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.

• Klíčová slova
• CRAC, EF‐hand, STIM1, STIM1 C‐terminus,
• MeSH
• kanály aktivované uvolněním vápníku * MeSH
• membránové proteiny metabolismus   MeSH
• protein ORAI1 MeSH
• protein STIM1 metabolismus   MeSH
• proteiny STIM * metabolismus   MeSH
• vápník metabolismus   MeSH
• vápníková signalizace fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• kanály aktivované uvolněním vápníku * MeSH
• membránové proteiny MeSH
• protein ORAI1 MeSH
• protein STIM1 MeSH
• proteiny STIM * MeSH
• vápník 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
• abnormální erytrocyty MeSH
• aminokyseliny metabolismus   MeSH
• dyslexie MeSH
• endoplazmatické retikulum metabolismus   MeSH
• ichtyóza MeSH
• kanály aktivované uvolněním vápníku * genetika   MeSH
• membránové proteiny * metabolismus   MeSH
• migréna MeSH
• mióza MeSH
• mutace MeSH
• myši MeSH
• protein ORAI1 metabolismus   MeSH
• protein STIM1 genetika   MeSH
• slezina abnormality   MeSH
• svalová únava MeSH
• trombocytopatie 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
• Názvy látek
• aminokyseliny MeSH
• kanály aktivované uvolněním vápníku * MeSH
• membránové proteiny * MeSH
• protein ORAI1 MeSH
• protein STIM1 MeSH
• Stim1 protein, mouse MeSH Prohlížeč
• vápník MeSH
• vápníkové kanály 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.

• 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

The protein stromal interaction molecule 1 (STIM1) plays a pivotal role in mediating store-operated calcium entry (SOCE) into cells, which is essential for adaptive immunity. It acts as a calcium sensor in the endoplasmic reticulum (ER) and extends into the cytosol, where it changes from an inactive (tight) to an active (extended) oligomeric form upon calcium store depletion. NMR studies of this protein are challenging due to its membrane-spanning and aggregation properties. Therefore follow the divide-and-conquer approach, focusing on individual domains first is in order. The cytosolic part is predicted to have a large content of coiled-coil (CC) structure. We report the 1H, 13C, 15N chemical shift assignments of the CC3 domain. This domain is crucial for the stabilisation of the tight quiescent form of STIM1 as well as for activating the ORAI calcium channel by direct contact, in the extended active form.

• Klíčová slova
• CRAC, Calcium channel, Coiled-coil structure, Store-operated calcium entry,
• MeSH
• lidé MeSH
• nádorové proteiny * chemie   metabolismus   MeSH
• nukleární magnetická rezonance biomolekulární * MeSH
• protein STIM1 * chemie   metabolismus   MeSH
• proteinové domény * MeSH
• sekvence aminokyselin MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• nádorové proteiny * MeSH
• protein STIM1 * MeSH
• STIM1 protein, human MeSH Prohlížeč

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
• Názvy látek
• kanály aktivované uvolněním vápníku MeSH
• nádorové proteiny MeSH
• ORAI1 protein, human MeSH Prohlížeč
• protein ORAI1 MeSH
• protein STIM1 MeSH
• STIM1 protein, human MeSH Prohlížeč

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.

• Klíčová slova
• Ca(2+) channel, Gating, Orai1, Pore, STIM1,
• MeSH
• gating iontového kanálu * MeSH
• protein ORAI1 genetika   metabolismus   MeSH
• protein STIM1 metabolismus   MeSH
• síra MeSH
• vápníkové kanály * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• komentáře MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protein ORAI1 MeSH
• protein STIM1 MeSH
• síra MeSH
• vápníkové kanály * 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.

• Klíčová slova
• Ca2+, EF-hand, MITF, Orai, SOCE, STIM, TFEB, hydrophobic pocket, tubular aggregate myopathy,
• MeSH
• autofagie * MeSH
• kationty dvojmocné metabolismus   MeSH
• konformace proteinů, alfa-helix MeSH
• lidé MeSH
• motivy EF-ruky MeSH
• mutace MeSH
• myopatie strukturální vrozené genetika   metabolismus   MeSH
• nádorové proteiny chemie   genetika   metabolismus   MeSH
• protein STIM1 chemie   genetika   metabolismus   MeSH
• rozbalení proteinů MeSH
• simulace molekulární dynamiky MeSH
• vápník metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kationty dvojmocné MeSH
• nádorové proteiny MeSH
• protein STIM1 MeSH
• STIM1 protein, human MeSH Prohlížeč
• vápník MeSH

