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Zeng, Bo. "Pharmacological regulation and function of store-operated calcium channels". Thesis, University of Hull, 2012. http://hydra.hull.ac.uk/resources/hull:6432.
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Store-operated Ca²⁺ entry (SOCE) is an important Ca²⁺ influx pathway existing in almost all types of mammalian cell. STIM1, ORAI and TRPC have been regarded as the molecular basis of SOCE. Once the endoplasmic reticulum (ER) Ca²⁺ store is depleted, STIM1 proteins move to the plasma membrane and activate ORAI and TRPC channels to allow Ca²⁺ influx. In this thesis, the pharmacological aspects and regulatory mechanisms of SOCE were investigated using HEK293 cells overexpressing STIM1, ORAI or TRPC genes. The expression and function of TRPC channels and their spliced variants in native cells were also examined. Using live-cell imaging, the cytosolic clustering of STIM1-EYFP was observed after challenging with the compounds including 2-APB, flufenamic acid, 4-chloro- 3-ethylphenol, U73122 and FCCP. The aggregation of STIM1 in the cytosol coud be a novel mechanism for the inhibition of SOCE, and the process did not rely on the depletion of ER Ca²⁺ store. The ryanodine receptor agonist 4-chloro-3-ethylphenol not only caused Ca²⁺ release and cytosolic STIM1 clustering, but also nonselectively inhibited ORAI1/2/3 and TRPC3/4/5/6 channels. The sensitivity of TRP channels to metal ions was also investigated using patch clamp. Micromolar Cu²⁺ significantly increased the currents of TRPC3/4/5/6 channels. The glutamic acid (E542/E543) and cysteine (C554) residues in the extracellular pore region of TRPC4 were involved in the channel opening by Cu²⁺. Moreover, Cu²⁺ showed inhibitory effect on TRPM2 channel. TRPC1/3/4/6 and multiple alternatively spliced variants were detected in the human ovarian adenocarcinoma-derived SKOV3 cells. Blockade of TRPC channel activity by 2-APB, SKF-96365, TRPC pore-blocking antibodies or transfection with TRPC siRNA significantly inhibited SKOV3 cell proliferation. Overexpression of TRPC genes promoted colony growth of SKOV3 cells. It is concluded that cytosolic STIM1 movement could be a new pharmacological target for SOCE. 4-Chloro-3-ethylphenol and Cu²⁺ are new modulators of ORAI and TRP channels. TRPC channels and their spliced variants are important for cancer cell growth. These findings provide novel insights into the pharmacology and pathophysiology of store-operated channels.
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Bonds, Timetria. "Store-Operated Calcium Channels in the Function of Intracardiac Neurons". Scholar Commons, 2012. http://scholarcommons.usf.edu/etd/3983.
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Proper autonomic regulation of mammalian cardiac function is dependent upon very complex and precise communication among the intracardiac ganglia and individual neurons within the ganglia. An array of neuromodulators is found within the ganglia that direct neuronal activity by modulating the movement of calcium. The current study determines that opioidergic agonists, which have been found to contribute to severe cardiac disease states and intracellular calcium mobilization, are also responsible for changes in the function of the intracardiac neuron via their effects on store-operated calcium channels (SOCs).
Previous studies suggest that phosphorylation plays a role in SOC regulation. Using Fura-2 calcium fluorometry, we determined that protein kinase A (PKA), protein kinase C (PKC), and cyclic adenosine monophosphate (cAMP) had no effect on store-operated calcium entry in the presence of antagonists, phorbol 12, 13 dibutyrate (PDBu), forskolin, and 8-Br cAMP, respectively. We also found pharmacologically that using both electrophysiology and calcium imaging that μ-opioid agonists, met-enkephalin (ME) and endomorphin (EM) depress SOC activity in intracardiac neurons. Arachidonic acid (AA), which has been found to depress SOC function in rat liver cells and μ-opioid receptor activation (MOR), blocked both store-operated calcium entry (SOCE) and the calcium release-activated current (ICRAC) significantly. Contrastingly, AA metabolites, prostaglandin E2)(PGE2) and prostaglandin D2 (PGD2), do not significantly influence SOCE which suggests that the effects of AA may be direct. The block elicited by EM was partially reversed by pertussis toxin (PTX), indicative of activation of a PTX-sensitive G-protein following MOR activation. Similarly, PLA2 inhibitors, OBAA and AACOCF3, decreased the percent block of SOCE due to opioid agonist-induced inhibition.
Using the perforated-patch method of I-clamp electrophysiology, we demonstrated that gadolinium, at low micromolar concentrations, reversibly reduced action potential firing. Importantly, these results suggest that SOCs may influence action potential firing in mammalian intracardiac neurons. Similarly, AA and EM depressed action potential firing. Taken together, these experiments suggest that a pathway involving EM and AA influences repetitive firing through SOC inhibition.
The importance of SOCs in the maintenance of action potential firing and more specifically, the expression and biophysical functionality of the individual pore-forming subunits (Orai1, 2, and 3) in any neuronal cell type has previously not been explored. Quantitative RT-PCR along with I-clamp electrophysiology revealed that Orai3 was exclusive to repetitively firing neurons. As a result, we hypothesize that robust Ca2+-dependent fast inactivation, also associated Orai3, is a factor in the maintenance of repetitive action potential firing.
Using Fura-2 calcium fluorometry and patch-clamp electrophysiology, we determined pharmacologically that μ-opioid receptor activation precedes an intracellular cascade that is dependent on a PTX-sensitive G-protein and AA but independent of prostaglandin and protein kinase activity. Finally, we used RT-PCR to determine the Orai subunits expressed in the intracardiac neurons and their influence on neuronal firing patterns. This study is the first to determine the role expressed subunits has in the maintenance of the electrical activity of the neuron.
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Covington, Elizabeth D. "Oligomerization and dynamic clustering underlying activity of store-operated calcium channels /". May be available electronically:, 2009. http://proquest.umi.com/login?COPT=REJTPTU1MTUmSU5UPTAmVkVSPTI=&clientId=12498.
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Arunachalam, Sasi. "The Role of store operated calcium channels in human carcinoid cell lines". University of Toledo Health Science Campus / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=mco1279216983.
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Douglas, Sophie Georgina. "Regulation of CRAC channels and agonist-induced Ca2+ signals". Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:ae94ca14-ac95-4ea6-b14a-14f9f7bafd63.
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Calcium ions (Ca2+) are extremely important intracellular messengers, activating a plethora of cellular processes. Growing evidence now points to a major role for the local Ca2+ signal in driving specific cellular responses. The simplest and most fundamental local Ca2+ signal is the Ca2+ microdomain, which rapidly forms when Ca2+ permeable ion channels open. In non-excitable cells the dominant Ca2+ entry channels are store-operated Ca2+ channels (SOCCs). The best characterised is the Ca2+ release activated Ca2+ (CRAC) channel. How local Ca2+ entry through CRAC channels impacts on channel function however is unclear. I have investigated the interaction between the Ca2+ binding protein calmodulin and CRAC channel activity and subsequent agonist-induced Ca2+ signals. Furthermore, I have investigated a role for mitofusin 2 (a protein that is known to tether the ER and mitochondria) on these Ca2+ signals. Using three different calmodulin mutant constructs with alterations to their Ca2+ binding sensitivities, I have shown that calmodulin facilitates CRAC channel dependent Ca2+ entry and maintains agonist-induced cytosolic Ca2+ oscillations in a lobe-specific manner. Calmodulin has four Ca2+ binding sites, two on the N-lobe and two on the C-lobe. I found a dominant negative calmodulin mutant (CAM4M, where all four binding sites had been mutated), or one where the C-lobe could not bind Ca2+ (CAM2C), impaired both Ca2+ influx through CRAC channels and maintenance of cytosolic Ca2+ oscillations. In contrast, a Ca2+-insensitive N-lobe mutant had little effect, (CAM2N). Knockdown of the mitochondrial Ca2+ uniporter regulator (MICU1) or mitochondrial membrane depolarization had similar effects to those seen with CAM4M or CAM2C, suggesting that at least in part, the action of calmodulin was through regulation of mitochondrial Ca2+ dynamics. This was confirmed by directly measuring the mitochondrial matrix Ca2+ concentration in intact RBL-1 cells using the mitochondrial targeted, fluorescent protein, pericam. Both CAM4M and disruption of mitochondrial Ca2+ buffering impaired agonist-induced mitochondrial Ca2+ uptake, suggesting that the modulation of CRAC channels occurred through Ca2+-calmodulin facilitation of mitochondrial Ca2+ uptake. Using a mutant Orai1 (A73E) that cannot bind calmodulin, I have shown that calmodulin tethered to the CRAC channel provides a major source of calmodulin for effective mitochondrial Ca2+ uptake. Physiological relevance of my proposed pathway was provided from experiments where I showed knockdown of MICU1 impaired agonist-induced CRAC channel dependent NFAT-1-driven gene expression. In addition, I establish a crucial role for mitochondrial MFN2 and presumably its ability to properly link the mitochondria and ER in the control of CRAC channels and agonist-induced Ca2+ signals.
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Luik, Riina M. "Molecular mechanisms of store-operated calcium signaling : local activation of CRAC channels by STIM1, the ER calcium sensor /". May be available electronically:, 2007. http://proquest.umi.com/login?COPT=REJTPTU1MTUmSU5UPTAmVkVSPTI=&clientId=12498.
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Bose, Diptiman Dipen. "Study of pharmacological and physiological factors regulating store operated calcium channels in a neuronal cell line". Scholarly Commons, 2006. https://scholarlycommons.pacific.edu/uop_etds/2650.
