolism   MeSH
• Genes, Reporter MeSH
• Molecular Dynamics Simulation MeSH
• Amino Acid Substitution MeSH
• Calcium metabolism   MeSH
• Calcium Signaling * MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Green Fluorescent Proteins genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 1-palmitoyl-2-oleoylphosphatidylcholine MeSH Browser
• Bacterial Proteins MeSH
• enhanced cyan fluorescent protein MeSH Browser
• Phosphatidylcholines MeSH
• Liposomes MeSH
• Luminescent Proteins MeSH
• Neoplasm Proteins MeSH
• ORAI1 protein, human MeSH Browser
• ORAI1 Protein MeSH
• Stromal Interaction Molecule 1 MeSH
• Recombinant Proteins MeSH
• STIM1 protein, human MeSH Browser
• Calcium MeSH
• yellow fluorescent protein, Bacteria MeSH Browser
• Green Fluorescent Proteins MeSH

The Ca2+ sensor STIM1 and the Ca2+ channel Orai1 that form the store-operated Ca2+ (SOC) channel complex are key targets for drug development. Selective SOC inhibitors are currently undergoing clinical evaluation for the treatment of auto-immune and inflammatory responses and are also deemed promising anti-neoplastic agents since SOC channels are linked with enhanced cancer cell progression. Here, we describe an investigation of the site of binding of the selective inhibitor Synta66 to the SOC channel Orai1 using docking and molecular dynamics simulations, and live cell recordings. Synta66 binding was localized to the extracellular site close to the transmembrane (TM)1 and TM3 helices and the extracellular loop segments, which, importantly, are adjacent to the Orai1-selectivity filter. Synta66-sensitivity of the Orai1 pore was, in fact, diminished by both Orai1 mutations affecting Ca2+ selectivity and permeation of Na+ in the absence of Ca2+. Synta66 also efficiently blocked SOC in three glioblastoma cell lines but failed to interfere with cell viability, division and migration. These experiments provide new structural and functional insights into selective drug inhibition of the Orai1 Ca2+ channel by a high-affinity pore blocker.

• Keywords
• Ca2+, SOCE, GBM, Orai, STIM, Synta66, binding, docking, glioblastoma multiforme, pocket, pore,
• Publication type
• Journal Article MeSH

Stromal interaction molecule 1 (STIM1) is a ubiquitously expressed Ca2+ sensor protein that induces permeation of Orai Ca2+ channels upon endoplasmic reticulum Ca2+-store depletion. A drop in luminal Ca2+ causes partial unfolding of the N-terminal STIM1 domains and thus initial STIM1 activation. We compared the STIM1 structure upon Ca2+ depletion from our molecular dynamics (MD) simulations with a recent 2D NMR structure. Simulation- and structure-based results showed unfolding of two α-helices in the canonical and in the non-canonical EF-hand. Further, we structurally and functionally evaluated mutations in the non-canonical EF-hand that have been shown to cause tubular aggregate myopathy. We found these mutations to cause full constitutive activation of Ca2+-release-activated Ca2+ currents (ICRAC) and to promote autophagic processes. Specifically, heterologously expressed STIM1 mutations in the non-canonical EF-hand promoted translocation of the autophagy transcription factors microphthalmia-associated transcription factor (MITF) and transcription factor EB (TFEB) into the nucleus. These STIM1 mutations additionally stimulated an enhanced production of autophagosomes. In summary, mutations in STIM1 that cause structural unfolding promoted Ca2+ down-stream activation of autophagic processes.

