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Donald L. Gill - One of the best experts on this subject based on the ideXlab platform.
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The STIM1-binding site nexus remotely controls Orai1 channel gating.
Nature communications, 2016Co-Authors: Yandong Zhou, Xizhuo Wang, Youjun Wang, Brad S Rothberg, Mohamed Trebak, Xiangyu Cai, Natalia A. Loktionova, Xianming Wang, Robert M. Nwokonko, Donald L. GillAbstract:The ubiquitously expressed Orai Ca2+ channels are gated through a unique process of intermembrane coupling with the Ca2+-sensing STIM proteins. Despite the significance of Orai1-mediated Ca2+ signals, how gating of Orai1 is triggered by STIM1 remains unknown. A widely held gating model invokes STIM1 binding directly to Orai1 pore-forming helix. Here we report that an Orai1 C-terminal STIM1-binding site, situated far from the N-terminal pore helix, alone provides the trigger that is necessary and sufficient for channel gating. We identify a critical ‘nexus’ within Orai1 connecting the peripheral C-terminal STIM1-binding site to the Orai1 core helices. Mutation of the nexus transforms Orai1 into a persistently open state exactly mimicking the action of STIM1. We suggest that the Orai1 nexus transduces the STIM1-binding signal through a conformational change in the inner core helices, and that STIM1 remotely gates the Orai1 channel without the necessity for direct STIM1 contact with the pore-forming helix. How plasma membrane Orai Ca2+ channels are activated by STIM proteins to activate Ca2+signals is still not fully known. Here the authors show that a nexus region located at the Orai1 C-terminus allows channel gating without a direct interaction of STIM1 with the channel pore.
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Molecular mechanisms underlying inhibition of STIM1-Orai1-mediated Ca2+ entry induced by 2-aminoethoxydiphenyl borate.
Pflugers Archiv : European journal of physiology, 2016Co-Authors: Yandong Zhou, Guolin Ma, Shuce Zhang, Shenyuan L. Zhang, Lian He, Lijuan Zhou, Donald L. GillAbstract:Store-operated Ca2+ entry (SOCE) mediated by STIM1 and Orai1 is crucial for Ca2+ signaling and homeostasis in most cell types. 2-Aminoethoxydiphenyl borate (2-APB) is a well-described SOCE inhibitor, but its mechanisms of action remain largely elusive. Here, we show that 2-APB does not affect the dimeric state of STIM1, but enhances the intramolecular coupling between the coiled-coil 1 (CC1) and STIM-Orai-activating region (SOAR) of STIM1, with subsequent reduction in the formation of STIM1 puncta in the absence of Orai1 overexpression. 2-APB also inhibits Orai1 channels, directly inhibiting Ca2+ entry through the constitutively active, STIM1-independent Orai1 mutants, Orai1-P245T and Orai1-V102A. When unbound from STIM1, the constitutively active Orai1-V102C mutant is not inhibited by 2-APB. Thus, we used Orai1-V012C as a tool to examine whether 2-APB can also inhibit the coupling between STIM1 and Orai1. We reveal that the functional coupling between STIM1 and Orai1-V102C is inhibited by 2-APB. This inhibition on coupling is indirect, arising from 2-APB's action on STIM1, and it is most likely mediated by functional channel residues in the Orai1 N-terminus. Overall, our findings on this two-site inhibition mediated by 2-APB provide new understanding on Orai1-activation by STIM1, important to future drug design.
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Molecular mechanisms underlying inhibition of STIM1-Orai1-mediated Ca 2+ entry induced by 2-aminoethoxydiphenyl borate.
Pflügers Archiv: European Journal of Physiology, 2016Co-Authors: Ming Wei, Yandong Zhou, Shuce Zhang, Shenyuan L. Zhang, Lijuan Zhou, Aomin Sun, Jin Liu, Donald L. GillAbstract:Store-operated Ca2+ entry (SOCE) mediated by STIM1 and Orai1 is crucial for Ca2+ signaling and homeostasis in most cell types. 2-Aminoethoxydiphenyl borate (2-APB) is a well-described SOCE inhibitor, but its mechanisms of action remain largely elusive. Here, we show that 2-APB does not affect the dimeric state of STIM1, but enhances the intramolecular coupling between the coiled-coil 1 (CC1) and STIM-Orai-activating region (SOAR) of STIM1, with subsequent reduction in the formation of STIM1 puncta in the absence of Orai1 overexpression. 2-APB also inhibits Orai1 channels, directly inhibiting Ca2+ entry through the constitutively active, STIM1-independent Orai1 mutants, Orai1-P245T and Orai1-V102A. When unbound from STIM1, the constitutively active Orai1-V102C mutant is not inhibited by 2-APB. Thus, we used Orai1-V012C as a tool to examine whether 2-APB can also inhibit the coupling between STIM1 and Orai1. We reveal that the functional coupling between STIM1 and Orai1-V102C is inhibited by 2-APB. This inhibition on coupling is indirect, arising from 2-APB's action on STIM1, and it is most likely mediated by functional channel residues in the Orai1 N-terminus. Overall, our findings on this two-site inhibition mediated by 2-APB provide new understanding on Orai1-activation by STIM1, important to future drug design.
