The Experts below are selected from a list of 5166 Experts worldwide ranked by ideXlab platform

Mohamed Trebak - One of the best experts on this subject based on the ideXlab platform.

  • Orai channel C-terminal peptides are key modulators of STIM-Orai coupling and calcium signal generation
    'Elsevier BV', 2021
    Co-Authors: James H. Baraniak, Yandong Zhou, Youjun Wang, Mohamed Trebak, Robert M Nwokonko, Michelle R. Jennette, Sarah A. Kazzaz, Jazmin M. Stenson, Abigale L. Whitsell, Donald L. Gill
    Abstract:

    Summary: Junctional coupling between endoplasmic reticulum (ER) Ca2+-sensor STIM proteins and plasma membrane (PM) Orai channels mediates Ca2+ signals in most cells. We reveal that PM-tethered, fluorescently tagged C-terminal M4x (fourth transmembrane helix contains a cytoplasmic C-terminal extension) peptides from Orai channels undergo a Leu-specific signature of direct interaction with the STIM1 Orai-activating region (SOAR), exactly mimicking STIM1 binding to gate Orai channels. The 20-amino-acid Orai3-M4x peptide associates avidly with STIM1 within ER-PM junctions, functions to competitively block native Ca2+ signals, and mediates a key modification of STIM-Orai coupling induced by 2-aminoethoxydiphenyl borate. By blocking STIM-Orai coupling, the Orai3-M4x peptide reveals the critical role of Orai channels in driving Ca2+ oscillatory signals and transcriptional control through NFAT. The M4x peptides interact independently with SOAR dimers consistent with unimolecular coupling between Orai subunits and STIM1 dimers. We reveal the critical role of M4x helices in defining the coupling interface between STIM and Orai proteins to mediate store-operated Ca2+ signals

  • multiple types of calcium channels arising from alternative translation initiation of the ORAI1 message
    Science Signaling, 2015
    Co-Authors: Pooja N Desai, Xuexin Zhang, Agnes K Janoshazi, Sunitha Bolimuntha, James W Putney, Mohamed Trebak
    Abstract:

    In mammals exclusively, the pore-forming Ca 2+ release–activated Ca 2+ (CRAC) channel subunit ORAI1 occurs in two forms because of alternative translation initiation. The longer, mammal-specific ORAI1α contains an additional 63 amino acids upstream of the conserved start site for ORAI1β, which occurs at methionine 64 in ORAI1α. ORAI1 participates in the generation of three distinct Ca 2+ currents, including two store-operated currents: I crac , which involves activation of ORAI1 channels by the Ca 2+ -sensing protein STIM1 (stromal interaction molecule 1), and I soc , which involves an interaction among ORAI1, the transient receptor potential (TRP) family member TRPC1 (TRP canonical 1), and STIM1. ORAI1 is also a pore-forming subunit of an arachidonic acid (or leukotriene C 4 )–regulated current I arc that involves interactions among ORAI1, Orai3, and STIM1. We evaluated the roles of the two ORAI1 forms in the Ca 2+ currents I crac , I soc , and I arc . We found that ORAI1α and ORAI1β were largely interchangeable for I crac and I soc , although ORAI1α exhibited stronger inhibition by Ca 2+ . Only the mammalian-specific ORAI1α functioned in the arachidonic acid–regulated current I arc . Thus, alternative translation initiation of the ORAI1 message produces at least three types of Ca 2+ channels with distinct signaling and regulatory properties.

  • leukotriene c4 synthase a critical enzyme in the activation of store independent ORAI1 orai3 channels is required for neointimal hyperplasia
    Journal of Biological Chemistry, 2015
    Co-Authors: Mohamed Trebak, Jose C Gonzalezcobos, Xuexin Zhang, Wei Zhang, Judith A Stolwijk, Khalid Matrougui
    Abstract:

    Leukotriene-C4 synthase (LTC4S) generates LTC4 from arachidonic acid metabolism. LTC4 is a proinflammatory factor that acts on plasma membrane cysteinyl leukotriene receptors. Recently, however, we showed that LTC4 was also a cytosolic second messenger that activated store-independent LTC4-regulated Ca2+ (LRC) channels encoded by ORAI1/Orai3 heteromultimers in vascular smooth muscle cells (VSMCs). We showed that Orai3 and LRC currents were up-regulated in medial and neointimal VSMCs after vascular injury and that Orai3 knockdown inhibited LRC currents and neointimal hyperplasia. However, the role of LTC4S in neointima formation remains unknown. Here we show that LTC4S knockdown inhibited LRC currents in VSMCs. We performed in vivo experiments where rat left carotid arteries were injured using balloon angioplasty to cause neointimal hyperplasia. Neointima formation was associated with up-regulation of LTC4S protein expression in VSMCs. Inhibition of LTC4S expression in injured carotids by lentiviral particles encoding shRNA inhibited neointima formation and inward and outward vessel remodeling. LRC current activation did not cause nuclear factor for activated T cells (NFAT) nuclear translocation in VSMCs. Surprisingly, knockdown of either LTC4S or Orai3 yielded more robust and sustained Akt1 and Akt2 phosphorylation on Ser-473/Ser-474 upon serum stimulation. LTC4S and Orai3 knockdown inhibited VSMC migration in vitro with no effect on proliferation. Akt activity was suppressed in neointimal and medial VSMCs from injured vessels at 2 weeks postinjury but was restored when the up-regulation of either LTC4S or Orai3 was prevented by shRNA. We conclude that LTC4S and Orai3 altered Akt signaling to promote VSMC migration and neointima formation.

  • 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, 2010
    Co-Authors: Rajender K Motiani, Iskandar F Abdullaev, Mohamed Trebak
    Abstract:

    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.

  • essential role for stim1 ORAI1 mediated calcium influx in pdgf induced smooth muscle migration
    American Journal of Physiology-cell Physiology, 2010
    Co-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 Trebak
    Abstract:

    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.

Michael D Cahalan - One of the best experts on this subject based on the ideXlab platform.

  • Store-dependent and-independent Modes Regulating Ca2 Release-activated Ca2 Channel Activity of Human ORAI1 and Orai3*□S
    2016
    Co-Authors: Shenyuan L. Zhang, Andriy V Yeromin, Ashot J Kozak, Aubin Penna, Weihua Jiang, Jing Chen, Wei Shen, Victor Chi, Michael D Cahalan
    Abstract:

    We evaluated currents induced by expression of human homologs of Orai together with STIM1 in human embryonic kidney cells. When co-expressed with STIM1, ORAI1 induced a large inwardly rectifying Ca2-selective current with Ca2-in-duced slow inactivation. A point mutation of ORAI1 (E106D) altered the ion selectivity of the induced Ca2 release-activated Ca2 (CRAC)-like currentwhile retaining an inwardly rectifying I-V characteristic. Expression of the C-terminal portion of STIM1 with ORAI1 was sufficient to generate CRAC current without store depletion. 2-APB activated a large relatively non-selective current inSTIM1andOrai3 co-expressing cells. 2-APB also inducedCa2 influx inOrai3-expressing cells without store depletion or co-expression of STIM1. The Orai3 current induced by 2-APB exhibited outward rectification and an inward component representing a mixed calcium and monova

  • nanoscale patterning of stim1 and ORAI1 during store operated ca2 entry
    Proceedings of the National Academy of Sciences of the United States of America, 2015
    Co-Authors: Stefano Perni, Andriy V Yeromin, Michael D Cahalan, Joseph L Dynes, Clara Franziniarmstrong
    Abstract:

    Stromal interacting molecule (STIM) and Orai proteins constitute the core machinery of store-operated calcium entry. We used transmission and freeze-fracture electron microscopy to visualize STIM1 and ORAI1 at endoplasmic reticulum (ER)-plasma membrane (PM) junctions in HEK 293 cells. Compared with control cells, thin sections of STIM1-transfected cells possessed far more ER elements, which took the form of complex stackable cisternae and labyrinthine structures adjoining the PM at junctional couplings (JCs). JC formation required STIM1 expression but not store depletion, induced here by thapsigargin (TG). Extended molecules, indicative of STIM1, decorated the cytoplasmic surface of ER, bridged a 12-nm ER-PM gap, and showed clear rearrangement into small clusters following TG treatment. Freeze-fracture replicas of the PM of ORAI1-transfected cells showed extensive domains packed with characteristic "particles"; TG treatment led to aggregation of these particles into sharply delimited "puncta" positioned upon raised membrane subdomains. The size and spacing of ORAI1 channels were consistent with the Orai crystal structure, and stoichiometry was unchanged by store depletion, coexpression with STIM1, or an ORAI1 mutation (L273D) affecting STIM1 association. Although the arrangement of ORAI1 channels in puncta was substantially unstructured, a portion of channels were spaced at ∼15 nm. Monte Carlo analysis supported a nonrandom distribution for a portion of channels spaced at ∼15 nm. These images offer dramatic, direct views of STIM1 aggregation and ORAI1 clustering in store-depleted cells and provide evidence for the interaction of a single ORAI1 channel with small clusters of STIM1 molecules.

  • mutations in ORAI1 transmembrane segment 1 cause stim1 independent activation of ORAI1 channels at glycine 98 and channel closure at arginine 91
    Proceedings of the National Academy of Sciences of the United States of America, 2011
    Co-Authors: Shenyuan L. Zhang, Andriy V Yeromin, Anna Amcheslavsky, Hongying Zheng, Michael D Cahalan
    Abstract:

    Stim and Orai proteins comprise the molecular machinery of Ca2+ release-activated Ca2+ (CRAC) channels. As an approach toward understanding the gating of ORAI1 channels, we investigated effects of selected mutations at two conserved sites in the first transmembrane segment (TM1): arginine 91 located near the cytosolic end of TM1 and glycine 98 near the middle of TM1. ORAI1 R91C, when coexpressed with STIM1, was activated normally by Ca2+-store depletion. Treatment with diamide, a thiol-oxidizing agent, induced formation of disulfide bonds between R91C residues in adjacent ORAI1 subunits and rapidly blocked STIM1-operated Ca2+ current. Diamide-induced blocking was reversed by disulfide bond-reducing agents. These results indicate that R91 forms a very narrow part of the conducting pore at the cytosolic side. Alanine replacement at G98 prevented STIM1-induced channel activity. Interestingly, mutation to aspartate (G98D) or proline (G98P) caused constitutive channel activation in a STIM1-independent manner. Both ORAI1 G98 mutants formed a nonselective Ca2+-permeable conductance that was relatively resistant to block by Gd3+. The double mutant R91W/G98D was also constitutively active, overcoming the normal inhibition of channel activity by tryptophan at the 91 position found in some patients with severe combined immunodeficiency (SCID), and the double mutant R91C/G98D was resistant to diamide block. These data suggest that the channel pore is widened and ion selectivity is altered by mutations at the G98 site that may perturb α-helical structure. We propose distinct functional roles for G98 as a gating hinge and R91 as part of the physical gate at the narrow inner mouth of the channel.

  • ORAI1 and stim1 move to the immunological synapse and are up regulated during t cell activation
    Proceedings of the National Academy of Sciences of the United States of America, 2008
    Co-Authors: Maria I Lioudyno, Shenyuan L. Zhang, Jack Roos, Olga Safrina, Ashot J Kozak, Kenneth A Stauderman, Aubin Penna, Debasish Sen, Michael D Cahalan
    Abstract:

    For efficient development of an immune response, T lymphocytes require long-lasting calcium influx through calcium release-activated calcium (CRAC) channels and the formation of a stable immunological synapse (IS) with the antigen-presenting cell (APC). Recent RNAi screens have identified Stim and Orai in Drosophila cells, and their corresponding mammalian homologs STIM1 and ORAI1 in T cells, as essential for CRAC channel activation. Here, we show that STIM1 and ORAI1 are recruited to the immunological synapse between primary human T cells and autologous dendritic cells. Both STIM1 and ORAI1 accumulated in the area of contact between either resting or super-antigen (SEB)-pretreated T cells and SEB-pulsed dendritic cells, where they were colocalized with T cell receptor (TCR) and costimulatory molecules. In addition, imaging of intracellular calcium signaling in T cells loaded with EGTA revealed significantly higher Ca2+ concentration near the interface, indicating Ca2+ influx localized at the T cell/dendritic cell contact area. Expression of a dominant-negative ORAI1 mutant blocked T cell Ca2+ signaling but did not interfere with the initial accumulation of STIM1, ORAI1, and CD3 in the contact zone. In activated T cell blasts, mRNA expression for endogenous STIM1 and all three human homologs of Orai was up-regulated, accompanied by a marked increase in Ca2+ influx through CRAC channels. These results imply a positive feedback loop in which an initial TCR signal favors up-regulation of STIM1 and Orai proteins that would augment Ca2+ signaling during subsequent antigen encounter.