• Keywords
• Ca2+, EF-hand, MITF, Orai, SOCE, STIM, TFEB, hydrophobic pocket, tubular aggregate myopathy,
• MeSH
• Autophagy * MeSH
• Cations, Divalent metabolism   MeSH
• Protein Conformation, alpha-Helical MeSH
• Humans MeSH
• EF Hand Motifs MeSH
• Mutation MeSH
• Myopathies, Structural, Congenital genetics   metabolism   MeSH
• Neoplasm Proteins chemistry   genetics   metabolism   MeSH
• Stromal Interaction Molecule 1 chemistry   genetics   metabolism   MeSH
• Protein Unfolding MeSH
• Molecular Dynamics Simulation MeSH
• Calcium metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cations, Divalent MeSH
• Neoplasm Proteins MeSH
• Stromal Interaction Molecule 1 MeSH
• STIM1 protein, human MeSH Browser
• Calcium MeSH

Ca2+ release-activated Ca2+ (CRAC) channels constitute the major Ca2+ entry pathway into the cell. They are fully reconstituted via intermembrane coupling of the Ca2+-selective Orai channel and the Ca2+-sensing protein STIM1. In addition to the Orai C terminus, the main coupling site for STIM1, the Orai N terminus is indispensable for Orai channel gating. Although the extended transmembrane Orai N-terminal region (Orai1 amino acids 73-91; Orai3 amino acids 48-65) is fully conserved in the Orai1 and Orai3 isoforms, Orai3 tolerates larger N-terminal truncations than Orai1 in retaining store-operated activation. In an attempt to uncover the reason for these isoform-specific structural requirements, we analyzed a series of Orai mutants and chimeras. We discovered that it was not the N termini, but the loop2 regions connecting TM2 and TM3 of Orai1 and Orai3 that featured distinct properties, which explained the different, isoform-specific behavior of Orai N-truncation mutants. Atomic force microscopy studies and MD simulations suggested that the remaining N-terminal portion in the non-functional Orai1 N-truncation mutants formed new, inhibitory interactions with the Orai1-loop2 regions, but not with Orai3-loop2. Such a loop2 swap restored activation of the N-truncation Orai1 mutants. To mimic interactions between the N terminus and loop2 in full-length Orai1 channels, we induced close proximity of the N terminus and loop2 via cysteine cross-linking, which actually caused significant inhibition of STIM1-mediated Orai currents. In aggregate, maintenance of Orai activation required not only the conserved N-terminal region but also permissive communication of the Orai N terminus and loop2 in an isoform-specific manner.

• Keywords
• atomic force microscopy (AFM), calcium release-activated calcium channel protein 1 (ORAI1), electrophysiology, signal transduction, stromal interaction molecule 1 (STIM1),
• MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Neoplasm Proteins chemistry   genetics   metabolism   MeSH
• ORAI1 Protein chemistry   genetics   metabolism   MeSH
• Stromal Interaction Molecule 1 chemistry   genetics   metabolism   MeSH
• Protein Domains MeSH
• Protein Structure, Secondary MeSH
• Calcium Channels chemistry   genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Neoplasm Proteins MeSH
• ORAI1 protein, human MeSH Browser
• Orai3 protein, human MeSH Browser
• ORAI1 Protein MeSH
• Stromal Interaction Molecule 1 MeSH
• STIM1 protein, human MeSH Browser
• Calcium Channels MeSH

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.

• MeSH
• Transcriptional Activation genetics   MeSH
• Arginine metabolism   MeSH
• Cell Membrane metabolism   MeSH
• Drosophila melanogaster MeSH
• Ion Channel Gating genetics   MeSH
• Genomics MeSH
• HCT116 Cells MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Patch-Clamp Techniques MeSH
• Mutation MeSH
• Neoplasm Proteins genetics   metabolism   MeSH
• Neoplasms metabolism   MeSH
• Muscular Diseases metabolism   MeSH
• ORAI1 Protein genetics   metabolism   MeSH
• Stromal Interaction Molecule 1 genetics   metabolism   MeSH
• Protein Structure, Secondary genetics   MeSH
• Molecular Dynamics Simulation MeSH
• Calcium metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Arginine MeSH
• Neoplasm Proteins MeSH
• ORAI1 protein, human MeSH Browser
• ORAI1 Protein MeSH
• Stromal Interaction Molecule 1 MeSH
• STIM1 protein, human MeSH Browser
• Calcium MeSH