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distinct orai coupling domains in STIM1 and stim2 define the orai activating site
Nature Communications, 2014Co-Authors: Xizhuo Wang, Yandong Zhou, Youjun Wang, Jonathan Soboloff, Eunan Hendron, Salvatore Mancarella, Mark Andrake, Brad S Rothberg, Donald L. GillAbstract:STIM1 and STIM2 are widely expressed endoplasmic reticulum (ER) Ca(2+) sensor proteins able to translocate within the ER membrane to physically couple with and gate plasma membrane Orai Ca(2+) channels. Although they are structurally similar, we reveal critical differences in the function of the short STIM-Orai-activating regions (SOAR) of STIM1 and STIM2. We narrow these differences in Orai1 gating to a strategically exposed phenylalanine residue (Phe-394) in SOAR1, which in SOAR2 is substituted by a leucine residue. Remarkably, in full-length STIM1, replacement of Phe-394 with the dimensionally similar but polar histidine head group prevents both Orai1 binding and gating, creating an Orai1 non-agonist. Thus, this residue is critical in tuning the efficacy of Orai activation. While STIM1 is a full Orai1-agonist, leucine-replacement of this crucial residue in STIM2 endows it with partial agonist properties, which may be critical for limiting Orai1 activation stemming from its enhanced sensitivity to store-depletion.
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Mechanism of Activation of Store-Operated Calcium Entry by 2-Aminoethoxydiphenyl Borate
Biophysical Journal, 2014Co-Authors: Yandong Zhou, Lucas Occhiena, Daniel Y. Chung, Xizhuo Wang, Youjun Wang, Donald L. GillAbstract:Store-operated Ca2+ entry (SOCE) is mediate by STIM-induced activation of Orai channels. The small molecule, 2-aminoethoxydiphenyl borate (2-APB), is known to have a biphasic effect on SOCE; lower 2-APB (≤10 μM) enhances, while higher levels of 2-APB (50-100 μM) are strongly inhibitory following a transient activation. But the mechanism by which 2-APB activates SOCe is still elusive. The effects of 2-APB were examined on coupling between various Orai1 mutants and STIM1, C-terminal (ct) STIM1 fragments, or the STIM-Orai activating region (SOAR) of STIM1, using a combination of imaging and whole-cell patch clamp analysis. Orai1 coupling with STIM1ct, STIM1ct-4EA and SOAR can be transiently activated by 2-APB at low levels (10 µM). The activation of SOCE by low 2-APB is induced by its action on the SOAR-Orai1 complex. We examined which domains/residues in Orai1 or in STIM1 are essential for the activating effect of 2-APB. Adjustment of cytosolic pH upward using nigericin abolished the action of 2-APB. In contrast, lower pH potentiated the activating effect of 2-APB. Our results reveal that the Orai1 C-terminus and N-terminus function in a concerted manner to mediate the activating effect of 2-APB on SOCE. Mutational analysis reveals that the negatively charged residues within the Orai1 C-terminal region are not required for the 2-APB-induced SOCE activation. Our studies provide new insights into the mechanism of 2-APB-induced activation of coupling between STIM and Orai and the triggering of SOCE.
Stefan Feske - One of the best experts on this subject based on the ideXlab platform.
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stim2 targets orai1 STIM1 to the akap79 signaling complex and confers coupling of ca2 entry with nfat1 activation
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Gayeon Son, Stefan Feske, Rajesh Bhardwaj, Krishna P Subedi, Hwei Ling Ong, Lucile Noyer, Hassan Saadi, Changyu Zheng, Indu S AmbudkarAbstract:The Orai1 channel is regulated by stromal interaction molecules STIM1 and STIM2 within endoplasmic reticulum (ER)-plasma membrane (PM) contact sites. Ca2+ signals generated by Orai1 activate Ca2+-dependent gene expression. When compared with STIM1, STIM2 is a weak activator of Orai1, but it has been suggested to have a unique role in nuclear factor of activated T cells 1 (NFAT1) activation triggered by Orai1-mediated Ca2+ entry. In this study, we examined the contribution of STIM2 in NFAT1 activation. We report that STIM2 recruitment of Orai1/STIM1 to ER-PM junctions in response to depletion of ER-Ca2+ promotes assembly of the channel with AKAP79 to form a signaling complex that couples Orai1 channel function to the activation of NFAT1. Knockdown of STIM2 expression had relatively little effect on Orai1/STIM1 clustering or local and global [Ca2+]i increases but significantly attenuated NFAT1 activation and assembly of Orai1 with AKAP79. STIM1ΔK, which lacks the PIP2-binding polybasic domain, was recruited to ER-PM junctions following ER-Ca2+ depletion by binding to Orai1 and caused local and global [Ca2+]i increases comparable to those induced by STIM1 activation of Orai1. However, in contrast to STIM1, STIM1ΔK induced less NFAT1 activation and attenuated the association of Orai1 with STIM2 and AKAP79. Orai1-AKAP79 interaction and NFAT1 activation were recovered by coexpressing STIM2 with STIM1ΔK. Replacing the PIP2-binding domain of STIM1 with that of STIM2 eliminated the requirement of STIM2 for NFAT1 activation. Together, these data demonstrate an important role for STIM2 in coupling Orai1-mediated Ca2+ influx to NFAT1 activation.
