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Tubular aggregate myopathy (TAM) is a rare myopathy characterized by muscle weakness and myalgia. Muscle fibers from TAM patients show characteristic accumulation of membrane tubules that contain proteins from the sarcoplasmic reticulum (SR). Gain-of-function mutations in STIM1 and ORAI1, the key proteins participating in the Store-Operated Ca2+ Entry (SOCE) mechanism, were identified in patients with TAM. Recently, the CASQ1 gene was also found to be mutated in patients with TAM. CASQ1 is the main Ca2+ buffer of the SR and a negative regulator of SOCE. Previous characterization of CASQ1 mutants in non-muscle cells revealed that they display altered Ca2+dependent polymerization, reduced Ca2+storage capacity and alteration in SOCE inhibition. We thus aimed to assess how mutations in CASQ1 affect calcium regulation in skeletal muscles, where CASQ1 is naturally expressed. We thus expressed CASQ1 mutants in muscle fibers from Casq1 knockout mice, which provide a valuable model for studying the Ca2+ storage capacity of TAM-associated mutants. Moreover, since Casq1 knockout mice display a constitutively active SOCE, the effect of CASQ1 mutants on SOCE inhibition can be also properly examined in fibers from these mice. Analysis of intracellular Ca2+ confirmed that CASQ1 mutants have impaired ability to store Ca2+and lose their ability to inhibit skeletal muscle SOCE; this is in agreement with the evidence that alterations in Ca2+entry due to mutations in either STIM1, ORAI1 or CASQ1 represents a hallmark of TAM. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum (ER) membrane contact sites (MCSs) are crucial regulatory hubs in cells, playing roles in signaling, organelle dynamics, and ion and lipid homeostasis. Previous work demonstrated that the highly conserved yeast Ltc/Lam sterol transporters localize and function at ER MCSs. Our analysis of the human family members, GRAMD1a and GRAMD2a, demonstrates that they are ER-PM MCS proteins, which mark separate regions of the plasma membrane (PM) and perform distinct functions in vivo. GRAMD2a, but not GRAMD1a, co-localizes with the E-Syt2/3 tethers at ER-PM contacts in a PIP lipid-dependent manner and pre-marks the subset of PI(4,5)P2-enriched ER-PM MCSs utilized for STIM1 recruitment. Data from an analysis of cells lacking GRAMD2a suggest that it is an organizer of ER-PM MCSs with pleiotropic functions including calcium homeostasis. Thus, our data demonstrate the existence of multiple ER-PM domains in human cells that are functionally specialized by GRAM-domain containing proteins.

The data generated here in relates to the research article “CaV1.3 enhanced store operated calcium promotes resistance to androgen deprivation in prostate cancer”. A model of prostate cancer (PCa) progression to castration resistance was employed, with untreated androgen sensitive LNCaP cell line alongside two androgen deprived (bicalutamide) sublines, either 10 days (LNCaP-ADT) or 2 years (LNCaP-ABL) treatment, in addition to androgen insensitive PC3. With this PCa model, qPCR was used to examined fold change in markers linked to androgen resistance, androgen receptor (AR) and neuron specific enolase (NSE), observing an increase under androgen deprivation. In addition, the gene expression of a range of calcium channels was measured, with only the L-type Voltage gated calcium channel, CACNA1D, demonstrating an increase during androgen deprivation. With CACNA1D knockdown the channel was found not to influence the gene expression of calcium channels, ORAI1 and STIM1. The calcium channel blocker (CCB), nifedipine, was employed to determine the impact of CaV1.3 on the observed store release and calcium entry measured via Fura-2AM ratiometric dye in our outlined PCa model. In both the presence and absence of androgen deprivation, nifedipine was found to have no impact on store release induced by thapsigargin (Tg) in 0mM Ca2+ nor store operated calcium entry (SOCE) following the addition of 2mM Ca2+. However, CACNA1D siRNA knockdown was able to reduce SOCE in PC3 cells. The effect of nifedipine on CaV1.3 in PCa biology was measured through cell proliferation assay, with no observed change in the presence of CCB. While siCACNA1D reduced PC3 cell proliferation. This data can be reused to inform new studies investigating altered calcium handling in androgen resistant prostate cancer. It provides insight into the mechanism of CaV1.3 and its functional properties in altered calcium in cancer, which can be of use to researchers investigating this channel in disease. Furthermore, it could be helpful in interpreting studies investigating CCB's as a therapeutic and in the development of future drugs targeting CaV1.3.

