Orai1
Dotaz
Zobrazit nápovědu
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.
Signal transduction by the high-affinity IgE receptor (FcεRI) depends on membrane lipid and protein compartmentalization. Recently published data show that cells treated with 1-heptanol, a cell membrane fluidizer, exhibit changes in membrane properties. However, the functional consequences of 1-heptanol-induced changes on mast cell signaling are unknown. This study shows that short-term exposure to 1-heptanol reduces membrane thermal stability and dysregulates mast cell signaling at multiple levels. Cells treated with 1-heptanol exhibited increased lateral mobility and decreased internalization of the FcεRI. However, this did not affect the initial phosphorylation of the FcεRI-β chain and components of the SYK/LAT1/PLCγ1 signaling pathway after antigen activation. In contrast, 1-heptanol inhibited SAPK/JNK phosphorylation and effector functions such as calcium response, degranulation, and cytokine production. Membrane hyperfluidization induced a heat shock-like response via increased expression of the heat shock protein 70, increased lateral diffusion of ORAI1-mCherry, and unsatisfactory performance of STIM1-ORAI1 coupling, as determined by flow-FRET. Furthermore, 1-heptanol inhibited the antigen-induced production of reactive oxygen species and potentiated stress-induced plasma membrane permeability by interfering with heat shock protein 70 activity. The combined data suggest that 1-heptanol-mediated membrane fluidization does not interfere with the earliest biochemical steps of FcεRI signaling, such as phosphorylation of the FcεRI-β chain and components of the SYK/LAT/PLCγ1 signaling pathway, instead inhibiting the FcεRI internalization and mast cell effector functions, including degranulation and cytokine production.
- MeSH
- cholesterol MeSH
- cytokiny MeSH
- heptanol MeSH
- mastocyty * MeSH
- signální transdukce * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Stormorken syndrome is a multiorgan hereditary disease caused by dysfunction of the endoplasmic reticulum (ER) Ca2+ sensor protein STIM1, which forms the Ca2+ release-activated Ca2+ (CRAC) channel together with the plasma membrane channel Orai1. ER Ca2+ store depletion activates STIM1 by releasing the intramolecular "clamp" formed between the coiled coil 1 (CC1) and CC3 domains of the protein, enabling the C terminus to extend and interact with Orai1. The most frequently occurring mutation in patients with Stormorken syndrome is R304W, which destabilizes and extends the STIM1 C terminus independently of ER Ca2+ store depletion, causing constitutive binding to Orai1 and CRAC channel activation. We found that in cis deletion of one amino acid residue, Glu296 (which we called E296del) reversed the pathological effects of R304W. Homozygous Stim1 E296del+R304W mice were viable and phenotypically indistinguishable from wild-type mice. NMR spectroscopy, molecular dynamics simulations, and cellular experiments revealed that although the R304W mutation prevented CC1 from interacting with CC3, the additional deletion of Glu296 opposed this effect by enabling CC1-CC3 binding and restoring the CC domain interactions within STIM1 that are critical for proper CRAC channel function. Our results provide insight into the activation mechanism of STIM1 by clarifying the molecular basis of mutation-elicited protein dysfunction and pathophysiology.
- MeSH
- aminokyseliny metabolismus MeSH
- endoplazmatické retikulum metabolismus MeSH
- kanály aktivované uvolněním vápníku * genetika MeSH
- membránové proteiny * metabolismus MeSH
- mutace MeSH
- myši MeSH
- protein ORAI1 metabolismus MeSH
- protein STIM1 genetika MeSH
- vápník metabolismus MeSH
- vápníkové kanály metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata 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 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.
Cardiac fibrotization is a well-known process characteristic of many cardiac pathological conditions. The key element is excessive activation of cardiac fibroblasts, their transdifferentiation into myofibroblasts, increased production, and accumulation of extracellular matrix proteins, resulting in cardiac stiffness. The exact cellular mechanisms and molecular components involved in the process are not fully elucidated, but the SOCE mechanism could play an important role. Its key molecules are the molecular sensor of calcium in ER/SR - STIM and the highly selective calcium channels Orai located in the plasma membrane. This study aims to evaluate selected SOCE-associated genes in the activation of HCF cell culture by several known substances (phenylephrine, isoprenaline) that represent cardiovascular overload. After cell cultivation, cell medium was collected to measure the soluble collagen content. From the harvested cells, qRT-PCR was performed to determine the mRNA levels of the corresponding genes. The activation of cells was based on changes in the relative expression of collagen genes as well as the collagen content in the medium of the cell culture. We detected an increase in the expression of the Orai2 isoform, a change in the Orai1/Orai3 ratio and also an increase in the expression of the STIM2 isoform. These results suggest an increased activation of the SOCE mechanism under stress conditions of fibroblasts, which supports the hypothesis of fibroblast activation in pathological processes by altering calcium homeostasis through the SOCE mechanism.