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store operated ca2 entry controls ameloblast cell function and enamel development
JCI insight, 2017Co-Authors: Miriam Eckstein, Martin Vaeth, Cinzia Fornai, Manikandan Vinu, Timothy G Bromage, Meerim K Nurbaeva, Jessica L Sorge, Paulo G Coelho, Youssef Idaghdour, Stefan FeskeAbstract:Loss-of-function mutations in stromal interaction molecule 1 (STIM1) impair the activation of Ca2+ release-activated Ca2+ (CRAC) channels and store-operated Ca2+ entry (SOCE), resulting in a disease syndrome called CRAC channelopathy that is characterized by severe dental enamel defects. The cause of these enamel defects has remained unclear given a lack of animal models. We generated STIM1/2K14cre mice to delete STIM1 and its homolog STIM2 in enamel cells. These mice showed impaired SOCE in enamel cells. Enamel in STIM1/2K14cre mice was hypomineralized with decreased Ca content, mechanically weak, and thinner. The morphology of SOCE-deficient ameloblasts was altered, showing loss of the typical ruffled border, resulting in mislocalized mitochondria. Global gene expression analysis of SOCE-deficient ameloblasts revealed strong dysregulation of several pathways. ER stress genes associated with the unfolded protein response were increased in STIM1/2-deficient cells, whereas the expression of components of the glutathione system were decreased. Consistent with increased oxidative stress, we found increased ROS production, decreased mitochondrial function, and abnormal mitochondrial morphology in ameloblasts of STIM1/2K14cre mice. Collectively, these data show that loss of SOCE in enamel cells has substantial detrimental effects on gene expression, cell function, and the mineralization of dental enamel.
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Diseases caused by mutations in ORAI1 and STIM1
Annals of the New York Academy of Sciences, 2015Co-Authors: Rodrigo S. Lacruz, Stefan FeskeAbstract:Ca(2+) release-activated Ca(2+) (CRAC) channels mediate a specific form of Ca(2+) influx called store-operated Ca(2+) entry (SOCE) that contributes to the function of many cell types. CRAC channels are composed of ORAI1 proteins located in the plasma membrane, which form its ion-conducting pore. ORAI1 channels are activated by stromal interaction molecule (STIM) 1 and STIM2 located in the endoplasmic reticulum. Loss- and gain-of-function gene mutations in ORAI1 and STIM1 in human patients cause distinct disease syndromes. CRAC channelopathy is caused by loss-of-function mutations in ORAI1 and STIM1 that abolish CRAC channel function and SOCE; it is characterized by severe combined immunodeficiency (SCID)-like disease, autoimmunity, muscular hypotonia, and ectodermal dysplasia, with defects in sweat gland function and dental enamel formation. The latter defect emphasizes an important role of CRAC channels in tooth development. By contrast, autosomal dominant gain-of-function mutations in ORAI1 and STIM1 result in constitutive CRAC channel activation, SOCE, and increased intracellular Ca(2+) levels that are associated with an overlapping spectrum of diseases, including nonsyndromic tubular aggregate myopathy (TAM) and York platelet and Stormorken syndromes. The latter two syndromes are defined, besides myopathy, by thrombocytopenia, thrombopathy, and bleeding diathesis. The fact that myopathy results from both loss- and gain-of-function mutations in ORAI1 and STIM1 highlights the importance of CRAC channels for Ca(2+) homeostasis in skeletal muscle function. The cellular dysfunction and clinical disease spectrum observed in mutant patients provide important information about the molecular regulation of ORAI1 and STIM1 proteins and the role of CRAC channels in human physiology.
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stim2 enhances receptor stimulated ca2 signaling by promoting recruitment of STIM1 to the endoplasmic reticulum plasma membrane junctions
Science Signaling, 2015Co-Authors: Hwei Ling Ong, Stefan Feske, Kwong Tai Cheng, Xibao Liu, Changyu Zheng, Lorena Brito De Souza, Corinne M Goldsmith, Indu S AmbudkarAbstract:A central component of receptor-evoked Ca(2+) signaling is store-operated Ca(2+) entry (SOCE), which is activated by the assembly of STIM1-Orai1 channels in endoplasmic reticulum (ER) and plasma membrane (PM) (ER-PM) junctions in response to depletion of ER Ca(2+). We report that STIM2 enhances agonist-mediated activation of SOCE by promoting STIM1 clustering in ER-PM junctions at low stimulus intensities. Targeted deletion of STIM2 in mouse salivary glands diminished fluid secretion in vivo and SOCE activation in dispersed salivary acinar cells stimulated with low concentrations of muscarinic receptor agonists. STIM2 knockdown in human embryonic kidney (HEK) 293 cells diminished agonist-induced Ca(2+) signaling and nuclear translocation of NFAT (nuclear factor of activated T cells). STIM2 lacking five carboxyl-terminal amino acid residues did not promote formation of STIM1 puncta at low concentrations of agonist, whereas coexpression of STIM2 with STIM1 mutant lacking the polybasic region STIM1ΔK resulted in co-clustering of both proteins. Together, our findings suggest that STIM2 recruits STIM1 to ER-PM junctions at low stimulus intensities when ER Ca(2+) stores are mildly depleted, thus increasing the sensitivity of Ca(2+) signaling to agonists.