Calsequestrin (CASQ) is the most abundant Ca2+ binding protein localized in the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle. The genome of vertebrates contains two genes, CASQ1 and CASQ2. CASQ1 and CASQ2 have a high level of homology, but show specific patterns of expression. Fast-twitch skeletal muscle fibers express only CASQ1, both CASQ1 and CASQ2 are present in slow-twitch skeletal muscle fibers, while CASQ2 is the only protein present in cardiomyocytes. Depending on the intraluminal SR Ca2+ levels, CASQ monomers assemble to form large polymers, which increase their Ca2+ binding ability. CASQ interacts with triadin and junctin, two additional SR proteins which contribute to localize CASQ to the junctional region of the SR (j-SR) and also modulate CASQ ability to polymerize into large macromolecular complexes. In addition to its ability to bind Ca2+ in the SR, CASQ appears also to be able to contribute to regulation of Ca2+ homeostasis in muscle cells. Both CASQ1 and CASQ2 are able to either activate and inhibit the ryanodine receptors (RyRs) calcium release channels, likely through their interactions with junctin and triadin. Additional evidence indicates that CASQ1 contributes to regulate the mechanism of store operated calcium entry in skeletal muscle via a direct interaction with the Stromal Interaction Molecule 1 (STIM1). Mutations in CASQ2 and CASQ1 have been identified, respectively, in patients with catecholamine-induced polymorphic ventricular tachycardia and in patients with some forms of myopathy. This review will highlight recent developments in understanding CASQ1 and CASQ2 in health and diseases. [ABSTRACT FROM AUTHOR]

In the adult, vascular smooth muscle cells (VSMC) are normally physiologically quiescent, arranged circumferentially in one or more layers within blood vessel walls. Remodelling of native VSMC to a proliferative state for vascular development, adaptation or repair is driven by platelet-derived growth factor (PDGF). A key effector downstream of PDGF receptors is store-operated calcium entry (SOCE) mediated through the plasma membrane calcium ion channel, ORAI1, which is activated by the endoplasmic reticulum (ER) calcium store sensor, stromal interaction molecule-1 (STIM1). This SOCE was shown to play fundamental roles in the pathological remodelling of VSMC. Exciting transgenic lineage-tracing studies have revealed that the contribution of the phenotypically-modulated VSMC in atherosclerotic plaque formation is more significant than previously appreciated, and growing evidence supports the relevance of ORAI1 signalling in this pathologic remodelling. ORAI1 has also emerged as an attractive potential therapeutic target as it is accessible to extracellular compound inhibition. This is further supported by the progression of several ORAI1 inhibitors into clinical trials. Here we discuss the current knowledge of ORAI1-mediated signalling in pathologic vascular remodelling, particularly in the settings of atherosclerotic cardiovascular diseases (CVDs) and neointimal hyperplasia, and the recent developments in our understanding of the mechanisms by which ORAI1 coordinates VSMC phenotypic remodelling, through the activation of key transcription factor, nuclear factor of activated T-cell (NFAT). In addition, we discuss advances in therapeutic strategies aimed at the ORAI1 target.