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Generation of Ca 2+ signals in cells involves regulation by multiple components controlling Ca 2+ release from the internal stores, Ca 2+ influx across the plasma membrane (PM), elicitation of Ca 2+ sensitive processes and finally the removal of Ca 2+ from the cell. One such mode of facilitating Ca 2+ entry is called store-operated Ca 2+ entry (SOCE) mediated by the store operated Ca 2+ channels (SOCs). SOCE, wherein the depletion of internal Ca 2+ stores triggers the influx of Ca 2+ across the PM, not only plays a vital role in refilling the Ca 2+ stores, but also regulates a multitude of downstream Ca 2+ regulated signalling events. Despite recent advances in elucidating the entry pathway, its molecular identity, biophysical properties and store-depletion signal remains undefined. The most potent inducer of SOCE, thapsigargin (TG), fails to induce Ca 2+ influx in the NG115-401L (401L) cells. This unusual phenotype of the cell makes it a useful model to study the mechanisms and components underlying the SOCE pathway. Although TG failed to induce SOCE in the 401L cells, we report that the activation of intracellular release channels such as the inositol-1,4,5-trisphosphate (fP3Rs) and ryanodine receptors (RyRs) were able to activate Ca 2+ influx upon store depletion. This is in keeping with mechanisms proposed to explain SOCE, namely the conformational coupling hypothesis, wherein depletion of the ER stores signals the release channels to physically interact with the PM SOCs. We found that disrupting the communication between the ER and the PM channels induced by actin disassembly affected both Ca 2+ release and influx. Our study shows that Ca 2+ release and influx is dependent on cortical actin organization and that the RyR mediated release is less regulated by cortical actin than the IP3R induced Ca 2+ release. Studies conducted using 2-aminoethoxy diphenylborate (2-APB), a commonly used SOC blocker, revealed that 2-APB stimulated Ca 2+ release in the 401L cells. This release of Ca 2+ was also found to be dependent on the conformational coupling between the ER and PM SOCs. We also studied the effect of overexpressing various isoforms of the transient receptor potential (TRP) channels. We found that protein kinase C (PKC) differentially regulated the activity of the TRP channels in the 401L cells. PKC activation prolonged the Ca 2+ influx in the wild type cells while attenuating the same in the TRP transfected cells. We also found that influx of surrogate cations (Ba 2+ ) is augmented in the TRPC transfected cells. Our studies reveal that the activation of ER release channels followed by conformational coupling with the PM channels may be a mechanism by which the 401L cells or neurons in general maintain a rigid control over intracellular Ca 2+ concentrations and thus regulate Ca 2+ homeostasis.
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Jones, Lynette. "The role of IP3 receptors 1 and 2 in the activation of store-operated calcium channels in rat liver cells /". Title page and abstract only, 2005. http://web4.library.adelaide.edu.au/theses/09SB/09sbj762.pdf.
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McElroy, Stuart Patrick. "Store operated calcium entry and TRPC channel expression in rat pulmonary artery smooth muscle cells". Thesis, University of Strathclyde, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.426332.
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Castro, Kraftchenko Joel, i kraf0005@flinders edu au. "STORE OPERATED Ca2+ CHANNELS IN LIVER CELLS: REGULATION BY BILE ACIDS AND A SUB-REGION OF THE ENDOPLASMIC RETICULUM". Flinders University. Medicine, 2008. http://catalogue.flinders.edu.au./local/adt/public/adt-SFU20080826.135311.
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Cholestasis is an important liver pathology. During cholestasis bile acids accumulate in the bile canaliculus affecting hepatocyte viability. The actions of bile acids require changes in the release of Ca2+ from intracellular stores and in Ca2+ entry. The target(s) of the Ca2+ entry pathway affected by bile acids is, however, not known. The overall objective of the work described in this thesis was to elucidate the target(s) and mechanism(s) of bile acids-induced modulation of hepatocytes calcium homeostasis.
First, it was shown that a 12 h pre-incubation with cholestatic bile acids (to mimic cholestasis conditions) induced the inhibition of Ca2+ entry through store-operated Ca2+ channels (SOCs), while the addition of choleretic bile acids to the incubation medium caused the reversible activation of Ca2+ entry through SOCs. Moreover, it was shown that incubation of liver cells with choleretic bile acids counteracts the inhibition of Ca2+ entry caused by pre-incubation with cholestatic bile acids. Thus, it was concluded that SOCs are the target of bile acids action in liver cells.
Surprisingly, despite the effect of choleretic bile acids in activating SOCs, the Ca2+ dye fura-2 failed to detect choleretic bile acid-induced Ca2+ release from intracellular stores in the absence of extracellular Ca2+. However, under the same conditions, when the sub-plasma membrane Ca2+ levels were measured using FFP-18 Ca2+ dye, choleretic bile acid induced a transient increase in FFP-18 fluorescence. This evidence suggested that choleretic bile acids-induced activation of Ca2+ entry through SOCs, involving the release of Ca2+ from a region of the endoplasmic reticulum (ER) located in the vicinity of the plasma membrane.
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Bonnefond, Marie-Laure. "Rôle de la signalisation calcique dans la sensibilisation des cancers ovariens chimiorésistants aux stratégies anti-Bcl-xL". Thesis, Normandie, 2017. http://www.theses.fr/2017NORMC424.
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Les cancers de l’ovaire représentent la première cause de décès par cancer gynécologique. L’échec des traitements, lié à l’apparition d’une chimiorésistance, implique de trouver de nouvelles stratégies thérapeutiques. Dans ce cadre, le laboratoire a mis en évidence la coopération de deux molécules anti-apoptotiques surexprimées dans les cancers de l’ovaire, Mcl-1 et Bcl-xL, qui empêchent alors le déclenchement de la mort cellulaire par apoptose. L’inhibition de ces cibles est alors mise en place. Un BH3-mimétique inhibiteur de Bcl-xL a été développé par la société Abbvie, l’ABT-737, qui possède un dérivé administrable par voie orale l’ABT-263 en essai clinique. En revanche, aucun inhibiteur de Mcl-1 n’est actuellement en clinique. L’inhibition de cet anti-apoptotique est donc l’un des objectifs du laboratoire. Sachant que Mcl-1 est extrêmement régulée, que l’inhibition de la voie de signalisation PI3K/Akt/mTOR conduit à l’inhibition de cet anti-apoptotique, et que le calcium est capable de moduler cette voie, nous nous sommes demandés si la modulation des flux calciques permettait l’inhibition de Mcl-1 dans nos cellules. Ces travaux de thèse ont pu montrer dans un premier temps que la chélation calcique par le BAPTA-AM permettait d’inhiber Mcl-1 via la voie mTORC1 et de sensibiliser les cellules à l’ABT-737. Dans un second temps, l’étude de l’effet d’un inhibiteur des flux calciques en essais cliniques, le carboxyamidotriazole a permis de mettre en évidence que l’inhibition des canaux calciques capacitifs pouvait entraîner l’inhibition de Mcl-1 de nouveau en inhibant mTORC1 et induire la mort cellulaire en combinaison avec l’ABT-737. Enfin des observations morphologiques ont montré que le CAI induisait un changement morphologique aboutissant à la mort des cellules. Ce type de mort semble être lié à une perturbation du métabolisme des cellules cancéreuses IGROV1-R10 et se rapprocherait d’une mort récemmement décrite dans la littérature : l’autosisOvarian cancer is the leading cause of death from gynecological cancer. There is an urgent need to find new therapeutic strategies due to failure of treatments associated to development of chemoresistance. In this context, the laboratory has shown the cooperation of two anti-apoptotic proteins overexpressed in ovarian cancer, Mcl-1 and Bcl-xL, for preventing apoptotic cell death. ABT-737, a Bcl-xL inhibitor BH3-mimetic, was developed by Abbvie and has a clinically derivative named ABT-263. In contrast, no Mcl-1 inhibitor is currently available in clinic. The inhibition of this anti-apoptotic protein is therefore one of the objectives of the laboratory. Since inhibition of PI3K / Akt / mTOR signaling pathway leads to inhibition of Mcl-1 expression and calcium is able to modulate this pathway, we wondered if the modulation of calcium fluxes allowed the inhibition of Mcl-1 in ovarian cancer cells. First we were able to show that calcium chelation by BAPTA-AM allowed to inhibit Mcl-1 expression via mTORC1 pathway and to sensitize the cells to ABT-737. In a second step, we investigated the effect of an inhibitor of calcium fluxes that is evaluated in clinical studies, carboxyamidotriazole. We show that the inhibition of capacitive calcium channels could lead to Mcl-1 down-expression via inhibition of mTORC1 and promote apoptosis in combination with ABT-737. Finally, we observed that CAI induces a morphological change resulting in cell death. This type of death seems to be related to disruption of metabolism in IGROV1-R10 cancer cells and would be close to a cell death recently described in the literature: autosis
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Terrie, Élodie. "Rôle de la signalisation calcique dépendante des Store-Operated Channels (SOC) dans les cellules souches neurales adultes et les cellules souches cancéreuses de glioblastomes". Thesis, Poitiers, 2019. http://www.theses.fr/2019POIT2322.
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Des cellules souches neurales (CSN) persistent dans le cerveau adulte et produisent des neurones et des cellules gliales tout au long de la vie de l’individu. Les CSN suscitent un intérêt considérable pour la médecine régénératrice mais leur utilisation thérapeutique potentielle nécessite au préalable d’approfondir les connaissances sur leurs mécanismes de régulation. Les glioblastomes, quant à eux, sont les tumeurs cérébrales les plus fréquentes chez l’adulte et les plus mortelles. Au sein de ces tumeurs, les cellules souches de glioblastomes (CSG) seraient issues de la transformation maligne des CSN et seraient responsable de l’initiation, de la propagation et de la résistance aux traitements des tumeurs. Des analyses transcriptomiques ont suggéré un rôle majeur de la signalisation calcique au sein des CSN et des CSG. Représentant une des voies principales d’entrée du calcium dans la cellule, les canaux calciques SOC (Store-Operated Channels) régulent de nombreux processus cellulaires, y compris la progression tumorale. L’objectif des travaux de cette thèse est d’évaluer le rôle des SOC dans les CSN et les CSG.Nous avons établi par des approches in vitro et in vivo, que les CSN de souris adulte expriment des SOC fonctionnels et que leur inhibition pharmacologique diminue la prolifération et l’autorenouvellement des CSN, propriété indispensable au maintien de la population souche. La deuxième partie de nos travaux montre que les CSG issues de cultures primaires de patients expriment des SOC dont l’inhibition altère la prolifération et l’autorenouvellement de ces cellules.Ainsi, les résultats obtenus lors de cette thèse mettent en évidence un rôle essentiel des SOC dans la régulation de l’autorenouvellement des CSN et des CSG. Les CSG étant responsables de la résistance aux traitements dans le glioblastome, ces travaux ouvrent des perspectives thérapeutiques ciblant les canaux calciques pour contrer cette pathologie au pronostic sombreNeural stem cells (NSC) persist in the brain of adult mammals and fuel the brain with new neurons and glial cells all lifelong. Recruited by brain injuries, NSC are considered with great interest by regenerative medicine. However, the development of new therapeutic approaches based on the use of NSC requires an in-depth knowledge of the mechanism regulating these cells. Glioblastomas are the most frequent and deadliest form of adult brain tumors. Within the tumor, glioblastoma stem cells (GSC) form a subpopulation of cells that is considered as responsible of tumor initiation, propagation and relapse, as these cells are particularly resistant to anti-tumoral treatments. GSC and NSC share key characteristics and numerous studies suggest that GSC arise from transformed NSC. Transcriptomic analysis of NSC and of GSC revealed an enrichment of calcium signaling transcripts in these two cell populations. Representing a major way of calcium influx into cells, Store-Operated Channels (SOC) are mobilized in response to a wide range of extracellular factors. SOC regulate many cellular processes and are often hijacked in cancer to promote tumor progression.The aim of this thesis is to evaluate potential SOC involvement in NSC and GSC regulation.The first part of this work, relying on in vitro and in vivo studies, demonstrates that NSC from adult mice express functional SOC whose inhibition by pharmacological agents reduces NSC proliferation and self-renewal. In the second part of this thesis, we demonstrate that GSC from primary cultures derived from patients express SOC, as do NSC, and that SOC inhibition reduces GSC ability to proliferate and self-renew.Accordingly, the results of this thesis demonstrate that SOC regulate NSC and GSC self-renewal, a property that is essential to maintain stem cells pool. As GSC are responsible for glioblastomas treatment resistance, our studies point to a potential new therapeutic way, via calcium channels, against this deadly pathology
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Batchelor, Helen R. "Characterisation of store-operated calcium entry in a vascular endothelial cell line and impact on the production of nitric oxide". Thesis, University of Oxford, 2014. https://ora.ox.ac.uk/objects/uuid:548a6062-2b2f-46ce-b91a-890561b5edff.