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Role Of STIM1 And Orai1 In Mammalian Oocyte Activation
Qatar Foundation Annual Research Conference Proceedings Volume 2014 Issue 1, 2014Co-Authors: M.sc Satanay Hubrack, Stefan Feske, Ethel Adap, Khaled MachacaAbstract:Upon fertilization, the mammalian oocyte undergoes a series of Ca2+ oscillations, which results in its activation and initiation of embryo development. It is believed that these periodic and extended Ca2+ responses are a consequence of intracellular Ca2+ release coupled to Ca2+ influx across the plasma membrane due to the activation of a process known as Store Operated Calcium Entry (SOCE). The underlying mechanism of SOCE is known to be controlled by Stim-Orai channels, where IP3-mediated emptying of ER calcium stores results in the aggregation of STIM1 molecules followed by interaction with Orai1 channels on the plasma membrane and activation of Ca2+ entry. STIM1 was shown to redistribute in the form of patches after mouse oocyte fertilization in a manner similar to its redistribution after pharmacological Ca2+ store depletion, while down-regulation of Orai1 expression in pig oocytes inhibited oscillations induced by fertilization. We have further shown that overexpression of STIM1 and Orai1 in mouse oocytes disrupts Ca2+ dynamics and inhibits egg activation. These data support a role for SOCE in Ca2+ signaling during fertilization. To further investigate the importance of STIM1 and Orai1 proteins in regulating events of egg activation, we are generating oocyte-specific STIM1 and Orai1 knockout mice by crossing STIM1-flox and Orai1-flox mice with ZP3-Cre transgenic mice. ZP3 is expressed only in oocytes leading to Cre expression only in this cell type and as such an oocyte-specific knockout. Studying rates of fertilization and Ca2+ entry in these knockout oocytes will provide insight into the role of these two proteins in the initiation and persistence of Ca2+oscillations after fertilization, and will help us determine whether there is an absolute requirement for STIM1 and/or Orai1 in regulating Ca2+ responses in early stages of fertilization.
Mohamed Trebak - One of the best experts on this subject based on the ideXlab platform.
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The STIM1-binding site nexus remotely controls Orai1 channel gating.
Nature communications, 2016Co-Authors: Yandong Zhou, Xizhuo Wang, Youjun Wang, Brad S Rothberg, Mohamed Trebak, Xiangyu Cai, Natalia A. Loktionova, Xianming Wang, Robert M. Nwokonko, Donald L. GillAbstract:The ubiquitously expressed Orai Ca2+ channels are gated through a unique process of intermembrane coupling with the Ca2+-sensing STIM proteins. Despite the significance of Orai1-mediated Ca2+ signals, how gating of Orai1 is triggered by STIM1 remains unknown. A widely held gating model invokes STIM1 binding directly to Orai1 pore-forming helix. Here we report that an Orai1 C-terminal STIM1-binding site, situated far from the N-terminal pore helix, alone provides the trigger that is necessary and sufficient for channel gating. We identify a critical ‘nexus’ within Orai1 connecting the peripheral C-terminal STIM1-binding site to the Orai1 core helices. Mutation of the nexus transforms Orai1 into a persistently open state exactly mimicking the action of STIM1. We suggest that the Orai1 nexus transduces the STIM1-binding signal through a conformational change in the inner core helices, and that STIM1 remotely gates the Orai1 channel without the necessity for direct STIM1 contact with the pore-forming helix. How plasma membrane Orai Ca2+ channels are activated by STIM proteins to activate Ca2+signals is still not fully known. Here the authors show that a nexus region located at the Orai1 C-terminus allows channel gating without a direct interaction of STIM1 with the channel pore.
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a novel native store operated calcium channel encoded by orai3 selective requirement of orai3 versus orai1 in estrogen receptor positive versus estrogen receptor negative breast cancer cells
Journal of Biological Chemistry, 2010Co-Authors: Rajender K Motiani, Iskandar F Abdullaev, Mohamed TrebakAbstract:Store-operated calcium (Ca2+) entry (SOCE) mediated by STIM/Orai proteins is a ubiquitous pathway that controls many important cell functions including proliferation and migration. STIM proteins are Ca2+ sensors in the endoplasmic reticulum and Orai proteins are channels expressed at the plasma membrane. The fall in endoplasmic reticulum Ca2+ causes translocation of STIM1 to subplasmalemmal puncta where they activate Orai1 channels that mediate the highly Ca2+-selective Ca2+ release-activated Ca2+ current (ICRAC). Whereas Orai1 has been clearly shown to encode SOCE channels in many cell types, the role of Orai2 and Orai3 in native SOCE pathways remains elusive. Here we analyzed SOCE in ten breast cell lines picked in an unbiased way. We used a combination of Ca2+ imaging, pharmacology, patch clamp electrophysiology, and molecular knockdown to show that native SOCE and ICRAC in estrogen receptor-positive (ER+) breast cancer cell lines are mediated by STIM1/2 and Orai3 while estrogen receptor-negative (ER−) breast cancer cells use the canonical STIM1/Orai1 pathway. The ER+ breast cancer cells represent the first example where the native SOCE pathway and ICRAC are mediated by Orai3. Future studies implicating Orai3 in ER+ breast cancer progression might establish Orai3 as a selective target in therapy of ER+ breast tumors.