The stromal interaction molecule 1 (STIM1) has two important functions, Ca2+ sensing within the endoplasmic reticulum and activation of the store-operated Ca2+ channel Orai1, enabling plasma-membrane Ca2+ influx. We combined molecular dynamics (MD) simulations with live-cell recordings and determined the sequential Ca2+-dependent conformations of the luminal STIM1 domain upon activation. Furthermore, we identified the residues within the canonical and noncanonical EF-hand domains that can bind to multiple Ca2+ ions. In MD simulations, a single Ca2+ ion was sufficient to stabilize the luminal STIM1 complex. Ca2+ store depletion destabilized the two EF hands, triggering disassembly of the hydrophobic cleft that they form together with the stable SAM domain. Point mutations associated with tubular aggregate myopathy or cancer that targeted the canonical EF hand, and the hydrophobic cleft yielded constitutively clustered STIM1, which was associated with activation of Ca2+ entry through Orai1 channels. On the basis of our results, we present a model of STIM1 Ca2+ binding and refine the currently known initial steps of STIM1 activation on a molecular level.

• MeSH
• algoritmy MeSH
• buněčná membrána metabolismus   MeSH
• endoplazmatické retikulum metabolismus   MeSH
• HEK293 buňky MeSH
• hydrofobní a hydrofilní interakce MeSH
• konfokální mikroskopie MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• motivy EF-ruky MeSH
• mutace MeSH
• nádorové buněčné linie MeSH
• nádorové proteiny chemie   genetika   metabolismus   MeSH
• protein ORAI1 chemie   metabolismus   MeSH
• protein STIM1 chemie   genetika   metabolismus   MeSH
• proteinové domény * MeSH
• rozbalení proteinů * MeSH
• simulace molekulární dynamiky * MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• nádorové proteiny MeSH
• protein ORAI1 MeSH
• protein STIM1 MeSH
• STIM1 protein, human MeSH Prohlížeč
• vápník 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.

• Klíčová slova
• Calcium, Gating, MD-simulations, Molecular dynamics, Orai, Orai1, Pore, STIM,
• MeSH
• lidé MeSH
• nádorové proteiny chemie   metabolismus   MeSH
• protein ORAI1 chemie   metabolismus   MeSH
• protein STIM1 chemie   metabolismus   MeSH
• simulace molekulární dynamiky * MeSH
• vápník metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• nádorové proteiny MeSH
• ORAI1 protein, human MeSH Prohlížeč
• protein ORAI1 MeSH
• protein STIM1 MeSH
• STIM1 protein, human MeSH Prohlížeč
• vápník MeSH

We report a new NMR-scale purification procedure for two recombinant wild type fragments of the stromal interaction molecule 1 (STIM1). This protein acts as a calcium sensor in the endoplasmic reticulum (ER) and extends into the cytosol accumulating at ER - plasma membrane (PM) junctions upon calcium store depletion ultimately leading to activation of the Orai/CRAC channel. The functionally relevant cytosolic part of STIM1 consists of three coiled coil domains, which are mainly involved in intra- and inter-molecular homomeric interactions as well as coupling to and gating of CRAC channels. The optimized one-step rapid purification procedure for two 15N,13C isotope-labeled cytosolic coiled coil fragments, which avoids the problems of previous approaches. The high yields of soluble well folded 15N,13C isotope-labeled cytosolic coiled coil fragments followed by detergent screening provide for initial NMR characterization of these domains. The longer 30.5 kDa fragment represents the largest STIM1 wild type fragment that has been recombinantly prepared and characterized in solution without need for mutation or refolding.

• Klíčová slova
• Calcium channel, Coiled coil, NMR spectroscopy, Oligomerization, Signal protein,
• MeSH
• chromatografie afinitní MeSH
• dynamický rozptyl světla MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• izotopové značení MeSH
• izotopy dusíku chemie   izolace a purifikace   MeSH
• izotopy uhlíku chemie   izolace a purifikace   MeSH
• lidé MeSH
• nádorové proteiny chemie   izolace a purifikace   MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• protein STIM1 chemie   izolace a purifikace   MeSH
• proteinové domény MeSH
• rekombinantní proteiny chemie   izolace a purifikace   MeSH
• rozpustnost MeSH
• sbalování proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Carbon-13 MeSH Prohlížeč
• izotopy dusíku MeSH
• izotopy uhlíku MeSH
• nádorové proteiny MeSH
• Nitrogen-15 MeSH Prohlížeč
• protein STIM1 MeSH
• rekombinantní proteiny MeSH
• STIM1 protein, human MeSH Prohlížeč