Calcium is a critical secondary messenger in microglia. In response to inflammation, microglia mobilize intracellular calcium and increase the expression of inducible nitric oxide synthase (iNOS), which produces nitric oxide (NO). This study set to explore whether NO regulates intracellular calcium dynamics through transient receptor potential (TRP) channels in primary wildtype (WT) and iNOS knockout (iNOS−/−) microglia, and the BV2 microglial cell line using calcium imaging and voltage-clamp recordings. Our results demonstrated that application of the NO-donor SNAP induced a biphasic calcium response in naïve murine microglia. Specifically, phase I was characterized by a rapid decline in calcium influx that was attenuated by pretreatment of the store operated calcium channel (SOCC) inhibitor 2APB, while phase II presented as a slow calcium influx that was abolished by pretreatment with the TRP vanilloid type 2 (TRPV2) channel inhibitor tranilast. Importantly, in the presence of a protein kinase G (PKG) inhibitor, the SNAP-mediated calcium decline in phase I persisted while the calcium influx in phase II was abolished. Application of thapsigargin to activate SOCCs caused a calcium influx through a nonselective cation conductance in BV2 microglia, which was abruptly attenuated by SNAP. Importantly, iNOS−/− microglia displayed a significantly larger calcium influx though SOCCs while expressing less stromal interaction molecule 1, Orai1, and TRP canonical type 1 and 3 mRNA, when compared to WT microglia. Together, these results demonstrate that NO signaling restricts calcium influx through SOCCs independent of PKG signaling and increases calcium influx through TRPV2 channels in a PKG-dependent mechanism in microglia. • Microglia mobilize intracellular calcium and produce nitric oxide. • Lack of inducible nitric oxide synthase enhanced store operated calcium channels. • Nitric oxide rapidly inhibited store operated calcium channels independent of PKG. • Nitric oxide induced a slow-onset calcium influx through TRPV2 channels. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are small membrane vesicles that serve as important intercellular signalling intermediaries in both malignant and non-malignant cells. For EVs formed by the plasma membrane, their biogenesis is characterized by an increase in intracellular calcium followed by successive membrane and cytoskeletal changes. EV-production is significantly higher in malignant cells relative to non-malignant cells and previous work suggests this is dependent on increased calcium mobilization and activity of calpain. However, calcium-signalling pathways involved in malignant and non-malignant EV biogenesis remain unexplored. Here we demonstrate; malignant cells have high basal production of plasma membrane EVs compared to non-malignant cells and this is driven by a calcium–calpain dependent pathway. Resting vesiculation in malignant cells occurs via mobilization of calcium from endoplasmic reticulum (ER) stores rather than from the activity of plasma membrane calcium channels. In the event of ER store depletion however, the store-operated calcium entry (SOCE) pathway is activated to restore ER calcium stores. Depleting both ER calcium stores and blocking SOCE, inhibits EV biogenesis. In contrast, calcium signalling pathways are not activated in resting non-malignant cells. Consequently, these cells are relatively low vesiculators in the resting state. Following cellular activation however, an increase in cytosolic calcium and activation of calpain increase in EV biogenesis. These findings contribute to furthering our understanding of extracellular vesicle biogenesis. As EVs are key mediators in the intercellular transfer of deleterious cancer traits such as cancer multidrug resistance (MDR), understanding the molecular mechanisms governing their biogenesis in cancer is the crucial first step in finding novel therapeutic targets that circumvent EV-mediated MDR. [ABSTRACT FROM AUTHOR]

Exercise promotes the formation of intracellular junctions in skeletal muscle between stacks of sarcoplasmic reticulum (SR) cisternae and extensions of transverse-tubules (TT) that increase co-localization of proteins required for store-operated Ca2+ entry (SOCE). Here, we report that SOCE, peak Ca2+ transient amplitude and muscle force production during repetitive stimulation are increased after exercise in parallel with the time course of TT association with SR-stacks. Unexpectedly, exercise also activated constitutive Ca2+ entry coincident with a modest decrease in total releasable Ca2+ store content. Importantly, this decrease in releasable Ca2+ store content observed after exercise was reversed by repetitive high-frequency stimulation, consistent with enhanced SOCE. The functional benefits of exercise on SOCE, constitutive Ca2+ entry and muscle force production were lost in mice with muscle-specific loss of Orai1 function. These results indicate that TT association with SR-stacks enhances Orai1-dependent SOCE to optimize Ca2+ dynamics and muscle contractile function during acute exercise.

Neointimal hyperplasia could be one of the most important complications after balloon angioplasty. Since calcium signaling has several physiologic effects on the regulation of the proliferation and migration of vascular smooth muscle cells (VSMCs), it was hypothesized that transmembrane protein 66 (TMEM66), a store operated calcium entry (SOCE)-associated regulatory factor, possesses vascular protection against balloon injury. The rat balloon-induced carotid artery injury model was performed. Histological analysis was used to check neointimal hyperplasia. TMEM66 expression was measured by PCR and immunoblotting. The results revealed that TMEM66 was expressed in the medial and neointimal layers of the injured artery, and the expression of TMEM66 was markedly decreased. TMEM66 overexpression attenuated neointimal hyperplasia via VSMC proliferation/migration inhibition, and restored expression of VSMC phenotypic markers. Moreover, TMEM66 overexpression reduced the increased expression of Stim1 and Orai1 and PDGF-BB treatment-enhanced [Ca2+]i. In conclusion, TMEM66 protects against balloon injury-induced neointimal hyperplasia, and may be a pharmacological target for the treatment of restenosis. [ABSTRACT FROM AUTHOR]