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essential role for STIM1 orai1 mediated calcium influx in pdgf induced smooth muscle migration
American Journal of Physiology-cell Physiology, 2010Co-Authors: Jonathan M Bisaillon, Marie Potier, Rajender K Motiani, Jose C Gonzalezcobos, Katharine Halligan, Wael F Alzawahra, Margarida Barroso, Harold A Singer, David Jourdheuil, Mohamed TrebakAbstract:We recently demonstrated that thapsigargin-induced passive store depletion activates Ca2+ entry in vascular smooth muscle cells (VSMC) through stromal interaction molecule 1 (STIM1)/Orai1, independently of transient receptor potential canonical (TRPC) channels. However, under physiological stimulations, despite the ubiquitous depletion of inositol 1,4,5-trisphosphate-sensitive stores, many VSMC PLC-coupled agonists (e.g., vasopressin and endothelin) activate various store-independent Ca2+ entry channels. Platelet-derived growth factor (PDGF) is an important VSMC promigratory agonist with an established role in vascular disease. Nevertheless, the molecular identity of the Ca2+ channels activated by PDGF in VSMC remains unknown. Here we show that inhibitors of store-operated Ca2+ entry (Gd3+ and 2-aminoethoxydiphenyl borate at concentrations as low as 5 μM) prevent PDGF-mediated Ca2+ entry in cultured rat aortic VSMC. Protein knockdown of STIM1, Orai1, and PDGF receptor-β (PDGFRβ) impaired PDGF-mediated Ca2+ influx, whereas Orai2, Orai3, TRPC1, TRPC4, and TRPC6 knockdown had no effect. Scratch wound assay showed that knockdown of STIM1, Orai1, or PDGFRβ inhibited PDGF-mediated VSMC migration, but knockdown of STIM2, Orai2, and Orai3 was without effect. STIM1, Orai1, and PDGFRβ mRNA levels were upregulated in vivo in VSMC from balloon-injured rat carotid arteries compared with noninjured control vessels. Protein levels of STIM1 and Orai1 were also upregulated in medial and neointimal VSMC from injured carotid arteries compared with noninjured vessels, as assessed by immunofluorescence microscopy. These results establish that STIM1 and Orai1 are important components for PDGF-mediated Ca2+ entry and migration in VSMC and are upregulated in vivo during vascular injury and provide insights linking PDGF to STIM1/Orai1 during neointima formation.
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evidence for STIM1 and orai1 dependent store operated calcium influx through icrac in vascular smooth muscle cells role in proliferation and migration
The FASEB Journal, 2009Co-Authors: Marie Potier, Iskandar F Abdullaev, Jonathan M Bisaillon, Jose C Gonzalez, Rajender K Motiani, Harold A Singer, Mohamed TrebakAbstract:The identity of store-operated calcium (Ca(2+)) entry (SOCE) channels in vascular smooth muscle cells (VSMCs) remains a highly contentious issue. Whereas previous studies have suggested that SOCE in VSMCs is mediated by the nonselective transient receptor potential canonical (TRPC) 1 protein, the identification of STIM1 and Orai1 as essential components of I(CRAC), a highly Ca(2+)-selective SOCE current in leukocytes, has challenged that view. Here we show that cultured proliferative migratory VSMCs isolated from rat aorta (called "synthetic") display SOCE with classic features, namely inhibition by 2-aminoethoxydiphenyl borate, ML-9, and low concentrations of lanthanides. On store depletion, synthetic VSMCs and A7r5 cells display currents with characteristics of I(CRAC). Protein knockdown of either STIM1 or Orai1 in synthetic VSMCs greatly reduced SOCE, whereas Orai2, Orai3, TRPC1, TRPC4, and TRPC6 knockdown had no effect. Orai1 knockdown reduced I(CRAC) in synthetic VSMCs and A7r5 cells. Synthetic VSMCs showed up-regulated STIM1/Orai1 proteins and SOCE compared with quiescent freshly isolated VSMC. Knockdown of STIM1 and Orai1 inhibited synthetic VSMC proliferation and migration, whereas STIM2, Orai2, and Orai3 knockdown had no effect. To our knowledge, these results are the first to show I(CRAC) in VSMCs and resolve a long-standing controversy by identifying CRAC as the elusive VSMC SOCE channel important for proliferation and migration.
