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