  • molecular identification of the crac channel by altered ion selectivity in a mutant of orai
    Nature, 2006
    Co-Authors: Andriy V Yeromin, Shenyuan L. Zhang, Olga Safrina, Michael D Cahalan, Weihua Jiang
    Abstract:

    Recent RNA interference screens have identified several proteins that are essential for store-operated Ca2+ influx and Ca2+ release-activated Ca2+ (CRAC) channel activity in Drosophila and in mammals, including the transmembrane proteins Stim (stromal interaction molecule) and Orai. Stim probably functions as a sensor of luminal Ca2+ content and triggers activation of CRAC channels in the surface membrane after Ca2+ store depletion. Among three human homologues of Orai (also known as olf186-F), ORAI1 on chromosome 12 was found to be mutated in patients with severe combined immunodeficiency disease, and expression of wild-type ORAI1 restored Ca2+ influx and CRAC channel activity in patient T cells. The overexpression of Stim and Orai together markedly increases CRAC current. However, it is not yet clear whether Stim or Orai actually forms the CRAC channel, or whether their expression simply limits CRAC channel activity mediated by a different channel-forming subunit. Here we show that interaction between wild-type Stim and Orai, assessed by co-immunoprecipitation, is greatly enhanced after treatment with thapsigargin to induce Ca2+ store depletion. By site-directed mutagenesis, we show that a point mutation from glutamate to aspartate at position 180 in the conserved S1-S2 loop of Orai transforms the ion selectivity properties of CRAC current from being Ca2+-selective with inward rectification to being selective for monovalent cations and outwardly rectifying. A charge-neutralizing mutation at the same position (glutamate to alanine) acts as a dominant-negative non-conducting subunit. Other charge-neutralizing mutants in the same loop express large inwardly rectifying CRAC current, and two of these exhibit reduced sensitivity to the channel blocker Gd3+. These results indicate that Orai itself forms the Ca2+-selectivity filter of the CRAC channel.

Richard S Lewis - One of the best experts on this subject based on the ideXlab platform.

  • alternative splicing converts stim2 from an activator to an inhibitor of store operated calcium channels
    Journal of Cell Biology, 2015
    Co-Authors: Anshul Rana, Chan Young Park, Michelle Yen, Amir M Sadaghiani, Seth Malmersjo, Ricardo E Dolmetsch, Richard S Lewis
    Abstract:

    Store-operated calcium entry (SOCE) regulates a wide variety of essential cellular functions. SOCE is mediated by STIM1 and STIM2, which sense depletion of ER Ca2+ stores and activate Orai channels in the plasma membrane. Although the amplitude and dynamics of SOCE are considered important determinants of Ca2+-dependent responses, the underlying modulatory mechanisms are unclear. In this paper, we identify STIM2β, a highly conserved alternatively spliced isoform of STIM2, which, in contrast to all known STIM isoforms, is a potent inhibitor of SOCE. Although STIM2β does not by itself strongly bind ORAI1, it is recruited to ORAI1 channels by forming heterodimers with other STIM isoforms. Analysis of STIM2β mutants and ORAI1-STIM2β chimeras suggested that it actively inhibits SOCE through a sequence-specific allosteric interaction with ORAI1. Our results reveal a previously unrecognized functional flexibility in the STIM protein family by which alternative splicing creates negative and positive regulators of SOCE to shape the amplitude and dynamics of Ca2+ signals.