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Store-operated calcium entry (SOCE) is a principal mechanism for extracellular calcium entry in non-excitable cell types, and is primarily facilitated by the calcium- release activated calcium (CRAC) channel; itself comprised of the pore-forming Orai-1 and calcium-sensing Stromal interaction molecule (STIM)-1 proteins. Depletion of endoplasmic reticulum (ER) calcium stores initiates STIM-1 translocation to defined ER-plasma membrane puncta, and subsequent Orai-STIM interaction and opening of Orai. The importance of this mechanism in calcium signalling in diverse tissue types is becoming increasingly clear. The vascular endothelium is a dynamic tissue, involved in the maintenance of vascular homeostasis and haemostasis. Many endothelium-derived bioactive agents, such as endothelin-1, prostaglandins, and the potent vasodilator nitric oxide (NO), are known to be produced via calcium- dependent mechanisms. However, the role of the CRAC channel in the vascular endothelium is poorly defined with little known about downstream targets of calcium influx through CRAC channels. The dysregulation of NO production by endothelial nitric oxide synthase (eNOS) is a major contributory factor in many vascular disease states, yet the calcium channel responsible for eNOS activation has yet to be identified. Within this thesis, I establish the endothelial cell line sEnd.1 as a new model system for studying CRAC channel signalling in the vascular endothelium, defining sEnd.1 SOCE as being CRAC channel-dependent. Inhibition of CRAC channels with an array of inhibitors, and knock-down of STIM-1, both reduced ATP- and TG-induced SOCE. The sEnd.1 model system was subsequently used to identify calcium entry through the CRAC channel as the elusive activation mechanism for eNOS. Through real-time imaging with the fluorescent NO dye DAF-2-DA, we established that NO production is non-linear, with a slow initial increase preceding a faster NO production phase. These kinetics, with a characteristic delay before fast production have, to our knowledge, not previously been reported. The time taken to reach the fast phase of NO production could be manipulated through changes in both local and bulk calcium rises, which indicated roles for both elements of calcium signalling in eNOS activation. eNOS regulation by calcium is complex, occurring not only through direct binding of calcium-calmodulin, but additionally through changing post-translational modifications, which in turn regulate the calcium-dependency of eNOS, such as phosphorylation of Ser1177. We propose that the delay in fast production of NO is due to the time taken to alter eNOS post-translational modifications, which thus remove inhibition on eNOS. Activation of CRAC channels increased phosphorylation of residue Ser1177 via calcium-calmodulin kinase II (CaMKII), with a similar time course to that required to reach the fast phase of NO production. Inhibition of CaMKII increased the time taken to reach fast activation. In conclusion this thesis presents a new model system for investigation of CRAC channel signalling in the endothelium. Furthermore, we clearly identify a critical endothelial pathway as being regulated by CRAC channels, by demonstrating the production of NO in response to both ATP and TG, which stimulate calcium entry through CRAC channels.
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Vanden, Abeele Fabien. "Caractérisation moléculaire et fonctionnelle des canaux calciques de type SOC (Store Operated Channel) : implication dans la cancerogénese prostatique". Lille 1, 2003. https://pepite-depot.univ-lille.fr/RESTREINT/Th_Num/2003/50376-2003-221.pdf.
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Seconde cause de mortalité par cancer chez l'homme, le cancer de la prostate presente une incidence croissante liée à l'augmentation de l'espérance de vie dans les pays développés. Ce cancer est, à l'origine, dépendant des androgènes, puis évolue vers un stade androgéno-indépendant pour lequel aucun traitement curatif n'existe. Le cancer androgéno-indépendant de la prostate se caractérise par l'àpparition de cellules tumorales surexprimant l'oncoprotéine Bcl-2 et de cellules cancéreuses différenciées en cellules neuroendocrines. Ces deux types cellulaires présentent un défaut d'apoptose rendant inefficaces les chimiothérapies. Il est maintenant bien établi que le calcium est un des facteurs majeurs impliqués dans l'apoptose. Cependant, les mécanismes exacts par lesquels le calcium participe à ce processus sont encore mal connus. Les canaux calciques de type SOC (Store Operated Channel) de la membrane cellulaire, activés par la vidange des réserves calciques intra-réticulaires, seraient des éléments clés, intervenant dans le contrôle de l'apoptose des cellules cancéreuses. Or, ces canaux n'ont jamais été étudiés dans les cellules cancéreuses prostatiques. Malgré des efforts de recherche intenses durant ces dix dernières années, deux questions principales restent encore posées concernant les canaux de type SOC: qu'elle est la nature du signal permettant l'activation spécifique de ces canaux et en quoi consiste leur nature moléculaire ? L'identification et la caractérisation électrophysiologique du courant SOC dans les cellules cancéreuses prostatiques LNCaP nous ont ensuite permis de détenniner la nature moléculaire et le mécanisme de couplage de ces canaux. De nombreuses recherches ont été dévolues ces dernières années aux canaux de type TRP "Transient Receptor Potential" et à leur relation possible avec les SOCs. Nos résultats montrent l'implication de trois protéines TRP dans la constitution des canaux SOC des cellules LNCaP. TRPC1, TRPC4 et TRPV6 seraient les Sous unités principales à la base de complexes hétéromultimériques encore non caractérisés et formant les canaux SOC. TRPC1 serait préférentiellement couplée à la vidange des stocks par un couplage de type conformationnel tandis que TRPC4 serait préférentiellement couplée via un facteur diffusible encore inconnu à ce jour. La protéine TRPV6, quant à elle, présente à la fois un couplage par un facteur diffusible et de type conformationnel. Nous avons mis en évidence une perturbation importante des mécanismes d'homéostasie calcique dans les cellules LNCaP rendues androgéno-indépendantes par surexpression de l'oncoprotéine Bcl-2 ou par différenciation neuroendocrine. Dans les deux cas, il s'ensuit une diminution importante de la concentration en calcium réticulaire et une inhibition du fonctionnement des canaux SOC. De ce fait, la résistance à l'apoptose observée dans les cellules androgéno-indépendantes serait en partie due à des perturbations de l'homéostasie calcique impliquant notamment les canaux SOC. Au cours de ma thèse, nous avons donc obtenu de nouveaux résultats qui pennettent d'expliquer le rôle des canaux SOC et du calcium réticulaire dans la régulation de l'apoptose des cellules cancéreuses de la prostate humaine.
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Vieira, Elaine. "Signal Transduction of Glucagon Secretion". Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6319.
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Kawasaki, Brian Takeshi. "Players in the regulation of calcium entry activation of the transient receptor potential channels by src tyrosine kinase and a distinct role for the IP3 receptor c-terminus in store- and receptor-operated calcium entry /". Diss., Restricted to subscribing institutions, 2005. http://proquest.umi.com/pqdweb?did=994232031&sid=9&Fmt=2&clientId=1564&RQT=309&VName=PQD.
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Florent, Romane. "Intérêt de la modulation pharmacologique des voies de signalisation calcique pour restaurer le contrôle de l'apoptose dans les cancers ovariens chimiorésistants Inhibition of store-operated channels by carboxyamidotriazole sensitizes ovarian carcinoma cells to anti-BclxL strategies through Mcl-1 down-regulation Drug Repositioning of the α1-Adrenergic Receptor Antagonist Naftopidil: A Potential New Anti-Cancer Drug? Bim, Puma and Noxa upregulation by Naftopidil sensitizes ovarian cancer to the BH3-mimetic ABT-737 and the MEK inhibitor Trametinib". Thesis, Normandie, 2020. http://www.theses.fr/2020NORMC413.
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Le sombre pronostic du cancer de l’ovaire s’explique notamment par une fréquence importante de résistance à la chimiothérapie conventionnelle présentée par les patientes. La mise en place de stratégies thérapeutiques alternatives à la chimiothérapie, mais aussi la découverte de biomarqueurs prédictifs de la réponse à ce traitement, représentent donc un enjeu majeur pour améliorer la prise en charge de cette pathologie. La chimiorésistance des cellules cancéreuses ovariennes s’explique principalement par leur résistance à l’apoptose, résultant d’un déséquilibre entre les membres pro- et anti-apoptotiques de la famille Bcl-2 qui contrôlent cette mort cellulaire. De ce fait, toutes stratégies capables de moduler le ratio [pro ]/[anti apoptotiques] en faveur des [pro-] restaure efficacement l’apoptose de ces cellules. Or, la signalisation calcique est connue pour réguler l’expression de ces protéines et apparait ainsi comme une cible pertinente pour restaurer l’apoptose des cellules cancéreuses ovariennes chimiorésistantes. Dans ce contexte, nous avons mis en évidence que trois modulateurs du signal calcique sont capables d’induire la mort de ces dernières en association à l’ABT-737, un BH3-mimétique ciblant l’activité de l’anti apoptotique Bcl-xL. Cette sensibilisation à l’ABT-737 est permise par le fait que le carboxyamidotriazole réprime l’expression de l’anti apoptotique Mcl-1 via l’inhibition des courants SOCE, le naftopidil augmente l’expression des protéines pro apoptotiques via l’induction d’un stress du RE ou l’activation de JNK et que la thapsigargine semble préparer à la mort cellulaire grâce à une augmentation de la concentration calcique intracellulaire via STIM1 et, peut-être, via l’induction de l’expression de Noxa. En outre, les acteurs de la signalisation calcique, connus pour subir un remodelage au cours des processus de cancérogenèse pourraient se révéler comme des outils de prédiction de réponse à la chimiothérapie. Dans ce contexte, nous avons mis en évidence que l’expression de la pompe calcique SERCA2 semble jouer le rôle de biomarqueur prédictif de la réponse à la chimiothérapie des patientes atteintes d’un cancer de l’ovaireThe poor prognosis of ovarian cancer is mainly explained by a high rate of resistance to conventional chemotherapy presented by patients. Therefore, discovery of both alternative therapeutic strategies to chemotherapy and predictive biomarkers for response to this treatment represent a major challenge for improving the management of this pathology. Chemoresistance of ovarian cancer cells is mainly due to their resistance to apoptosis, resulting from an imbalance between the pro- and anti-apoptotic members of the Bcl-2 family that control this type of cell death. Thus, all strategies able to modulate the [pro]/[anti-apoptotic] protein ratio in favor of [pro-] effectively restore apoptosis in these cells. However, calcium signaling is known to regulate the expression of these proteins and thus appears to be a relevant target for restoring apoptosis in chemoresistant ovarian cancer cells. In this context, we have shown that three calcium signal modulators are able to induce the death of these cells in association with ABT-737, a BH3-mimetic targeting the activity of the anti-apoptotic Bcl-xL. This sensitization to ABT-737 is enabled by the fact that carboxyamidotriazole represses the expression of the anti apoptotic Mcl-1 via the inhibition of SOCE currents, naftopidil increases pro-apoptotic protein expression via ER stress induction or JNK activation and thapsigargin seems to prepare cell death through increasing intracellular calcium concentration via STIM1 and, maybe, through Noxa expression induction. In addition, players of the calcium signaling toolkit, known to undergo remodeling during carcinogenesis could be proven as tools for predicting response to chemotherapy. In this context, we have shown that the expression of the calcium pump SERCA2 seems to play a role as a predictive biomarker for response to chemotherapy of patients with ovarian cancer
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Zhang, Changfeng. "Investigation of the endoplsmic reticulum calcium stores for their potential roles in neuroprotection using the NG115-401L neuronal cell line model". Scholarly Commons, 2014. https://scholarlycommons.pacific.edu/uop_etds/142.