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STIM1 and orai1 mediate crac currents and store operated calcium entry important for endothelial cell proliferation
Circulation Research, 2008Co-Authors: Iskandar F Abdullaev, Jonathan M Bisaillon, Marie Potier, Jose C Gonzalez, Rajender K Motiani, Mohamed TrebakAbstract:Recent breakthroughs in the store-operated calcium (Ca(2+)) entry (SOCE) pathway have identified STIM1 as the endoplasmic reticulum Ca(2+) sensor and Orai1 as the pore forming subunit of the highly Ca(2+)-selective CRAC channel expressed in hematopoietic cells. Previous studies, however, have suggested that endothelial cell (EC) SOCE is mediated by the nonselective canonical transient receptor potential channel (TRPC) family, TRPC1 or TRPC4. Here, we show that passive store depletion by thapsigargin or receptor activation by either thrombin or the vascular endothelial growth factor activates the same pathway in primary ECs with classical SOCE pharmacological features. ECs possess the archetypical Ca(2+) release-activated Ca(2+) current (I(CRAC)), albeit of a very small amplitude. Using a maneuver that amplifies currents in divalent-free bath solutions, we show that EC CRAC has similar characteristics to that recorded from rat basophilic leukemia cells, namely a similar time course of activation, sensitivity to 2-aminoethoxydiphenyl borate, and low concentrations of lanthanides, and large Na(+) currents displaying the typical depotentiation. RNA silencing of either STIM1 or Orai1 essentially abolished SOCE and I(CRAC) in ECs, which were rescued by ectopic expression of either STIM1 or Orai1, respectively. Surprisingly, knockdown of either TRPC1 or TRPC4 proteins had no effect on SOCE and I(CRAC). Ectopic expression of STIM1 in ECs increased their I(CRAC) to a size comparable to that in rat basophilic leukemia cells. Knockdown of STIM1, Stim2, or Orai1 inhibited EC proliferation and caused cell cycle arrest at S and G2/M phase, although Orai1 knockdown was more efficient than that of Stim proteins. These results are first to our knowledge to establish the requirement of STIM1/Orai1 in the endothelial SOCE pathway.
Peter B. Stathopulos - One of the best experts on this subject based on the ideXlab platform.
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Synergistic stabilization by nitrosoglutathione-induced thiol modifications in the stromal interaction molecule-2 luminal domain suppresses basal and store operated calcium entry
Scientific Reports, 2020Co-Authors: Matthew J. Novello, Qingping Feng, Mengqi Zhang, Peter B. StathopulosAbstract:Stromal interaction molecule−1 and −2 (STIM1/2) are endoplasmic reticulum (ER) membrane-inserted calcium (Ca^2+) sensing proteins that, together with Orai1-composed Ca^2+ channels on the plasma membrane (PM), regulate intracellular Ca^2+ levels. Recent evidence suggests that S -nitrosylation of the luminal STIM1 Cys residues inhibits store operated Ca^2+ entry (SOCE). However, the effects of thiol modifications on STIM2 during nitrosative stress and their role in regulating basal Ca^2+ levels remain unknown. Here, we demonstrate that the nitric oxide (NO) donor nitrosoglutathione (GSNO) thermodynamically stabilizes the STIM2 Ca^2+ sensing region in a Cys-specific manner. We uncovered a remarkable synergism in this stabilization involving the three luminal Cys of STIM2, which is unique to this paralog. S -Nitrosylation causes structural perturbations that converge on the face of the EF-hand and sterile α motif (EF-SAM) domain, implicated in unfolding-coupled activation. In HEK293T cells, enhanced free basal cytosolic Ca^2+ and SOCE mediated by STIM2 overexpression could be attenuated by GSNO or mutation of the modifiable Cys located in the luminal domain. Collectively, we identify the Cys residues within the N-terminal region of STIM2 as modifiable targets during nitrosative stress that can profoundly and cooperatively affect basal Ca^2+ and SOCE regulation.
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Structural elements of stromal interaction molecule function.
Cell Calcium, 2018Co-Authors: Matthew J. Novello, Qingping Feng, Mitsuhiko Ikura, Peter B. StathopulosAbstract:Abstract Stromal interaction molecule (STIM)-1 and -2 are multi-domain, single-pass transmembrane proteins involved in sensing changes in compartmentalized calcium (Ca2+) levels and transducing this cellular signal to Orai1 channel proteins. Our understanding of the molecular mechanisms underlying STIM signaling has been dramatically improved through available X-ray crystal and solution NMR structures. This high-resolution structural data has revealed that intricate intramolecular and intermolecular protein-protein interactions are involved in converting STIMs from the quiescent to activation-competent states. This review article summarizes the current high resolution structural data on specific EF-hand, sterile α motif and coiled-coil interactions which drive STIM function in the activation of Orai1 channels. Further, the work discusses the effects of post-translational modifications on the structure and function of STIMs. Future structural studies on larger STIM:Orai complexes will be critical to fully defining the molecular bases for STIM function and how post-translational modifications influence these mechanisms.
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missense mutation in immunodeficient patients shows the multifunctional roles of coiled coil domain 3 cc3 in STIM1 activation
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Mate Maus, Peter B. Stathopulos, Amit Jairaman, Martin Muik, Marc Fahrner, Carl Weidinger, Melina J Benson, Sebastian Fuchs, Stephan EhlAbstract:Store-operated Ca2+ entry (SOCE) is a universal Ca2+ influx pathway that is important for the function of many cell types. SOCE occurs upon depletion of endoplasmic reticulum (ER) Ca2+ stores and relies on a complex molecular interplay between the plasma membrane (PM) Ca2+ channel ORAI1 and the ER Ca2+ sensor stromal interaction molecule (STIM) 1. Patients with null mutations in ORAI1 or STIM1 genes present with severe combined immunodeficiency (SCID)-like disease. Here, we describe the molecular mechanisms by which a loss-of-function STIM1 mutation (R429C) in human patients abolishes SOCE. R429 is located in the third coiled-coil (CC3) domain of the cytoplasmic C terminus of STIM1. Mutation of R429 destabilizes the CC3 structure and alters the conformation of the STIM1 C terminus, thereby releasing a polybasic domain that promotes STIM1 recruitment to ER–PM junctions. However, the mutation also impairs cytoplasmic STIM1 oligomerization and abolishes STIM1–ORAI1 interactions. Thus, despite its constitutive localization at ER–PM junctions, mutant STIM1 fails to activate SOCE. Our results demonstrate multifunctional roles of the CC3 domain in regulating intra- and intermolecular STIM1 interactions that control (i) transition of STIM1 from a quiescent to an active conformational state, (ii) cytoplasmic STIM1 oligomerization, and (iii) STIM1–ORAI1 binding required for ORAI1 activation.
