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.

• Klíčová slova
• AND-gate, CRAC channel, Electrophysiology, Gating, Gating checkpoints, Opening-permissive conformation, Orai1, STIM1, Signal propagation,
• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• fosfatidylcholiny chemie   metabolismus   MeSH
• gating iontového kanálu genetika   MeSH
• genetické vektory chemie   metabolismus   MeSH
• HEK293 buňky MeSH
• interakční proteinové domény a motivy MeSH
• konformace proteinů, alfa-helix MeSH
• konformace proteinů, beta-řetězec MeSH
• lidé MeSH
• liposomy chemie   metabolismus   MeSH
• luminescentní proteiny genetika   metabolismus   MeSH
• metoda terčíkového zámku MeSH
• mutace MeSH
• nádorové proteiny chemie   genetika   metabolismus   MeSH
• protein ORAI1 chemie   genetika   metabolismus   MeSH
• protein STIM1 chemie   genetika   metabolismus   MeSH
• regulace genové exprese MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• reportérové geny MeSH
• simulace molekulární dynamiky MeSH
• substituce aminokyselin MeSH
• vápník metabolismus   MeSH
• vápníková signalizace * MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 1-palmitoyl-2-oleoylphosphatidylcholine MeSH Prohlížeč
• bakteriální proteiny MeSH
• enhanced cyan fluorescent protein MeSH Prohlížeč
• fosfatidylcholiny MeSH
• liposomy MeSH
• luminescentní proteiny MeSH
• nádorové proteiny MeSH
• ORAI1 protein, human MeSH Prohlížeč
• protein ORAI1 MeSH
• protein STIM1 MeSH
• rekombinantní proteiny MeSH
• STIM1 protein, human MeSH Prohlížeč
• vápník MeSH
• yellow fluorescent protein, Bacteria MeSH Prohlížeč
• zelené fluorescenční proteiny 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.

• Klíčová slova
• atomic force microscopy (AFM), calcium release-activated calcium channel protein 1 (ORAI1), electrophysiology, signal transduction, stromal interaction molecule 1 (STIM1),
• MeSH
• HEK293 buňky MeSH
• lidé MeSH
• nádorové proteiny chemie   genetika   metabolismus   MeSH
• protein ORAI1 chemie   genetika   metabolismus   MeSH
• protein STIM1 chemie   genetika   metabolismus   MeSH
• proteinové domény MeSH
• sekundární struktura proteinů MeSH
• vápníkové kanály chemie   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• nádorové proteiny MeSH
• ORAI1 protein, human MeSH Prohlížeč
• Orai3 protein, human MeSH Prohlížeč
• protein ORAI1 MeSH
• protein STIM1 MeSH
• STIM1 protein, human MeSH Prohlížeč
• vápníkové kanály 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
• aktivace transkripce genetika   MeSH
• arginin metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• Drosophila melanogaster MeSH
• gating iontového kanálu genetika   MeSH
• genomika MeSH
• HCT116 buňky MeSH
• HEK293 buňky MeSH
• lidé MeSH
• metoda terčíkového zámku MeSH
• mutace MeSH
• nádorové proteiny genetika   metabolismus   MeSH
• nádory metabolismus   MeSH
• nemoci svalů metabolismus   MeSH
• protein ORAI1 genetika   metabolismus   MeSH
• protein STIM1 genetika   metabolismus   MeSH
• sekundární struktura proteinů genetika   MeSH
• simulace molekulární dynamiky MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• arginin MeSH
• nádorové proteiny MeSH
• ORAI1 protein, human MeSH Prohlížeč
• protein ORAI1 MeSH
• protein STIM1 MeSH
• STIM1 protein, human MeSH Prohlížeč
• vápník MeSH

STIM1 (stromal interaction molecule 1) and Orai proteins are the essential components of Ca(2+) release-activated Ca(2+) (CRAC) channels. We focused on the role of cholesterol in the regulation of STIM1-mediated Orai1 currents. Chemically induced cholesterol depletion enhanced store-operated Ca(2+) entry (SOCE) and Orai1 currents. Furthermore, cholesterol depletion in mucosal-type mast cells augmented endogenous CRAC currents, which were associated with increased degranulation, a process that requires calcium influx. Single point mutations in the Orai1 amino terminus that would be expected to abolish cholesterol binding enhanced SOCE to a similar extent as did cholesterol depletion. The increase in Orai1 activity in cells expressing these cholesterol-binding-deficient mutants occurred without affecting the amount in the plasma membrane or the coupling of STIM1 to Orai1. We detected cholesterol binding to an Orai1 amino-terminal fragment in vitro and to full-length Orai1 in cells. Thus, our data showed that Orai1 senses the amount of cholesterol in the plasma membrane and that the interaction of Orai1 with cholesterol inhibits its activity, thereby limiting SOCE.

• MeSH
• biotinylace MeSH
• bodová mutace MeSH
• buněčná membrána metabolismus   MeSH
• buněčné linie MeSH
• cholesterol oxidasa metabolismus   MeSH
• cholesterol metabolismus   MeSH
• cirkulární dichroismus MeSH
• elektrofyziologické jevy MeSH
• fluorescenční spektrometrie MeSH
• HEK293 buňky MeSH
• histamin metabolismus   MeSH
• lidé MeSH
• mastocyty metabolismus   MeSH
• mutace MeSH
• peptidy metabolismus   MeSH
• protein ORAI1 MeSH
• rezonanční přenos fluorescenční energie MeSH
• signální transdukce MeSH
• terciární struktura proteinů MeSH
• vápník metabolismus   MeSH
• vápníkové kanály metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cholesterol oxidasa MeSH
• cholesterol MeSH
• histamin MeSH
• ORAI1 protein, human MeSH Prohlížeč
• peptidy MeSH
• protein ORAI1 MeSH
• vápník MeSH
• vápníkové kanály MeSH