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There is significant interest in the field of neuroscience to gain a better understanding of how neurons die in neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. We have used the neuronal cell line NG115-401L with unique calcium signaling characteristics to test the hypothesis that improving calcium loading into the endoplasmic reticulum (ER) to increase ER calcium levels acts as a possible neuroprotective response. We approached this problem using both pharmacological and genetic approaches targeting the central mediator of calcium uptake in the ER localized sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) enzyme. The pharmacological studies involved use of the ginger root compound 6-gingerol, which to date is the best documented agent for activating SERCA enzymes in heart and skeletal muscle. However, in our experiments, gingerol did not appear to activate NG115-401L SERCA pumps; indeed, the compound produced a response more like that of a SERCA inhibitor inducing a rapid ER calcium depletion. In addition, gingerol stimulated robust calcium influx responses, an unexpected result given the NG115-401L neural cell line is uniquely deficient in calcium influx pathways. Our genetic approach involved expressing the stromal interaction molecule 1 (STIM1) protein in the NG115-401L cell, which is also an ER localized protein that serves as a pivotal calcium influx channel regulator. NG115-40lL neurons present a native deficiency of STIM1 expression in a background phenotype with well characterized perturbations in ER calcium regulation and control of calcium influx pathways. Thus, STIM1 may be predicted to increase ER calcium levels, conferring protection against neuron cell death due to ER calcium store defects. STIM1 expression reconstituted the corrupted calcium influx pathway in NG115-401L neurons, which conferred neuroprotective responses to ER calcium perturbation, mitochondrial oxidative stress and subsequent cell death. Our results argue for unique and undiscovered regulatory effects of gingerol on the ER calcium circulation system, and suggest that the expression of STIM1 in these neurons protects against ER stress and oxidative stress via reconstruction of cellular calcium homeostasis.
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Ng, Lih Chyuan. "Store-operated channels in rat pulmonary artery". Thesis, University of Strathclyde, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.248256.
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Nash, Katherine Louise. "Store-operated calcium entry in human spermatozoa". Thesis, University of Birmingham, 2012. http://etheses.bham.ac.uk//id/eprint/3260/.
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The contribution of store-operated calcium entry (SOCE) in the response of human sperm to progesterone, a steroid secreted from the cumulus cells surrounding the oocyte, has not yet been elucidated. The aim of this study was to investigate the presence of SOCE proteins in human sperm and examine the effects of pharmacological modulation of SOCE on the progesterone-induced biphasic intracellular calcium concentration ([Ca\(^{2+}\)]i) response. STIM (stromal interacting molecule) and Orai, proteins of the SOCE system were detected in human sperm in a similar location to intracellular Ca\(^{2+}\) stores. 2-aminoethyldiphenyl borate (2-APB; SOCE modulator) altered SOCE in human sperm in a bimodal manner as seen in other cell types. Furthermore, 5\(\mu\)M 2-APB potentiated the initial progesterone-induced [Ca\(^{2+}\)]i transient within the neck and midpiece, but not in the flagellum. In the sustained phase of the progesterone-induced [Ca\(^{2+}\)]i response both 5\(\mu\)M 2-APB and 10\(\mu\)M loperamide (another modulator of SOCE) potentiated the [Ca\(^{2+}\)]i response. Higher doses of 2-APB (50-200\(\mu\)M) didn’t potentiate the transient [Ca\(^{2+}\)]i and inhibited the sustained response consistent with reported actions on SOCE. Ryanodine receptors were localised to the neck/midpiece region which suggested that they may mobilise intracellular Ca\(^{2+}\) stores in response to progesterone, leading to activation of STIM/Orai and initiating SOCE.
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Di, Capite Joseph Lister. "Store-operated calcium entry and mast cell function". Thesis, University of Oxford, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.509920.
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Bailey, Marc Aaron. "Store operated calcium entry in abdominal aortic aneurysm". Thesis, University of Leeds, 2016. http://etheses.whiterose.ac.uk/13678/.
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Background: Abdominal Aortic Aneurysm (AAA) is a focal dilatation of the abdominal aorta that progresses over time and eventually ruptures. Surgery is risky but a medical therapy is lacking. Vascular smooth muscle cells (VSMC) are apoptotic in end-stage AAA. There is evidence that they undergo pathological remodelling in early disease. My novel hypothesis is that reduction of this pathological vascular remodelling will be beneficial. Platelet derived growth factor (PDGF) drives VSMC remodelling through IP3 generation, ER Ca2+ store release and store operated Ca2+ entry (SOCE) via the plasma membrane Orai1 channel. In this thesis I will explore the possibilities of small-molecule Orai1 inhibition to attenuate VSMC remodelling and AAA growth. Methods & Results: A novel Orai1 blocker, JPIII is used throughout the thesis. Orai1 was functionally expressed in AAA VSMC and could be inhibited by JPIII which had nanomolar potency against SOCE and reduced downstream proliferation, migration and apoptosis. JPIII was also effective in rodent VSMC against SOCE and the downstream NFAT activation and was selective for Orai1 in a Cerep selectivity screen. In vivo, three murine models of AAA were used (AngII, CaCl2 and PPE); VSMC remodelling and Orai1 upregulation assessed histologically were apparent in all three models. JPIII treatment reduced AAA VSMC remodelling (by histology) and AAA lumen dimensions (by histology and in vivo ultrasound). JPIII was also effective against AAA progression in mice with established AAA. No major toxic flags were demonstrated but JPIII was a pan CYP450 inhibitor. It was possible to improve the PK of the molecule without loss of the AAA effect. Conclusion: These data suggest it is possible to modify the VSMC remodelling response in AAA through Orai1 inhibition with a small molecule blocker with no major off target effects in mice. This is a promising starting point for translational development of a therapy for human patients.
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Djillani, Alaeddine. "Caractérisation des canaux calciques dans les polynucléaires neutrophiles : rôle dans la phagocytose et la production des radicaux libres oxygénés". Phd thesis, Université Paris Sud - Paris XI, 2013. http://tel.archives-ouvertes.fr/tel-01069097.
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Les polynucléaires neutrophiles représentent 50-70% des leucocytes sanguins et possèdent un rôle majeur dans la défense de l'organisme contre les pathogènes. Le Ca2+ est un second messager qui joue un rôle primordial dans le chimiotactisme, la phagocytose, la dégranulation et la production de formes réactives de l'oxygène (FRO) afin de neutraliser l'agent pathogène. Dans ces cellules, l'influx calcique de type SOCE est essentiel pour l'homéostasie calcique. Il est peu étudié en raison du manque d'outils pharmacologiques spécifiques d'où l'importance dans un premier temps de chercher de nouvelles molécules. Les cellules T Jurkat dont le SOCE est largement caractérisé servent de modèle pour la caractérisation initiale de ces molécules. Le 2-APB est parmi les molécules les plus largement utilisées dans la caractérisation du SOCE en raison de sa double activité sur le SOCE avec une potentialisation à [1-10 μM] et une inhibition à [> 20 μM]. En revanche, ce produit manque de spécificité et agit sur d'autres cibles cellulaires comme les récepteurs à l'inositol (1,4,5)-trisphosphate (InsP3Rs). La 1ère étape est de sélectionner à partir d'analogues commerciaux du 2-APB (Methoxy-APB, Dimethoxy-APB, Cyclic-APB, Benzothienyl-APB, Thienyl-APB et MDEB), des composés plus spécifiques et également plus efficaces que la molécule mère. Deux molécules se sont distinguées : le MDEB comme uniquement potentialisant du SOCE et le Benzothienyl-APB comme un puissant inhibiteur. En revanche, tous les analogues du 2-APB inhibent les InsP3Rs à l'exception du MDEB qui semble plus spécifique du SOCE. L'effet du MDEB sur le courant calcique, ICRAC, a été étudié grâce à la technique du patch-clamp. Il augmente d'environ 4 fois l'amplitude de ICRAC par rapport à celle enregistrée dans les cellules contrôle. Par ailleurs, le MDEB ralentie l'inactivation rapide de ICRAC due au Ca2+. Sur le plan physiologique, le MDEB à des concentrations croissantes inhibe la synthèse de l'IL-2 dans les cellules Jurkat stimulées et ceci malgré son effet potentialisant du SOCE. Cette activité est liée à son effet pro-apoptotique dans les cellules Jurkat stimulées. Le MDEB et le Benzothienyl-APB caractérisés dans la 1ère partie nous ont servi d'outils potentiels afin d'étudier le SOCE des cellules PLB-985 différenciées en cellules proches de neutrophiles. Le SOCE a été induit soit par un traitement des cellules avec la thapsigargine (Tg) soit de manière physiologique avec les peptides fMLF et le WKYMVm deux chimioattractants, ligands des récepteurs aux peptides formylés FPR et FPRL1 respectivement. En plus, le SOCE induit par la Tg est modulable par le 2-APB, potentialisé par le MDEB et inhibé par le Benzothienyl-APB. La phagocytose des levures par les cellules PLB-985 différenciées ainsi que la production de FRO intraphagosomales ont été inhibées par le MDEB et le Benzothienyl-APB. Les FRO extracellulaires ont été également inhibées par Benzothienyl-APB en revanche à cause de la forte interférence du MDEB avec la technique de mesure nous n'avons pas pu étudier ses activités. En conclusion, le MDEB et le Benzothienyl-APB sont de nouveaux outils pharmacologiques potentialisant ou inhibant le SOCE des leucocytes, qui nous permettront dans l'avenir une meilleure compréhension de l'entrée calcique et ses rôles dans ces cellules.