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STIM1 orai1 coiled coil interplay in the regulation of store operated calcium entry
Nature Communications, 2013Co-Authors: Peter B. Stathopulos, Le Zheng, Rainer Schindl, Christoph Romanin, Martin Muik, Marc Fahrner, Genevieve M C Gasmiseabrook, Mitsuhiko IkuraAbstract:Orai1 calcium channels in the plasma membrane are activated by stromal interaction molecule-1 (STIM1), an endoplasmic reticulum calcium sensor, to mediate store-operated calcium entry (SOCE). The cytosolic region of STIM1 contains a long putative coiled-coil (CC)1 segment and shorter CC2 and CC3 domains. Here we present solution nuclear magnetic resonance structures of a trypsin-resistant CC1-CC2 fragment in the apo and Orai1-bound states. Each CC1-CC2 subunit forms a U-shaped structure that homodimerizes through antiparallel interactions between equivalent α-helices. The CC2:CC2' helix pair clamps two identical acidic Orai1 C-terminal helices at opposite ends of a hydrophobic/basic STIM-Orai association pocket. STIM1 mutants disrupting CC1:CC1' interactions attenuate, while variants promoting CC1 stability spontaneously activate Orai1 currents. CC2 mutations cause remarkable variability in Orai1 activation because of a dual function in binding Orai1 and autoinhibiting STIM1 oligomerization via interactions with CC3. We conclude that SOCE is activated through dynamic interplay between STIM1 and Orai1 helices.
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auto inhibitory role of the ef sam domain of stim proteins in store operated calcium entry
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Le Zheng, Peter B. Stathopulos, Rainer Schindl, Christoph Romanin, Guangyao Li, Mitsuhiko IkuraAbstract:Stromal interaction molecules (STIM)s function as endoplasmic reticulum calcium (Ca2+) sensors that differentially regulate plasma membrane Ca2+ release activated Ca2+ channels in various cells. To probe the structural basis for the functional differences between STIM1 and STIM2 we engineered a series of EF-hand and sterile α motif (SAM) domain (EF-SAM) chimeras, demonstrating that the STIM1 Ca2+-binding EF-hand and the STIM2 SAM domain are major contributors to the autoinhibition of oligomerization in each respective isoform. Our nuclear magnetic resonance (NMR) derived STIM2 EF-SAM structure provides a rationale for an augmented stability, which involves a 54° pivot in the EF-hand:SAM domain orientation permissible by an expanded nonpolar cleft, ionic interactions, and an enhanced hydrophobic SAM core, unique to STIM2. Live cells expressing “super-unstable” or “super-stable” STIM1/STIM2 EF-SAM chimeras in the full-length context show a remarkable correlation with the in vitro data. Together, our data suggest that divergent Ca2+- and SAM-dependent stabilization of the EF-SAM fold contributes to the disparate regulation of store-operated Ca2+ entry by STIM1 and STIM2.
Mitsuhiko Ikura - One of the best experts on this subject based on the ideXlab platform.
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Structural elements of stromal interaction molecule function.
Cell Calcium, 2018Co-Authors: Matthew J. Novello, Qingping Feng, Mitsuhiko Ikura, Peter B. StathopulosAbstract:Abstract Stromal interaction molecule (STIM)-1 and -2 are multi-domain, single-pass transmembrane proteins involved in sensing changes in compartmentalized calcium (Ca2+) levels and transducing this cellular signal to Orai1 channel proteins. Our understanding of the molecular mechanisms underlying STIM signaling has been dramatically improved through available X-ray crystal and solution NMR structures. This high-resolution structural data has revealed that intricate intramolecular and intermolecular protein-protein interactions are involved in converting STIMs from the quiescent to activation-competent states. This review article summarizes the current high resolution structural data on specific EF-hand, sterile α motif and coiled-coil interactions which drive STIM function in the activation of Orai1 channels. Further, the work discusses the effects of post-translational modifications on the structure and function of STIMs. Future structural studies on larger STIM:Orai complexes will be critical to fully defining the molecular bases for STIM function and how post-translational modifications influence these mechanisms.