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
The calcium release activated calcium channel is activated by the endoplasmic reticulum-resident calcium sensor protein STIM1. On activation, STIM1 C terminus changes from an inactive, tight to an active, extended conformation. A coiled-coil clamp involving the CC1 and CC3 domains is essential in controlling STIM1 activation, with CC1 as the key entity. The nuclear magnetic resonance-derived solution structure of the CC1 domain represents a three-helix bundle stabilized by interhelical contacts, which are absent in the Stormorken disease-related STIM1 R304W mutant. Two interhelical sites between the CC1α1 and CC1α2 helices are key in controlling STIM1 activation, affecting the balance between tight and extended conformations. Nuclear magnetic resonance-directed mutations within these interhelical interactions restore the physiological, store-dependent activation behavior of the gain-of-function STIM1 R304W mutant. This study reveals the functional impact of interhelical interactions within the CC1 domain for modifying the CC1-CC3 clamp strength to control the activation of STIM1.
- MeSH
- abnormální erytrocyty MeSH
- dyslexie genetika MeSH
- HEK293 buňky MeSH
- ichtyóza genetika MeSH
- kanály aktivované uvolněním vápníku metabolismus MeSH
- klonování DNA MeSH
- konformace nukleové kyseliny MeSH
- lidé MeSH
- magnetická rezonanční spektroskopie MeSH
- metoda terčíkového zámku MeSH
- migréna genetika MeSH
- mióza genetika MeSH
- molekulární modely MeSH
- mutace genetika MeSH
- nádorové proteiny genetika MeSH
- protein ORAI1 genetika MeSH
- protein STIM1 genetika MeSH
- slezina abnormality MeSH
- svalová únava genetika MeSH
- trombocytopatie genetika MeSH
- Check Tag
- lidé 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
Store-operated calcium entry (SOCE) is one of regulatory mechanisms which regulates Ca2+ cycling in the heart. SOCE alterations in pathological conditions contribute to progression of heart failure and cardiac hypertrophy by multiple signaling pathways such as Cn/NFAT and CaMKII/MEF2. Several components mediating SOCE have been identified, such as STIM and Orai. Different isoforms of both Orai and STIM have been detected in animal studies, exhibiting distinct functional properties. This study is focused on the analysis of STIM and Orai isoforms expression in the end-stage human failing myocardium. Left ventricle samples isolated from 43 explanted hearts from patients undergoing heart transplant and from 5 healthy donor hearts were used to determine the mRNA levels of Orai1, Orai2 and Orai3, STIM1, STIM2 and STIM2.1 by qRT-PCR. The expression was further analyzed for connection with gender, related co-morbidities, pathoetiology, clinical data and biochemical parameters. We show that Orai1 expression is decreased by 30 % in failing myocardium, even though we detected no significant changes in expression of Orai2 or Orai3. Interestingly, this decrease in Orai1 was gender-specific and was present only in men, with no change in women. The ratio Orai1/Orai3 was significantly lower in males as well. The novel STIM2.1 isoform was detected both in healthy and failing human myocardium. In the end-stage heart failure, the expression of STIM2.1 was significantly decreased. The lower ratio of STIM2.1/STIM2 in failing hearts indicates a switch from SOCE-inhibiting STIM2.1 isoform to stimulatory STIM2.2. STIM1 mRNA levels were not significantly changed. These observed alterations in Orai and STIM expression were independent of functional heart parameters, clinical or biochemical patient characteristics. These results provide detailed insight into the alterations of SOCE regulation in human failing myocardium. Gender-specific change in Orai1 expression might represent a possible mechanism of cardioprotective effects of estrogens. The switch from STIM2.1 to STIM2.2 indicates an amplification of SOCE and could contribute to the hypertrophy development in the filing heart.
- MeSH
- dospělí MeSH
- kanály aktivované uvolněním vápníku metabolismus MeSH
- lidé středního věku MeSH
- lidé MeSH
- myokard metabolismus MeSH
- pohlavní dimorfismus MeSH
- protein - isoformy metabolismus MeSH
- proteiny STIM metabolismus MeSH
- remodelace komor MeSH
- srdeční selhání metabolismus MeSH
- studie případů a kontrol MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