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Hao, Baixia, i 郝佰侠. "Regulatory and functional studies of store-operated calcium entry". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hdl.handle.net/10722/196486.
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Ca2+ signaling is essential for a wide variety of cellular activities, ranging from short term activities, such as synaptic and muscle contraction, to long term processes, such as proliferation and differentiation. Store-operated Ca2+ entry (SOCE), an important Ca2+ influx pathway in non-excitable cells, well coordinates Ca2+ release from ER and Ca2+ influx through plasma membrane. STIM1 and Orai1, serving as ER Ca2+ sensor and pore forming subunit, respectively, are the two essential components of SOCE machinery. In addition to activate Orai1 channel, studies have shown that STIM1 regulates other plasma membrane Ca2+ channels and senses a variety of cellular stresses to regulate SOCE. Therefore, it is of great interests to investigate the mechanisms and physiological functions of STIM1 and Orai1 mediated SOCE.
Here, we performed tandem affinity purification to identify STIM1 associated proteins in Hela cells stably expressing STIM1-His6-3×Flag. Four candidate proteins, including GRP78, HSP70, IQGAP1, and Actin, were identified by mass spectrometry analyses. Surprisingly, IQGAP1 failed to affect the activity of SOCE. Interestingly, GRP78 knockdown only affected the inactivation phase while exerted no effect on the activation phase of SOCE. In addition, GRP78 knockdown markedly induced cell apoptosis and dramatically increased the ER Ca2+ concentration. Moreover, GRP78 was involved in the regulation of SOCE by the ER stress. These data indicate that GRP78 is an important regulator of SOCE to prevent Ca2+ overload in cells. HSP70, however, significantly reduced the activity of SOCE by inhibiting STIM1 translocation to ER-PM junctions. Future studies will explore the mechanism of GRP78 and HSP70 in regulating SOCE by confocal and TIRF imaging. Embryonic stem (ES) cells proliferate unlimitedly and can differentiate into all fetal and adult cell types. This property endows ES cells to be the promising candidates in the therapy of neurodegenerative diseases. Thus, it is important to identify novel signaling molecules or events that could play a role in the neural commitment of ES cells. Accumulated evidences have documented the role of STIM1 and Orai1 mediated SOCE in neuronal activities. Yet, the role of SOCE in early neural development remains to be determined. Here we examined the role of STIM1 and Orai1 during neural differentiation of mouse ES cells. Both of STIM1 and Orai1 were expressed and functionally active in ES cells, and expressions of STIM1 and Orai1 were dynamically regulated during neural differentiation of mouse ES cells. STIM1 knockdown inhibited the differentiation of mouse ES cells into neural progenitors, neurons, and astrocytes. In addition, STIM1 knockdown caused severe cell death and markedly suppressed the proliferation of neural progenitors. Surprisingly, Orai1 knockdown had little effect on neural differentiation of mouse ES cells, but the neurons derived from Orai1 knockdown ES cells, like those from STIM1 knockdown cells, had defective SOCE. Taken together, our data indicate that STIM1 is required for neural differentiation of mouse ES cells independent of Orai1-mediated SOCE.published_or_final_version
Physiology
Doctoral
Doctor of Philosophy
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Lur, Gyorgy. "STIM1, Orai1 and store operated calcium entry in pancreatic acinar cells". Thesis, University of Liverpool, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.539501.
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Harper, A. G. S. "The mechanisms underlying the activation of store-operated calcium entry in human platelets". Thesis, University of Cambridge, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.603727.
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This thesis examines the mechanisms underlying the activation of the store-operated calcium entry (SOCE) pathway in human platelets and how they relate to the Ca2+ signals elicited in platelets stimulated with physiological agonists and phorbol esters. A novel role for an increase in the activity of the cysteine protease calpain in the activation of SOCE is demonstrated. Data is also presented dissociating the role of the actin cytoskeleton in the store-operated Ca2+ entry from the store-operated Mn2+ and Na+ entries and demonstrate that the role of the cytoskeleton is not in the activation of the store-operated channel but in regulating Ca2+ entry via the Na+/Ca2+ exchanger (NCX). These data therefore provide strong evidence against the de novo conformational coupling hypothesis in human platelets. During further examination of the role of the NCX in SOCE in human platelets, it is demonstrated that SOCE elicits dense granule secretion in human platelets and inhibiting the platelets response to secreted autocrine messengers greatly reduces TG-evoked Ca2+ entry, suggesting an important role of autocrine modulation of SOCEI. These results therefore question the specificity of using thapsigargin as a specific activator of this Ca2+ entry pathway. The results also suggest the presence of two pharmacologically-distinct NCX subpopulations in human platelets. Results from experiments investigating the autocrine modulation of the Ca2+ responses of platelets stimulated with physiological agonists such as ADP and thrombin are also presented. These experiments lead to the suggestion that a rise in [Na2+]i may play an important role in potentiating the activation of human platelets. Lastly data is presented suggesting the presence of the TRPV1 ion channel in human platelets and provides preliminary evidence towards a role for this ion channel in modulating the secretion of serotonin from these cells.
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Whitworth, Claire Leanne. "Phenotype-specific store-operated calcium entry and the differentiation response in neuroblastoma cells". Thesis, University of Newcastle upon Tyne, 2016. http://hdl.handle.net/10443/3404.
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Understanding the fundamental molecular mechanisms that control the proliferationdifferentiation cellular switch and maintenance of the differentiated state is needed to fully harness the therapeutic potential for highly detrimental diseases such as cancer and neurodegenerative disorders. Intracellular free Ca2+ plays an essential role in the differentiation process and, more specifically, the ubiquitous Ca2+ signalling pathway; storeoperated Ca2+ entry (SOCE) is altered with differentiation. The SH-SY5Y neuroblastoma cancer cell line was utilised in this study to investigate the role of SOCE in phenotype-specific differentiation responses using morphological, biochemical and functional single cell, Ca2+ imaging techniques. Neuroblastoma is a paediatric malignancy of the sympathetic nervous system that is comprised of immature neural crest cells. Retinoic acid is used to treat neuroblastoma patients however many respond poorly, leading to aggressive disease progression. The SHSY5Y neuroblastoma-derived cell line consists of three morphologically distinct phenotypes; immature neuroblastic N-type cells, non-neuronal S-type cells and putative intermediate Itype cells, which exhibit variable tumourgenicity and can be induced to differentiate using 9- cis-retinoic acid (9cRA). The 9cRA-induced differentiation response of N-type and S-type populations involved morphological changes accompanied by an uncoupling of SOCE from Ca2+ store release that could be observed from the first day of 9cRA treatment. SOCE down-regulation was attributed to changes in expression and localisation of the CRAC channel protein Orai1 and the Ca2+ sensing protein STIM1. The extent of SOCE uncoupling was influenced in N-type and I-type cells but not S-type cells by the predominant background cell environment. Conditioned media from proliferating and differentiating N-type and S-type populations was also able to influence cell phenotype and the associated SOCE responses. This study describes how the 9cRA-induced differentiation response occurred in a multi-step manner in N-type populations and in gradual manner in S-type populations and raises the possibility that SOCE proteins could potentially be utilised as drug targets in neuroblastoma treatment or neurodegenerative disease therapy.
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McIvor, Emma. "Modelling store operated calcium entry : creating a three dimensional spatio-temporal model to predict local calcium signals". Thesis, University of Nottingham, 2018. http://eprints.nottingham.ac.uk/55110/.
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Calcium is a signalling messenger that is crucial to cellular function, controlling a diverse range of processes such as apoptosis, cell proliferation and muscle contraction. Store operated calcium entry (SOCE) is a specific pathway coupling depletion of the calcium stores within the endoplasmic reticulum (ER) to calcium influx through Orai channels on the plasma membrane. SOCE occurs in small sub-cellular regions called 'ER-PM junctions' which are typically less than $300$nm in diameter. The small size of these domains prevent direct measurement of the calcium signals as current calcium imaging techniques cannot resolve the local signals within ER-PM junctions. The calcium signals associated with SOCE control many downstream cellular processes, such as gene expression and immune responses. There is substantial evidence demonstrating that the placement of the calcium signalling machinery, including Orai channels and SERCA pumps, is vital to the generation of spatially distinct calcium signals which then enhance the selectivity of the calcium signal. However, experimental techniques cannot investigate the local calcium dynamics occurring on a spatial scale of micrometres so mathematical modelling techniques can be used to close this gap in understanding how the local calcium dynamics affect the experimentally observed global calcium dynamics. In this thesis, we construct a three dimensional spatio-temporal model of calcium dynamics and investigate the relationship between the placement of core components of the calcium signalling machinery, e.g. Orai channels and SERCA pumps, and the spatial calcium profiles generated as well as the rates of ER refilling observed. The model includes a spatially extended ER-PM junction to examine the spatial signature of the calcium profiles generated and a spatially extended sub-PM ER to examine the impact of Orai channel and SERCA pump placement on ER refilling dynamics. The model is the first to include spatially extended versions of both the ER-PM junction and sub-PM ER. In this thesis, we first focus on the construction of the spatio-temporal model and the solution techniques used to solve the model. We implement a semi-analytical solution using Green's functions to calculate the analytical solution of the spatial component of the diffusion equation and use numerical time stepping methods in MATLAB to evolve the spatial calcium profile over time. We compare the predictions of the model to expected biological outcomes and then use the model to investigate how the placement of Orai channels, and in particular how clustering of Orai channels, creates spatially distinct calcium profiles. We then examine whether the spatial calcium profile affects ER refilling and what factors control ER refilling. We find that Orai channel clustering creates spatially distinct calcium profiles within the ER-PM junction but does not enhance ER refilling. ER refilling is more strongly controlled by the proximity of SERCA pumps to Orai channels. In fact, the placement of SERCA2b pumps weakly affects ER refilling but the major regulator of ER refilling is the placement of SERCA2a pumps within the ER-PM junction. However, ER refilling continues, albeit at reduced rates, regardless of Orai channel and SERCA pump placement which suggests that other factors, such as the geometry of the ER-PM junction, could be important regulators of ER refilling. This work is relevant to experimental biologists and mathematicians within the calcium signalling community as the calcium signals generated within the ER-PM junction are crucial for advancing the understanding of how calcium signals regulate cellular function. The local calcium dynamics are important regulators of whole cell calcium dynamics and so mathematical methods allowing rigorous investigation of the mechanisms controlling local calcium signalling will be invaluable to furthering our understanding of how SOCE regulates cell function.
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Sharmeen, Cynthia. "Involvement of Beta-arrestin 1 and Beta-arrestin 2 in store operated calcium entry". Mémoire, Université de Sherbrooke, 2016. http://hdl.handle.net/11143/9499.