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STIM1 orai1 coiled coil interplay in the regulation of store operated calcium entry
Nature Communications, 2013Co-Authors: Peter B. Stathopulos, Le Zheng, Rainer Schindl, Christoph Romanin, Martin Muik, Marc Fahrner, Genevieve M C Gasmiseabrook, Mitsuhiko IkuraAbstract:Orai1 calcium channels in the plasma membrane are activated by stromal interaction molecule-1 (STIM1), an endoplasmic reticulum calcium sensor, to mediate store-operated calcium entry (SOCE). The cytosolic region of STIM1 contains a long putative coiled-coil (CC)1 segment and shorter CC2 and CC3 domains. Here we present solution nuclear magnetic resonance structures of a trypsin-resistant CC1-CC2 fragment in the apo and Orai1-bound states. Each CC1-CC2 subunit forms a U-shaped structure that homodimerizes through antiparallel interactions between equivalent α-helices. The CC2:CC2' helix pair clamps two identical acidic Orai1 C-terminal helices at opposite ends of a hydrophobic/basic STIM-Orai association pocket. STIM1 mutants disrupting CC1:CC1' interactions attenuate, while variants promoting CC1 stability spontaneously activate Orai1 currents. CC2 mutations cause remarkable variability in Orai1 activation because of a dual function in binding Orai1 and autoinhibiting STIM1 oligomerization via interactions with CC3. We conclude that SOCE is activated through dynamic interplay between STIM1 and Orai1 helices.
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auto inhibitory role of the ef sam domain of stim proteins in store operated calcium entry
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Le Zheng, Peter B. Stathopulos, Rainer Schindl, Christoph Romanin, Guangyao Li, Mitsuhiko IkuraAbstract:Stromal interaction molecules (STIM)s function as endoplasmic reticulum calcium (Ca2+) sensors that differentially regulate plasma membrane Ca2+ release activated Ca2+ channels in various cells. To probe the structural basis for the functional differences between STIM1 and STIM2 we engineered a series of EF-hand and sterile α motif (SAM) domain (EF-SAM) chimeras, demonstrating that the STIM1 Ca2+-binding EF-hand and the STIM2 SAM domain are major contributors to the autoinhibition of oligomerization in each respective isoform. Our nuclear magnetic resonance (NMR) derived STIM2 EF-SAM structure provides a rationale for an augmented stability, which involves a 54° pivot in the EF-hand:SAM domain orientation permissible by an expanded nonpolar cleft, ionic interactions, and an enhanced hydrophobic SAM core, unique to STIM2. Live cells expressing “super-unstable” or “super-stable” STIM1/STIM2 EF-SAM chimeras in the full-length context show a remarkable correlation with the in vitro data. Together, our data suggest that divergent Ca2+- and SAM-dependent stabilization of the EF-SAM fold contributes to the disparate regulation of store-operated Ca2+ entry by STIM1 and STIM2.
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Stromal interaction molecule (STIM) 1 and STIM2 calcium sensing regions exhibit distinct unfolding and oligomerization kinetics.
The Journal of biological chemistry, 2008Co-Authors: Peter B. Stathopulos, Le Zheng, Mitsuhiko IkuraAbstract:Abstract Stromal interaction molecules (STIM) 1 and STIM2 are regulators of store-operated calcium (Ca2+) entry as well as basal cytoplasmic Ca2+ levels in human cells. Despite a high sequence similarity (>65%) and analogous sequence-based domain architectures, STIM1 and STIM2 differentially influence these phenomena. Among all eukaryotes, the endoplasmic reticulum luminal portion of STIM proteins minimally encode EF-hand and sterile α-motif (SAM) domains (EF-SAM), which are responsible for sensing changes in Ca2+ levels and initiating oligomerization. STIM oligomerization is a key induction step in the activation of Ca2+-permeable channels on the plasma membrane. Here, we show that the kinetic half-time of conversion from a monomeric to a steady oligomeric state is >70× shorter for STIM1 EF-SAM than STIM2 under similar conditions. Urea-induced rates of unfolding for STIM1 EF-SAM are >3× quicker when compared with STIM2, coherent with partial unfolding-coupled aggregation. Additionally, we demonstrate that the isoform-specific N-terminal residues beyond EF-SAM can influence the stability of this region. We postulate that distinct oligomerization dynamics of STIM isoforms have evolved to adapt to differential roles in Ca2+ homeostasis and signaling.
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structural and mechanistic insights into STIM1 mediated initiation of store operated calcium entry
Cell, 2008Co-Authors: Peter B. Stathopulos, Le Zheng, Michael J Plevin, Mitsuhiko IkuraAbstract:Summary Stromal interaction molecule-1 (STIM1) activates store-operated Ca 2+ entry (SOCE) in response to diminished luminal Ca 2+ levels. Here, we present the atomic structure of the Ca 2+ -sensing region of STIM1 consisting of the EF-hand and sterile α motif (SAM) domains (EF-SAM). The canonical EF-hand is paired with a previously unidentified EF-hand. Together, the EF-hand pair mediates mutually indispensable hydrophobic interactions between the EF-hand and SAM domains. Structurally critical mutations in the canonical EF-hand, "hidden" EF-hand, or SAM domain disrupt Ca 2+ sensitivity in oligomerization via destabilization of the entire EF-SAM entity. In mammalian cells, EF-SAM destabilization mutations within full-length STIM1 induce punctae formation and activate SOCE independent of luminal Ca 2+ . We provide atomic resolution insight into the molecular basis for STIM1-mediated SOCE initiation and show that the folded/unfolded state of the Ca 2+ -sensing region of STIM is crucial to SOCE regulation.