  • single molecule analysis of diffusion and trapping of stim1 and ORAI1 at endoplasmic reticulum plasma membrane junctions
    Molecular Biology of the Cell, 2014
    Co-Authors: Elizabeth D Covington, Richard S Lewis
    Abstract:

    Following endoplasmic reticulum (ER) Ca(2+) depletion, STIM1 and ORAI1 complexes assemble autonomously at ER-plasma membrane (PM) junctions to trigger store-operated Ca(2+) influx. One hypothesis to explain this process is a diffusion trap in which activated STIM1 diffusing in the ER becomes trapped at junctions through interactions with the PM, and STIM1 then traps ORAI1 in the PM through binding of its calcium release-activated calcium activation domain. We tested this model by analyzing STIM1 and ORAI1 diffusion using single-particle tracking, photoactivation of protein ensembles, and Monte Carlo simulations. In resting cells, STIM1 diffusion is Brownian, while ORAI1 is slightly subdiffusive. After store depletion, both proteins slow to the same speeds, consistent with complex formation, and are confined to a corral similar in size to ER-PM junctions. While the escape probability at high STIM:Orai expression ratios is <1%, it is significantly increased by reducing the affinity of STIM1 for ORAI1 or by expressing the two proteins at comparable levels. Our results provide direct evidence that STIM-Orai complexes are trapped by their physical connections across the junctional gap, but also reveal that the complexes are surprisingly dynamic, suggesting that readily reversible binding reactions generate free STIM1 and ORAI1, which engage in constant diffusional exchange with extrajunctional pools.

  • single molecule analysis of diffusion and trapping of stim1 and ORAI1 at er plasma membrane junctions
    Molecular Biology of the Cell, 2014
    Co-Authors: Elizabeth D Covington, Richard S Lewis
    Abstract:

    Following ER Ca depletion, STIM1 and ORAI1 complexes assemble autonomously at ERplasma membrane (PM) junctions to trigger store-operated Ca influx. One hypothesis to explain this process is a diffusion trap in which activated STIM1 diffusing in the ER becomes trapped at junctions through interactions with the PM, and STIM1 then traps ORAI1 in the PM through binding of its CRAC activation domain. We tested this model by analyzing STIM1 and ORAI1 diffusion using single-particle tracking, photoactivation of protein ensembles, and Monte Carlo simulations. In resting cells, STIM1 diffusion is Brownian while ORAI1 is slightly subdiffusive. After store depletion both proteins slow to the same speeds, consistent with complex formation, and are confined to a corral similar in size to ER-PM junctions. While the escape probability at high STIM:Orai expression ratios is <1%, it is significantly increased by reducing the affinity of STIM1 for ORAI1 or by expressing the two proteins at comparable levels. Our results provide direct evidence that STIM-Orai complexes are trapped by their physical connections across the junctional gap, but also reveal that the complexes are surprisingly dynamic, suggesting that readily reversible binding reactions generate free STIM1 and ORAI1 which engage in constant diffusional exchange with extrajunctional pools.

Rajender K Motiani - One of the best experts on this subject based on the ideXlab platform.

  • mechanisms of stim1 activation of store independent leukotriene c4 regulated ca2 channels
    Molecular and Cellular Biology, 2013
    Co-Authors: Xuexin Zhang, Martin Muik, Marc Fahrner, Rainer Schindl, Jonathan M Bisaillon, Rajender K Motiani, Jose C Gonzalezcobos, Brian Ruhle, Wei Zhang, Margarida Barroso
    Abstract:

    We recently showed, in primary vascular smooth muscle cells (VSMCs), that the platelet-derived growth factor activates canonical store-operated Ca(2+) entry and Ca(2+) release-activated Ca(2+) currents encoded by ORAI1 and STIM1 genes. However, thrombin activates store-independent Ca(2+) selective channels contributed by both Orai3 and ORAI1. These store-independent Orai3/ORAI1 channels are gated by cytosolic leukotriene C4 (LTC4) and require STIM1 downstream LTC4 action. However, the source of LTC4 and the signaling mechanisms of STIM1 in the activation of this LTC4-regulated Ca(2+) (LRC) channel are unknown. Here, we show that upon thrombin stimulation, LTC4 is produced through the sequential activities of phospholipase C, diacylglycerol lipase, 5-lipo-oxygenease, and leukotriene C4 synthase. We show that the endoplasmic reticulum-resident STIM1 is necessary and sufficient for LRC channel activation by thrombin. STIM1 does not form sustained puncta and does not colocalize with ORAI1 either under basal conditions or in response to thrombin. However, STIM1 is precoupled to Orai3 and Orai3/ORAI1 channels under basal conditions as shown using Forster resonance energy transfer (FRET) imaging. The second coiled-coil domain of STIM1 is required for coupling to either Orai3 or Orai3/ORAI1 channels and for LRC channel activation. We conclude that STIM1 employs distinct mechanisms in the activation of store-dependent and store-independent Ca(2+) entry pathways.

  • store independent ORAI1 3 channels activated by intracrine leukotrienec4 role in neointimal hyperplasia
    Circulation Research, 2013
    Co-Authors: Jose C Gonzalezcobos, Martin Muik, Rainer Schindl, Jonathan M Bisaillon, Rajender K Motiani, Xuexin Zhang, Brian Ruhle, Wei Zhang, Amy M Spinelli, Arti V Shinde
    Abstract:

    Rationale: Through largely unknown mechanisms, Ca 2+ signaling plays important roles in vascular smooth muscle cell (VSMC) remodeling. ORAI1-encoded store-operated Ca 2+ entry has recently emerged as an important player in VSMC remodeling. However, the role of the exclusively mammalian Orai3 protein in native VSMC Ca 2+ entry pathways, its upregulation during VSMC remodeling, and its contribution to neointima formation remain unknown. Objective: The goal of this study was to determine the agonist-evoked Ca 2+ entry pathway contributed by Orai3; Orai3 potential upregulation and role during neointima formation after balloon injury of rat carotid arteries. Methods and Results: Ca 2+ imaging and patch-clamp recordings showed that although the platelet-derived growth factor activates the canonical Ca 2+ release-activated Ca 2+ channels via store depletion in VSMC, the pathophysiological agonist thrombin activates a distinct Ca 2+ -selective channel contributed by ORAI1, Orai3, and stromal interacting molecule1 in the same cells. Unexpectedly, Ca 2+ store depletion is not required for activation of ORAI1/3 channel by thrombin. Rather, the signal for ORAI1/3 channel activation is cytosolic leukotrieneC 4 produced downstream thrombin receptor stimulation through the catalytic activity of leukotrieneC 4 synthase. Importantly, Orai3 is upregulated in an animal model of VSMC neointimal remodeling, and in vivo Orai3 knockdown inhibits neointima formation. Conclusions: These results demonstrate that distinct native Ca 2+ -selective Orai channels are activated by different agonists/pathways and uncover a mechanism whereby leukotrieneC 4 acts through hitherto unknown intracrine mode to elicit store-independent Ca 2+ signaling that promotes vascular occlusive disease. Orai3 and Orai3-containing channels provide novel targets for control of VSMC remodeling during vascular injury or disease.

  • stim1 and ORAI1 mediate crac channel activity and are essential for human glioblastoma invasion
    Pflügers Archiv: European Journal of Physiology, 2013
    Co-Authors: Iskandar F Abdullaev, Rajender K Motiani, Maria C Hyzinskigarcia, Xuexin Zhang, Matthew M Henkel, Yuhung Kuo
    Abstract:

    The Ca2+ sensor stromal interacting molecule 1 (STIM1) and the Ca2+ channel ORAI1 mediate the ubiquitous store-operated Ca2+ entry (SOCE) pathway activated by depletion of internal Ca2+ stores and mediated through the highly Ca2+-selective, Ca2+ release-activated Ca2+ (CRAC) current. Furthermore, STIM1 and ORAI1, along with Orai3, encode store-independent Ca2+ currents regulated by either arachidonate or its metabolite, leukotriene C4. Orai channels are emerging as important contributors to numerous cell functions, including proliferation, migration, differentiation, and apoptosis. Recent studies suggest critical involvement of STIM/Orai proteins in controlling the development of several cancers, including malignancies of the breast, prostate, and cervix. Here, we quantitatively compared the magnitude of SOCE and the expression levels of STIM1 and ORAI1 in non-malignant human primary astrocytes (HPA) and in primary human cell lines established from surgical samples of the brain tumor glioblastoma multiforme (GBM). Using Ca2+ imaging, patch-clamp electrophysiology, pharmacological reagents, and gene silencing, we established that in GBM cells, SOCE and CRAC are mediated by STIM1 and ORAI1. We further found that GBM cells show upregulation of SOCE and increased ORAI1 levels compared to HPA. The functional significance of SOCE was evaluated by studying the effects of STIM1 and ORAI1 knockdown on cell proliferation and invasion. Utilizing Matrigel assays, we demonstrated that in GBM, but not in HPA, downregulation of STIM1 and ORAI1 caused a dramatic decrease in cell invasion. In contrast, the effects of STIM1 and ORAI1 knockdown on GBM cell proliferation were marginal. Overall, these results demonstrate that STIM1 and ORAI1 encode SOCE and CRAC currents and control invasion of GBM cells. Our work further supports the potential use of channels contributed by Orai isoforms as therapeutic targets in cancer.

  • 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, 2010
    Co-Authors: Rajender K Motiani, Iskandar F Abdullaev, Mohamed Trebak
    Abstract:

    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.

  • essential role for stim1 ORAI1 mediated calcium influx in pdgf induced smooth muscle migration
    American Journal of Physiology-cell Physiology, 2010
    Co-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 Trebak
    Abstract:

    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.

Donald L. Gill - One of the best experts on this subject based on the ideXlab platform.

  • Orai channel C-terminal peptides are key modulators of STIM-Orai coupling and calcium signal generation
    'Elsevier BV', 2021
    Co-Authors: James H. Baraniak, Yandong Zhou, Youjun Wang, Mohamed Trebak, Robert M Nwokonko, Michelle R. Jennette, Sarah A. Kazzaz, Jazmin M. Stenson, Abigale L. Whitsell, Donald L. Gill
    Abstract:

    Summary: Junctional coupling between endoplasmic reticulum (ER) Ca2+-sensor STIM proteins and plasma membrane (PM) Orai channels mediates Ca2+ signals in most cells. We reveal that PM-tethered, fluorescently tagged C-terminal M4x (fourth transmembrane helix contains a cytoplasmic C-terminal extension) peptides from Orai channels undergo a Leu-specific signature of direct interaction with the STIM1 Orai-activating region (SOAR), exactly mimicking STIM1 binding to gate Orai channels. The 20-amino-acid Orai3-M4x peptide associates avidly with STIM1 within ER-PM junctions, functions to competitively block native Ca2+ signals, and mediates a key modification of STIM-Orai coupling induced by 2-aminoethoxydiphenyl borate. By blocking STIM-Orai coupling, the Orai3-M4x peptide reveals the critical role of Orai channels in driving Ca2+ oscillatory signals and transcriptional control through NFAT. The M4x peptides interact independently with SOAR dimers consistent with unimolecular coupling between Orai subunits and STIM1 dimers. We reveal the critical role of M4x helices in defining the coupling interface between STIM and Orai proteins to mediate store-operated Ca2+ signals

  • distinct orai coupling domains in stim1 and stim2 define the orai activating site
    Nature Communications, 2014
    Co-Authors: Xizhuo Wang, Yandong Zhou, Youjun Wang, Jonathan Soboloff, Eunan Hendron, Salvatore Mancarella, Mark Andrake, Brad S Rothberg, Donald L. Gill
    Abstract:

    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.