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Résumé : La variation de la [Ca2+] intracellulaire participe à nombreux de processus biologiques. Les cellules eucaryotes expriment à la membrane plasmique une variété de canaux par lesquelles le calcium peut entrer. Dans les cellules non excitables, deux mécanismes principaux permettent l'entrée calcique; l'entrée capacitative de Ca2+ via Orai1 (SOCE) et l'entrée calcique activé par un récepteur (ROCE). Plusieurs protéines clés sont impliquées dans la régulation de ces voies d'entrée calcique, ainsi que dans l'homéostasie calcique. TRPC6 est un canal calcique impliquée dans l'entrée calcique dans les cellules à la suite d’une stimulation d’un récepteur hormonal. TRPC6 transloque à la membrane cellulaire et il y demeure jusqu'à ce que le stimulus soit retiré. Les mécanismes qui régulent le trafic et l'activation de TRPC6 sont cependant encore peu connus. Des découvertes récentes ont démontré qu'il y a un rôle potentiel de Rho kinase dans l'activité de TRPC6. Rho kinase est activée par la petite protéine G RhoA qui peut être activée par les protéines G hétérotrimériques Gα12 et Gα13. En plus de Gα12 et Gα13, les protéines de désensibilisation des GPCR β -arrestin 1 et / ou β-arrestin 2 peuvent aussi activer RhoA. Le but de notre étude est d'examiner la participation des protéines Gα12/13 et β-arrestin 1/ β-arrestin 2 dans l'activation de TRPC6 et de la protéine Orai1. Nous avons utilisé des ARN interférant (siRNA) spécifiques pour induire une réduction de l'expression de Gα12/13 ou β-arrestin 1/β-arrestin 2. La conséquence sur l’entrée de Ca2+ dans les cellules a été ensuite déterminée par imagerie calcique en temps réel suite à une stimulation par la vasopressine (AVP), thapsigargin ou carbachol. Nous avons donc identifié que dans des cellules A7r5, une lignée cellulaire de musculaires lisses vasculaires où le canal TRPC6 exprimé de manière endogène, la diminution de l’expression des protéines Gα12 ou Gα13 ne semble pas modifier l’entrée Ca2+ induit par l’AVP par rapport aux cellules témoins. D'autre part, la diminution de l’expression β-arrestin 1 ou β-arrestin 2 dans des cellules HEK 293 ainsi que des cellules HEK 293 exprimant de façon stable TRPC6 (cellules T6.11) ont augmenté l’entrée de Ca2+ induite par thapsigargin, un activateur pharmacologique de SOCE. Des études de co-immunoprécipitation démontrent une interaction entre la β-arrestin 1 et STIM1, alors qu'aucune interaction n'a été observée entre les β-arrestin 1 et Orai1. Nous avons de plus montré à l'aide d'analyse en microscopie confocale que la diminution de l’expression β-arrestin 1 ou β-arrestin 2 n’influence pas la quantité d’Orai1 à la périphérie cellulaire. Cependant, des résultats préliminaires indiquent que la diminution de l’expression β-arrestin 1 ou β-arrestin 2 augmente la quantité de STIM1-YFP dans l'espace intracellulaire et diminue sa quantité à la périphérie cellulaire. En conclusion, nous avons montré que les β-arrestin 1 ou β-arrestin 2 sont impliquées dans l'entrée capacitative de Ca2+ (SOCE) et contrôlent la quantité de STIM1 dans le réticulum endoplasmique.Abstract : In an organism, intracellular [Ca2+] takes part in many biological processes. Eukaryotic cells express a variety of channels in the plasma membrane through which calcium can enter. In non-excitable cells, two main mechanisms allow calcium entry; the store-operated calcium entry via Orai1 (SOCE) and receptor-operated calcium entry (ROCE). Several key proteins are involved in the regulation of these calcium entry pathways as well as in calcium homeostasis. TRPC6 is a calcium channel implied in calcium entrance into the cells following hormonal stimulation and translocates to the plasma membrane. TRPC6 channel appear to the plasma membrane until the stimulus is present. Although, the mechanisms that regulate the trafficking and activation of TRPC6 are still little known. Recent findings have demonstrated that there is a potential role of Rho kinase in activity of TRPC6. Rho kinase is activated by the small G protein RhoA that itself can be activated by the heterotrimeric G proteins Gα12 and Gα13. In addition to Gα12 and Gα13 proteins, cytosolic GPCR desensitizing proteins β-arrestin 1 and/or β-arrestin 2 could also activate RhoA. The purpose of our study is to investigate the involvement of the proteins Gα12/13 and β-arrestin 1/β-arrestin 2 in the activation of TRPC6 and Orai1 protein. We used siRNA specific to Gα12/13 or β-arrestin 1/β-arrestin 2 to knockdown their endogenous expression. Then, calcium imaging in real time was performed in order to see the quantity of calcium entered into the cell following stimulation by vasopressin (AVP), thapsigargin, or carbachol. We hence identified that in A7r5 cell, vascular smooth muscle cell where TRPC6 channel expressed endogenously; reduced expression of Gα12 or Gα13 proteins does not seem to modify the AVP-induced Ca2+ entry compared to control cells. On the other hand, calcium imaging experiment in knocked down β-arrestin 1 or β-arrestin 2 in HEK 293 cells as well as HEK 293 cells stably transfected with TRPC6 (T6.11 cells) resulted in an increased thapsigargin-induced calcium entry. The co-immunoprecipitation studies demonstrate an interaction between β-arrestin 1 and STIM1, a calcium sensor in SOCE influx, while no interaction was observed between β-arrestin 1 and Orai1.We moreover showed by confocal microscopy that reduced expression of β-arrestin 1/ β-arrestin 2 does not influence the quantity of Orai1 at the cell periphery. Preliminary results showed that reduced expression of β-arrestin 1 or β-arrestin 2 increases the quantity of STIM1-YFP in the intracellular space and less it’s in peri-membrane space. In conclusion, we showed that β-arrestin 1 or β-arrestin 2 are involved in the store-operated calcium entry (SOCE) and control the quantity of STIM1 in the endoplasmic reticulum.
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Wallace, Patrick. "Excitation/contraction coupling in mouse anococcygeus smooth muscle : a role for store-operated calcium entry". Thesis, King's College London (University of London), 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.424303.
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Nassrallah, Wissam. "Store-Operated Response in CA1 Pyramidal Neurons Exhibits Features of Homeostatic Synaptic Plasticity". Thesis, Université d'Ottawa / University of Ottawa, 2015. http://hdl.handle.net/10393/33357.
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Homeostatic synaptic plasticity (HSP) regulates synaptic strength in response to changing neuronal firing patterns. This form of plasticity is defined by neurons’ ability to sense and over time integrate their level of firing activity, and to actively maintain it within a defined range. For instance, a compensatory increase in synaptic strength occurs when neuronal activity is chronically attenuated. However, the underpinning cellular mechanisms of this fundamental neural process remain poorly understood. We previously found that during activity deprivation, HSP leads to an increase in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) receptor function as well as a shift in subunit composition from Ca2+-impermeable GluA2-containing AMPA receptors to Ca2+-permeable GluA2-lacking AMPA receptors not only at synapses, but also at extrasynaptic sites. Neurons therefore appear to be actively enhancing Ca2+ entry, possibly as a compensatory mechanism in response to a prolonged Ca2+ deficit. To test whether the homeostatic response may, at least in part, be mediated by internal Ca2+ stores, we depleted endoplasmic reticulum (ER) Ca2+ stores by using the Sarco/endoplasmic reticulum Ca2+ ATPases (SERCA) pump blocker cyclopiazonic acid (CPA) for a prolonged period. Interestingly, we have found that prolonged Ca2+-store depletion leads not only to an increase in synaptic strength per se, but also a cell-wide increase in synaptic Ca2+-permeable GluA2-lacking AMPARs. This increase in Ca2+ influx following periods of inactivity is conceptually highly reminiscent of a store-operated response, whereby cells re-establish their calcium levels following Ca2+ store depletion using cell surface Ca2+ channels. Our results suggest that neurons use synaptic receptors as means to regulate store Ca2+ levels, thus significantly expanding our understanding of the repertoire used by neurons to modulate cellular excitability.
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Blackman, Sarah Kathryn. "Contribution of Calcium Entry through Non-Voltage Operated Calcium Channels to the Contractile Response of Vascular Smooth Muscle to Agonists". Thesis, King's College London (University of London), 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.487202.
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Depletion of Ca2+ stores in the sarcoplasmic reticulum results in the opening of store operated cation channels (SOCCs) on the plasma membrane, allowing Ca2 + influx into the cell and refilling of the stores. The work described in this thesis set out to determine the extent to which Ca2 + entry through the store operated pathway contributes to agonist-induced contraction. in aortic smooth mUSfle. Three major techniques were used to study agonist responses in aortae from C57BL6 mice and Wistar rats. Aortic rings were mounted in organ baths to study the pharmacology of contractile responses to noradrenaline (NA), phenylephrine (PE) thapsigargin (Tg), arg-vasopressin (AVP) and potassium chloride (KCI). These whole tissue experiments were supplemented with electrophysiological and Fura-2 microfluorimetry recordings of cells isolated from the whole tissue by enzymatic digestion. Mouse aortic ring experiments revealed that contraction in response to NA is dependent on verapamil-insensitive ion channels that can be distinguished from those activated by store depletion with Tg by their sensitivity to l-lOmM GdCh or LaCL3 • The ability of low concentrations of 2-APB to enhance contraction induced by NA suggests a role for STTh11 in mediating contraction to NA in the mouse aorta. Single cell experiments designed to further investigate SOCCs in the mouse aorta confirmed the· presence of a Ca2 +-activated chloride channel but did not confirm the presence of the SOCC reported by Trepakova et al (2001) . . Rat aortic ring experiments were carried out to investigate the role of nitric oxide (NO) in the reciprocal regulation model of receptor and store operated calcium entry. No evidence was obtained for the involvement of smooth muscle NO in modulating contractions induced by phenylephrine or AVP in whole tissues.
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Endo, Yukari. "Dominant mutations in ORAI1 cause tubular aggregate myopathy with hypocalcemia via constitutive activation of store-operated Ca2+ channels". Kyoto University, 2015. http://hdl.handle.net/2433/198937.
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Boutry, Maxime. "Dysfonctions des lysosomes et neurodégénérescence : l'exemple de la paraplégie spastique de type SPG11". Thesis, Paris 6, 2017. http://www.theses.fr/2017PA066295/document.
