Orai1 Dotaz Zobrazit nápovědu
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
Calcium influx through plasma membrane calcium release-activated calcium (CRAC) channels, which are formed of hexamers of Orai1, is a potent trigger for many important biological processes, most notably in T cell-mediated immunity. Through a bioinformatics-led cell biological screen, we have identified Orai1 as a substrate for the rhomboid intramembrane protease RHBDL2. We show that RHBDL2 prevents stochastic calcium signaling in unstimulated cells through conformational surveillance and cleavage of inappropriately activated Orai1. A conserved disease-linked proline residue is responsible for RHBDL2's recognizing the active conformation of Orai1, which is required to sharpen switch-like signaling triggered by store-operated calcium entry. Loss of RHBDL2 control of CRAC channel activity causes severe dysregulation of downstream CRAC channel effectors, including transcription factor activation, inflammatory cytokine expression, and T cell activation. We propose that this surveillance function may represent an ancient activity of rhomboid proteases in degrading unwanted signaling proteins.
- MeSH
- aktivace lymfocytů MeSH
- buněčná membrána metabolismus MeSH
- Drosophila melanogaster MeSH
- gating iontového kanálu MeSH
- HEK293 buňky MeSH
- konformace proteinů MeSH
- lidé MeSH
- membránové proteiny metabolismus MeSH
- mutace MeSH
- proteasy chemie MeSH
- protein ORAI1 chemie MeSH
- serinové endopeptidasy metabolismus MeSH
- signální transdukce MeSH
- stochastické procesy MeSH
- vápník metabolismus MeSH
- vápníková signalizace fyziologie MeSH
- vápníkové kanály chemie MeSH
- vazba proteinů MeSH
- výpočetní biologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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
- 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
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.
- 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
The Ca(2+) release-activated Ca(2+) channel mediates Ca(2+) influx in a plethora of cell types, thereby controlling diverse cellular functions. The channel complex is composed of stromal interaction molecule 1 (STIM1), an endoplasmic reticulum Ca(2+)-sensing protein, and Orai1, a plasma membrane Ca(2+) channel. Channels composed of STIM1 and Orai1 mediate Ca(2+) influx even at low extracellular Ca(2+) concentrations. We investigated whether the activity of Orai1 adapted to different environmental Ca(2+) concentrations. We used homology modeling and molecular dynamics simulations to predict the presence of an extracellular Ca(2+)-accumulating region (CAR) at the pore entrance of Orai1. Furthermore, simulations of Orai1 proteins with mutations in CAR, along with live-cell experiments, or simulations and electrophysiological recordings of the channel with transient, electrostatic loop3 interacting with loop1 (the site of CAR) determined that CAR enhanced Ca(2+) permeation most efficiently at low external Ca(2+) concentrations. Consistent with these results, cells expressing Orai1 CAR mutants exhibited impaired gene expression stimulated by the Ca(2+)-activated transcription factor nuclear factor of activated T cells (NFAT). We propose that the Orai1 channel architecture with a close proximity of CAR to the selectivity filter, which enables Ca(2+)-selective ion permeation, enhances the local extracellular Ca(2+) concentration to maintain Ca(2+)-dependent gene regulation even in environments with relatively low Ca(2+)concentrations.
- MeSH
- Drosophila melanogaster MeSH
- genetická transkripce fyziologie MeSH
- HEK293 buňky MeSH
- iontový transport fyziologie MeSH
- lidé MeSH
- membránové proteiny * chemie genetika metabolismus MeSH
- permeabilita buněčné membrány fyziologie MeSH
- proteiny Drosophily * chemie genetika metabolismus MeSH
- sekundární struktura proteinů MeSH
- vápník metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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.
- Publikační typ
- časopisecké články MeSH
- Publikační typ
- abstrakt z konference MeSH
The store-operated calcium channels Orai1-3 form extraordinary long and funnel like pores, in stark contrast to a classical pore loop architecture. A hydrophobic segment centrally located in the Orai pore controls gating. Here, we comment on a recent work that describes decisive binding between three residues that controls the open and closed conformation of Orai channels.
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.
- 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
Highly Ca2+ selective channels trigger a large variety of cellular signaling processes in both excitable and non-excitable cells. Among these channels, the Orai channel is unique in its activation mechanism and its structure. It mediates Ca2+ influx into the cytosol with an extremely small unitary conductance over longer time-scales, ranging from minutes up to several hours. Its activation is regulated by the Ca2+ content of the endoplasmic reticulum (ER). Depletion of luminal [Ca2+]ER is sensed by the STIM1 single transmembrane protein that directly binds and gates the Orai1 channel. Orai mediated Ca2+ influx increases cytosolic Ca2+ from 100 nM up to low micromolar range close to the pore and thereby forms Ca2+ microdomains. Hence, these features of the Orai channel can trigger long-term signaling processes without affecting the overall Ca2+ content of a single living cell. Here we focus on the architecture and dynamic conformational changes within the Orai channel. This review summarizes current achievements of molecular dynamics simulations in combination with live cell recordings to address gating and permeation of the Orai channel with molecular precision.