  • distinct roles of stim1 and stim2 c terminal orai coupling domains
    Biophysical Journal, 2013
    Co-Authors: Xizhuo Wang, Youjun Wang, Eunan Hendron, Donald L. Gill
    Abstract:

    Store-operated Ca2+ entry (SOCe) is essential for Ca2+ homeostasis and signaling. SOCe is mediated by STIM proteins which function as ER Ca2+ store sensors, coupling with and activating PM Orai Ca2+ channels. While STIM1-ORAI1 coupling is well characterized, the coupling between STIM2 and ORAI1 shows some important functional differences from STIM1. The molecular basis of these differences remains uncharacterized. We examined the STIM2 C-terminal (S2-Ct) region that has considerable homology with the known functional coupling domains of S1-Ct. We examined the comparative functions of STIM1 and STIM2 fragments using a combination of Ca2+ imaging, patch-clamp current analysis, and analysis of the pharmacological modifier, 2-APB. Deletion of the “variable” C-terminal region (534-833) immediately downstream from the STIM-Orai activating region of STIM2 (SOAR2; 435-533) from either whole STIM2 or S2-Ct, had little effect on the activation of ORAI1 channels. Similarly, deletion from S2-Ct of the N-terminal region (325-433) upstream from SOAR2, had little effect on ORAI1-activation by S2-Ct. Thus the cytosolic regions outside SOAR2 seem to be less important for mediating STIM2 coupling to and activate ORAI1. Interestingly, SOAR2 expression alone is sufficient to mimic some of the different coupling properties that distinguish full length STIM2 from STIM1, including the poor intrinsic coupling to activate ORAI1 and the strong enhancement of ORAI1 activation induced by 2-APB. To gain further insights on how the two SOAR domains couple and activate ORAI1, we constructed a series of SOAR1 and SOAR2 chimeras. Using these chimeras, our results reveal that the Sα1-Sα3 helices in the SOAR molecules are important for defining the distinct ORAI1 activating properties of STIM1 and STIM2.

  • the calcium store sensor stim1 reciprocally controls orai and cav1 2 channels
    Science, 2010
    Co-Authors: Youjun Wang, Jonathan Soboloff, Xiang D. Tang, Eunan Hendron, Salvatore Mancarella, Xiaoxiang Deng, Satoru Eguchi, Donald L. Gill
    Abstract:

    Calcium signals, pivotal in controlling cell function, can be generated by calcium entry channels activated by plasma membrane depolarization or depletion of internal calcium stores. We reveal a regulatory link between these two channel subtypes mediated by the ubiquitous calcium-sensing STIM proteins. STIM1 activation by store depletion or mutational modification strongly suppresses voltage-operated calcium (CaV1.2) channels while activating store-operated Orai channels. Both actions are mediated by the short STIM-Orai activating region (SOAR) of STIM1. STIM1 interacts with CaV1.2 channels and localizes within discrete endoplasmic reticulum/plasma membrane junctions containing both CaV1.2 and ORAI1 channels. Hence, STIM1 interacts with and reciprocally controls two major calcium channels hitherto thought to operate independently. Such coordinated control of the widely expressed CaV1.2 and Orai channels has major implications for Ca2+ signal generation in excitable and nonexcitable cells.

  • the short n terminal domains of stim1 and stim2 control the activation kinetics of ORAI1 channels
    Journal of Biological Chemistry, 2009
    Co-Authors: Yandong Zhou, Donald L. Gill, Youjun Wang, Salvatore Mancarella, Chanyu Yue, Michael F Ritchie, Jonathan Soboloff
    Abstract:

    STIM1 and STIM2 are dynamic transmembrane endoplasmic reticulum Ca2+ sensors, coupling directly to activate plasma membrane Orai Ca2+ entry channels. Despite extensive sequence homology, the STIM proteins are functionally distinct. We reveal that the short variable N-terminal random coil sequences of STIM1 and STIM2 confer profoundly different activation properties. Using ORAI1-expressing HEK293 cells, chimeric replacement of the 43-amino-acid STIM1 N terminus with that of STIM2 attenuates ORAI1-mediated Ca2+ entry and drastically slows store-induced ORAI1 channel activation. Conversely, the 55-amino-acid STIM2 terminus substituted within STIM1 strikingly enhances both ORAI1-mediated Ca2+ entry and constitutive coupling to activate ORAI1 channels. Hence, STIM N termini are powerful coupling modifiers, functioning in STIM2 to “brake” the otherwise constitutive activation of ORAI1 channels afforded by its high sensitivity to luminal Ca2+.