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.

• Klíčová slova
• Ca2+, SOCE, GBM, Orai, STIM, Synta66, binding, docking, glioblastoma multiforme, pocket, pore,
• Publikační typ
• časopisecké články 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

STIM1 and Orai1 are key components of the Ca2+-release activated Ca2+ (CRAC) current. Orai1, which represents the subunit forming the CRAC channel complex, is activated by the ER resident Ca2+ sensor STIM1. The genetically inherited Stormorken syndrome disease has been associated with the STIM1 single point R304W mutant. The resulting constitutive activation of Orai1 mainly involves the CRAC-activating domain CAD/SOAR of STIM1, the exposure of which is regulated by the molecular interplay between three cytosolic STIM1 coiled-coil (CC) domains. Here we present a dual mechanism by which STIM1 R304W attains the pathophysiological, constitutive activity eliciting the Stormorken syndrome. The R304W mutation induces a helical elongation within the CC1 domain, which together with an increased CC1 homomerization, destabilize the resting state of STIM1. This culminates, even in the absence of store depletion, in structural extension and CAD/SOAR exposure of STIM1 R304W leading to constitutive CRAC channel activation and Stormorken disease.

• MeSH
• abnormální erytrocyty metabolismus   patologie   MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• bodová mutace * MeSH
• dyslexie genetika   metabolismus   patologie   MeSH
• exprese genu MeSH
• HEK293 buňky MeSH
• ichtyóza genetika   metabolismus   patologie   MeSH
• interakční proteinové domény a motivy MeSH
• iontový transport MeSH
• konformace proteinů, alfa-helix MeSH
• lidé MeSH
• luminescentní proteiny genetika   metabolismus   MeSH
• metoda terčíkového zámku MeSH
• migréna genetika   metabolismus   patologie   MeSH
• mióza genetika   metabolismus   patologie   MeSH
• molekulární modely MeSH
• multimerizace proteinu 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
• sekvence aminokyselin MeSH
• slezina abnormality   metabolismus   patologie   MeSH
• substituce aminokyselin MeSH
• svalová únava genetika   MeSH
• trombocytopatie genetika   metabolismus   patologie   MeSH
• vápník chemie   metabolismus   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
• bakteriální proteiny MeSH
• Cyan Fluorescent Protein MeSH Prohlížeč
• 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

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

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
• protein ORAI1 MeSH
• protein STIM1 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
• Názvy látek
• membránové proteiny * MeSH
• olf186-F protein, Drosophila MeSH Prohlížeč
• protein ORAI1 MeSH
• protein STIM1 MeSH
• proteiny Drosophily * MeSH
• Stim protein, Drosophila MeSH Prohlížeč
• vápník MeSH