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Les lysosomes sont importants pour la survie et la fonction des cellules du système nerveux central et en particulier des neurones. Le mécanisme de la reformation des lysosomes est crucial pour maintenir une quantité adéquate de lysosomes fonctionnels dans les cellules. La spatacsine, qui joue un rôle dans le ce mécanisme est impliquée dans la paraplégie spastique de type SPG11 ; une maladie caractérisée par des troubles moteurs et cognitifs sévères. L’utilisation de modèles cellulaires de cette pathologie permet d’étudier les mécanismes physiopathologiques à l’origine d’altérations de la reformation des lysosomes. J’ai montré que la perte de fonction de la spatacsine est responsable de l’accumulation de lipides dans les lysosomes. Ces accumulations sont constituées de gangliosides et de cholestérol et sont présentes dans les autolysosomes perturbant leur recyclage en lysosomes, notamment en empêchant le recrutement de protéines impliquées dans le mécanisme. Les accumulations de gangliosides rendent les neurones à l’exposition au glutamate ce qui suggère que ces altérations pourraient avoir un rôle dans la neurodégénérescence. J’ai aussi montré que l’absence de spatacsine provoque une dérégulation de l’import de Ca2+ extracellulaire par le « store-operated calcium entry » ce qui conduit à altération de l’homéostasie calcique. L’inhibition de l’import de calcium par le SOCE permet de réduire les accumulations de lipides et de rétablir partiellement le recyclage des lysosomes. Ainsi, l’absence de spatacsine induit une altération de l’homéostasie calcique qui participe à l’accumulation de lipides dans le système lysosomal ce qui est délétère pour la survie des neuronesLysosomal dysfunctions are involved in a large number of neurodegenerative diseases highlightingthe crucial function of lysosomes in neuron survival and function. The mechanism of lysosomereformation from autolysosomes allow cells to maintain the ool of functional lysosomes.Disruptions of this rocess are involved in athologies affecting the central nervous system. Inparticular, spatacsin that lays a role in lysosome recycling is implicated in hereditary spasticparaplegia type SPG11, a severe disease characterized by motors and cognitive alterations. Thispathology is caused by loss of function mutations in SPG11, encoding spatacsin. The study ofSPG11 cellular models gives the opportunity to decipher the hysiopathological mechanismsunderlying lysosomal reformation disruptions.During my thesis, I showed that loss of spatacsin function induces lipid accumulation in lysosomesand articularly in autolysosomes both in fibroblasts and neurons from Spg11-/- mice. Gangliosidesand cholesterol are among lipids that accumulate in autolysosomes impairing lysosomal membranerecycling by disrupting the recruitment of keys roteins. Neurons with ganglioside accumulationsare more sensitive to glutamate induced neuronal death, suggesting that these accumulations areinvolved in neurodegeneration. These results could be of great importance since accumulations ofgangliosides in lysosomes arise in many diseases.I also showed that loss of spatacsin disrupts extracellular calcium import by the store-operatedcalcium entry (SOCE) leading to an increase in cytosolic calcium levels. Lysosomal calcium contentis also increased in Spg11-/- cells and calcium release from lysosome by TRPML1 is reduced.Inhibiting SOCE and stimulating lysosomal calcium release by TRPML1 reduced lipidsaccumulations in lysosomes and artially restored lysosome reformation.Our data suggest that absence of spatacsin is responsible for a disruption of calcium homeostasisthat contributes to lipid accumulation in autolysosomes, disturbing reformation of lysosomes fromautolysosomes. Inhibiting gangliosides synthesis could be used as a therapeutic strategy. However,understanding how loss of function of spatacsin alters these cellular athways will allow thedevelopment of targeted therapeutic approaches
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Nakano, Makoto. "Frequency-dependent requirement for calcium store-operated mechanisms in induction of homosynaptic long-term depression at hippocampus CA1 synapses". Kyoto University, 2004. http://hdl.handle.net/2433/145290.
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Sawamura, Seishirou. "Elucidation of signal regulation by interacting molecules and proteins of Ca2+ influx channels". 京都大学 (Kyoto University), 2016. http://hdl.handle.net/2433/215579.
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Deng, Xiaoxiang. "Dissecting the mechanism of STIM coupling to Orai". Diss., Temple University Libraries, 2011. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/213111.
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BiochemistryPh.D.
Store-operated Ca2+ entry (SOCE) triggered by the depletion of endoplasmic reticulum (ER) luminal Ca2+ stores is a major Ca2+ entry pathway in non-excitable cells and is essential in physiological Ca2+ signaling and homeostasis. STIM proteins are sensors of ER luminal Ca2+, which translocate to ER-plasma membrane (PM) junctional regions to activate the family of Orai channels mediating Ca2+ entry. This study is focused on dissecting the mechanism of STIM interacting with Orai. A powerful modifier of SOCE, 2-aminoethoxydiphenyl borate (2-APB) is utilized. First, the action of 2-APB on the mammalian Orai homologues are characterized using the DT40 STIM knockout cells. 50 ìM 2-APB directly activates Orai3 but not Orai1 or Orai2. Second, while it stimulates the STIM2-mediated constitutive Ca2+ entry through Orai, 2-APB also induces the cytosolic STIM C-terminus fragments to translocate to the PM and activate Orai1. These data reveal 50 ìM 2-APB enhances STIM-Orai coupling. Further, this enhanced binding of STIM and Orai leads to a conformational change within the STIM-Orai complex, which is possibly the underlying mechanism for the 50 ìM 2-APB inhibitory effect on SOCE. Finally, six residues (344-349) at the N-terminus of the STIM-Orai activation region (SOAR) prove to be critical for this inhibitory action. These same six amino acid region also constitutes an ancillary Orai binding site within SOAR, in addition to the main polybasic region. The deletion of this ancillary site abolishes the ability of SOAR to bind to and activate Orai1, but can be compensated for by the STIM-Orai binding enhancing effect of 50 ìM 2-APB. The majority of STIM1 is located on the ER membrane, while a small proportion of STIM1 is on the PM. Using an extracellularly applied STIM1 antibody, the PM STIM1 can be aggregated to exert an influence on the ER STIM1. Although the PM STIM1 is not obligatory for STIM1-mediated Orai activation, it nevertheless may have a functional presence in the PM. Lastly, a regulatory link between voltage-gated Ca2+ channels (Cav channels) and the STIM proteins is established. After activation by store depletion, STIM strongly suppresses the Cav1.2 channels. There is a biochemical interaction between STIM1 and the Cav1.2 pore subunit á1C. This inhibitory effect is independent of Orai1 activation. Hence, STIM1 interacts with and reciprocally controls two major Ca2+ channels.
Temple University--Theses
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Beesetty, Pavani. "Consequences of TRPM7 kinase inactivation in immune cells". Wright State University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=wright1526406780596661.
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Xu, Yanjun [Verfasser], Ronald P. [Akademischer Betreuer] [Gutachter] Kühnlein i Ahmed [Gutachter] Mansouri. "Regulation of Drosophila melanogaster body fat storage by store-operated calcium entry / Yanjun Xu ; Gutachter: Ronald P. Kühnlein, Ahmed Mansouri ; Betreuer: Ronald P. Kühnlein". Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2017. http://d-nb.info/1132813042/34.
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Tian, Geng. "On the Generation of cAMP Oscillations and Regulation of the Ca2+ Store-operated Pathway in Pancreatic Islet α- and β-cells". Doctoral thesis, Uppsala universitet, Institutionen för medicinsk cellbiologi, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-191852.
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Insulin and glucagon are released in pulses from pancreatic β- and α-cells, respectively. Both cell types are electrically excitable, and elevation of the cytoplasmic Ca2+ concentration ([Ca2+]i) due to depolarization with voltage-dependent entry of the cation is the main trigger of hormone secretion. Store-operated Ca2+ entry (SOCE) also contributes to the [Ca2+]i elevation and this process has been suggested to be particularly important for glucagon secretion. cAMP is another important messenger that amplifies Ca2+-triggered secretion of both hormones, but little is known about cAMP dynamics in islet cells. In type-2 diabetes, there is deteriorated β-cell function associated with elevated concentrations of fatty acids, but the underlying mechanisms are largely unknown. To clarify the processes that regulate insulin and glucagon secretion, cAMP signalling and the store-operated pathway were investigated in β- and α-cells, primarily within their natural environment in intact mouse and human islets of Langerhans. Fluorescent biosensors and total internal reflection microscopy were used to investigate signalling specifically at the plasma membrane (PM). Adrenaline increased and decreased the sub-PM cAMP concentration ([cAMP]pm) in immuno-identified α-cells and β-cells, respectively, which facilitated cell identification. Glucagon elicited [cAMP]pm oscillations in α- and β-cells, demonstrating both auto- and paracrine effects of the hormone. Whereas glucagon-like peptide 1 (GLP-1) consistently elevated [cAMP]pm in β-cells, only few α-cells responded, indicating that GLP-1 regulates glucagon secretion without changes of α-cell [cAMP]pm. Both α- and β-cells responded to glucose with pronounced oscillations of [cAMP]pm that were partially Ca2+-dependent and synchronized among islet β-cells. The glucose-induced cAMP formation was mediated by plasma membrane-bound adenylyl cyclases. Several phosphodiesterases (PDEs), including the PDE1, -3, -4, and -8 families, were required for shaping the [cAMP]pm signals and pulsatile insulin secretion. Prolonged exposure of islets to the fatty acid palmitate deteriorated glucose-stimulated insulin secretion with loss of pulsatility. This defect was associated with impaired cAMP generation, while [Ca2+]i signalling was essentially unaffected. Stromal interacting molecule 1 (STIM1) is critical for activation of SOCE by sensing the Ca2+ concentration in the endoplasmic reticulum (ER). ER Ca2+ depletion caused STIM1 aggregation, co-clustering with the PM Ca2+ channel protein Orai1 and SOCE activation. Glucose, which inhibits SOCE by filling the ER with Ca2+, reversed the PM association of STIM1. Consistent with a role of the store-operated pathway in glucagon secretion, this effect was maximal at the low glucose concentrations that inhibit glucagon release, whereas considerably higher concentrations were required in β-cells. Adrenaline induced STIM1 translocation to the PM in α-cells and the reverse process in β-cells, partially reflecting the opposite effects of adrenaline on cAMP in the two cell types. However, cAMP-induced STIM1 aggregates did not co-cluster with Orai1 or activate SOCE, indicating that STIM1 translocation can occur independently of Orai1 clustering and SOCE.
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Galitzine, Marie. "Redistribution des phospholipides membranaires : caractérisation des mécanismes déficients dans le syndrome hémoragique de Scott". Paris 7, 2005. http://www.theses.fr/2005PA077022.
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Henke, Nadine Verfasser], Axel [Akademischer Betreuer] Methner i Dieter [Akademischer Betreuer] [Willbold. "How Stromal Interaction Molecule 1 (STIM1) and Store Operated Calcium Entry (SOCE) affect mitochondrial energy metabolism and neuronal function / Nadine Henke. Gutachter: Axel Methner ; Dieter Willbold". Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2013. http://d-nb.info/1031074910/34.
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Henke, Nadine [Verfasser], Axel Akademischer Betreuer] Methner i Dieter [Akademischer Betreuer] [Willbold. "How Stromal Interaction Molecule 1 (STIM1) and Store Operated Calcium Entry (SOCE) affect mitochondrial energy metabolism and neuronal function / Nadine Henke. Gutachter: Axel Methner ; Dieter Willbold". Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2013. http://d-nb.info/1031074910/34.
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Goswamee, Priyodarshan. "The Role of Orai-Mediated Ca2+ Entry in Migration in a Gastroenteropancreatic Neuroendocrine Tumor Model". University of Toledo Health Science Campus / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=mco1438280470.
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Lorenzo, Moldero Ivan. "Localization and regulation of trpv4 channels in CILIATED epithelia". Doctoral thesis, Universitat Pompeu Fabra, 2008. http://hdl.handle.net/10803/7185.
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La neteja del moc i dels patògens dels pulmons, i el transport de gàmets i embrions en els òrgans reproductius de les femelles són funcions clau en els epitelis ciliats, tals com aquells que es troben presents en les vies respiratòries i l'oviducte. La taxa de transport mucociliar és funció de la freqüència de batut ciliar (CBF) i aquesta freqüència és augmentada per increments en la concentració de Ca2+ intracelul·lar. El canal catiònic "transient potential vanilloid 4" (TRPV4) intervé en l'entrada de Ca2+ en resposta a estímuls mecànics i osmòtics. L'expressió del TRPV4 en l'epiteli ciliat de les vies respiratòries i de l'oviducte és confirmada mitjançant la localització per immunofluorescència del canal iònic a la membrana apical de l'epiteli ciliat i polaritzat, allà on la senyalització de Ca2+ és requerida per la regulació de la CBF. Cèl·lules ciliades de la tràquea de ratolins TRPV4-/- no expressen el canal TRPV4, no responen a l'activador específic del TRPV4, el 4α-phorbol 12,13-didecanoate (4α-PDD) i presenten respostes de Ca2+ reduïdes a temperatures mitjanes (~25ºC- 8ºC), un altre estímul dels canals TRPV4. L'activació dels canals TRPV4 per solucions altament viscoses i per hypotonicitat depèn de l'activació de la via de la fosfolipasa A2(PLA2)i la subseqüent producció de àcid epoxieicosatrienoic (EET). En condicions de baixa activació de la PLA2, estímuls mecànics i hipotònics alliberen ATP per a l'activació de la via de la fosfolipasa C (PLC)-inositol trifosfat (IP3) per contribuir a l'activació dels canals TRPV4. Descrivim que el metabòlit IP3 sense ser un agonista per ell mateix, sensibilitza el TRPV4 per a l'activació de EET, essent aquest un mecanisme general. L'acoblament funcional entre els canals TRPV4 de la membrana plasmàtica i els receptors de IP3 (IP3R) és necessari tant per iniciar com mantenir la senyalització oscil·latòria del Ca2+ desencadenada per estímuls viscosos i hipotònics. Un dels principals activadors de la CBF, la adenosina-5'-trifosfat (ATP), desencadena una resposta cel·lular mediada per Ca2+ en la que es desencadena tant l'alliberament de Ca2+ des dels dipòsits intracel·lulars com l'entrada de Ca2+. És destacable la contribució de el TRPV4 en l'augment de la CBF mediada per ATP. És més, el nostre treball implica als canals TRPV4 exclusivament en l'entrada de Ca2+ activada per receptor (ROCE). Tot plegat, aquesta tesi doctoral mostra el paper dels canals TRPV4 en l'acoblament d'estímuls fisiològics tipus mecànic, osmòtic i químic a la regulació de la CBF en l'epiteli ciliat destinat al transport mucociliar.Clearance of mucus and pathogenic agents from lungs and the transport of gametes and embryos in the female reproductive organs are key functions of ciliated epithelia such as those present in the airways and the oviduct. The rate of mucociliary transport is a function of ciliary beat frequency (CBF) and this, in turn, is increased by increases in intracellular calcium. Transient potential vanilloid 4 (TRPV4)cation channel mediates Ca2+ influx in response to mechanical and osmotic stimuli. TRPV4 expression in ciliated epithelia from airways and oviduct is confirmed by immunofluorescence localization of the channel at the apical membrane of the polarized ciliated epithelia, where the Ca2+ signalling is required for CBF regulation. Ciliated tracheal cells from TRPV4-/-mice show no TRPV4 expression, neither increases in intracellular Ca2+ and CBF in response to the TRPV4-specific activator 4α- phorbol 12,13- idecanoate (4α-PDD), and reduced responses to mild temperatures (~25ºC - 38ºC), another TRPV4-activating stimulus. TRPV4 gating by high viscous loads and hypotonicity depends on phospholipase A2 (PLA2) pathway activation and subsequent production of epoxyeicosatrienoic acid (EET). Under conditions of low PLA2 activation, mechanical and hypotonic stimuli use extracellular ATP release-mediated activation of phospholipase C (PLC)-inositol triphosphate(IP3)signalling to support TRPV4 gating. We describe that IP3, without being an agonist itself, sensitizes TRPV4 to EET activation. Besides, the functional coupling between plasma membrane TRPV4 channels and IP3 receptors (IP3R) is required to initiate and maintain the cellular oscillatory Ca2+ signal triggered by high viscous loads and hypotonic stimuli. One of the main CBF activators, adenosine-5'-triphosphate (ATP), triggers both Ca2+ release from intracellular Ca2+ stores and Ca2+ entry. Interestingly, TRPV4 contributes to ATP-induced increase in CBF. Furthermore, our work implicates TRPV4 channel exclusively in receptor-operated Ca2+ entry. Collectively, this PhD thesis shows the role of TRPV4 channels coupling physiologically relevant mechanical, hypotonic and chemical stimuli to CBF regulation in motile ciliary epithelia.
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Saul, Stephanie [Verfasser], i Markus [Akademischer Betreuer] Hoth. "Ca2+ and reactive oxygen species as determinands of immune and skin cell function$dA central role for Orai- and STIM-mediated store-operated calcium entry / Stephanie Saul. Betreuer: Markus Hoth". Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2016. http://d-nb.info/1099952107/34.
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Bennett, Orville R. "Expressing human Orai3 in insect cells for pharmacological studies". Wright State University / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=wright1326846462.
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Carreras, Sureda Amado 1986. "Modulation of T lymphocite activation by ORMDL3". Doctoral thesis, Universitat Pompeu Fabra, 2014. http://hdl.handle.net/10803/319714.
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Genome wide association studies (GWAS) have pointed out ORMDL3 gene as a risk factor for several proinflammatory and autoimmune diseases. The protein encoded by this gene belongs to a family of transmembrane proteins of the endoplasmic reticulum involved in calcium homeostasis and cellular lipid metabolism. The driving force behind this work was the compelling idea of finding out a precise mechanism for the pathological association of this protein. This thesis explores the potential role of ORMDL3 in T lymphocytes focusing on the activation process. Thus, we have demonstrated that calcium signaling and activation of T cells are influenced by the expression levels of ORMDL3. Besides, we have shown that inherited components of our genome modify ORMDL3 expression levels and lymphocyte physiology. Finally, we have characterized the molecular complex formed by ORMDL proteins. Altogether, this work allows a better understanding of the pathophysiology associated to ORMDL3 and its linkage with the immune system.Estudis d’associació genètica ample han apuntat cap al gen ORMDL3 com a factor de risc per diverses malalties pro-inflamatòries i autoimmunes. La proteïna codificada per aquest gen pertany a una família de proteïnes transmembrana del reticle endoplàsmic involucrada en l’homeòstasi de calci i en el metabolisme lipidic cel•lular. El motiu que ens va impulsar a dur a terme aquest treball era la idea de trobar el mecanisme darrere les associacions a patologia per aquesta proteïna. Aquesta tesis explora el rol potencial d’ORMDL3 en limfòcits T, amb èmfasi al procés d’activació. Així doncs hem demostrat que la senyalització de calci i la activació de cèl•lules T es veu influenciada pels nivells d’expressió d’ORMDL3. A més hem demostrat que components del nostre genoma modifiquen els nivells d’expressió d’ORMDL3 i la fisiologia limfocitària. Per acabar hem caracteritzat el complex molecular de les proteïnes ORMDL. En conjunt, aquest treball permet una millor comprensió de la fisiopatologia associada a ORMDL3 i la seva relació amb el sistema immune
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Gao, Yadong [Verfasser]. "The role of calcium-activated potassium channels and store-operated calcium channels in human macrophages / vorgelegt von Yadong Gao". 2007. http://d-nb.info/986359440/34.
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Tsai, Yu-Chieh, i 蔡雨潔. "The role of store-operated calcium channels in differentiation of rat mesenchymal stem cells". Thesis, 2012. http://ndltd.ncl.edu.tw/handle/20221505338518925501.
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碩士慈濟大學
生理暨解剖醫學碩士班
100
Stem cells have certain features such as self-renewal and differentiation potential. Adult stem cells are minor populations found in adult organs, they cannot give rise to an organism and only differentiate to specific cell lineages, mesenchymal stem cells (MSC) belong to this group. MSC can differentiate into several lineages, including osteocytes, chondrocytes and adipocytes. Recently, unexpected plasticity has been attributed to mesenchymal stem cells, as they have been demonstrated to differentiate into a number of non-mesoderm-type cells such as cardiomyocytes and neuron cells. In this study, we induce rat MSC (rMSC) to differentiate into neuron-like cells and adipocytes to investigate the correlation between calcium and cell differentiation. Calcium, a ubiquitous second messenger, regulates many cellular functions, including cell growth, differentiation, and apoptosis. Intracellular calcium homeostasis is maintained via calcium channels, calcium pumps and calcium exchangers. Store-operated calcium entry (SOCE) are the major source of intracellar calcium in non-excitable cells. Therefore, the aim of this study is to investigate the role of SOC channels in rMSC differentiation. rMSC is induced to differentiate into adipocytes and neuron-like cells. We measure the difference of concentration between Control and differentiation groups using microspectrofluorometry. We find that calcium influx through SOC channels and calcium depletion is significantly increase in differentiated rMSC. Therefore, we use Western blot and Q-PCR to determene that whether the expressions of STIM-1, Orai-1 and TRPC1 is differences between Control and differentiation groups. Our finding show that the levels of STIM-1 expression is significantly increase, but the levels of Orai-1 and TRPC1 expression have no significant difference between Control and differentiation groups. Inhibition of SOCE by a treatment with 2APB can blocked rMSC differentiation. Next, we measured the expression of IP3R1, IP3R2 and IP3R3 and find a significantly increase in IP3R2 after differentiation. Our results suggest that STIM-1 may play a critical role in the differentiation process.