[Display omitted] [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum (ER) membrane contact sites (MCSs) are crucial regulatory hubs in cells, playing roles in signaling, organelle dynamics, and ion and lipid homeostasis. Previous work demonstrated that the highly conserved yeast Ltc/Lam sterol transporters localize and function at ER MCSs. Our analysis of the human family members, GRAMD1a and GRAMD2a, demonstrates that they are ER-PM MCS proteins, which mark separate regions of the plasma membrane (PM) and perform distinct functions in vivo. GRAMD2a, but not GRAMD1a, co-localizes with the E-Syt2/3 tethers at ER-PM contacts in a PIP lipid-dependent manner and pre-marks the subset of PI(4,5)P2-enriched ER-PM MCSs utilized for STIM1 recruitment. Data from an analysis of cells lacking GRAMD2a suggest that it is an organizer of ER-PM MCSs with pleiotropic functions including calcium homeostasis. Thus, our data demonstrate the existence of multiple ER-PM domains in human cells that are functionally specialized by GRAM-domain containing proteins.

Proper skeletal muscle function is controlled by intracellular Ca2+ levels and by efficient production of energy (ATP). In the last 15 years our laboratory collected the following results: a. Short-term (7-15 days) denervation of muscle fibers causes misplacement and damage of membrane structures involved in EC coupling (calcium release units, CRUs) and of the mitochondrial network;1 b. Sedentary ageing causes partial disarray/damage of CRUs and of calcium entry units (CEUs, structures involved in SOCE) and loss/misplacement of mitochondria; c. Re-innervation and regular exercise promote rescue/maintenance of the proper architecture of CRUs, CEUs, and mitochondria in both denervation and ageing. All these structural changes were accompanied by related functional changes, i.e. loss/decay in function caused by denervation and ageing, and improved function following re-innervation and exercise. These data suggested that integrity and proper disposition of intracellular organelles deputed to Ca2+ handling and aerobic generation of ATP is challenged by inactivity (or reduced activity) and improved following recover of movements. To definitely test this hypothesis, we generated a model of short-term inactivity, and randomly assigned mice to two experimental groups: i) immobilized adult mice, in which inactivity is obtained for 6 days by unilateral immobilization of a hind limb; ii) rehabilitation group, in which mice underwent 15 days of treadmill running after the immobilization procedure. The results collected in this study indicate that: i) even short-term inactivity caused significant alterations to the membrane system and organelles previously discussed; and ii) 2 weeks rehabilitation rescued proper intracellular organization of CRUs, CEUs, and mitochondria. [ABSTRACT FROM AUTHOR]

Aims: Cigarette smoke (CS) in active smokers and second-hand smoke exposure exacerbate respiratory disorders such as asthma and chronic bronchitis. While women are known to experience a more asthmatic response to CS than emphysema in men, there is limited information on the mechanisms of CS-induced airway dysfunction. We hypothesize that CS interferes with a normal (protective) bronchodilatory role of estrogens, thus worsening airway contractility. Methods: We tested effects of cigarette smoke extract (CSE) on 17β-estradiol (E2) signaling in enzymatically-dissociated bronchial airway smooth muscle (ASM) obtained from lung samples of non-smoking female patients undergoing thoracic surgery. Results: In fura-2 loaded ASM cells, CSE increased intracellular calcium ([Ca2+]i) responses to 10µM histamine. Acute exposure to physiological concentrations of E2 decreased [Ca2+]i responses. However, in 24h exposed CSE cells, although expression of estrogen receptors was increased, the effect of E2 on [Ca2+]i was blunted. Acute E2 exposure also decreased store-operated Ca2+ entry and inhibited stromal interaction molecule 1 (STIM1) phosphorylation: effects blunted by CSE. Acute exposure to E2 increased cAMP, but less so in 24h CSE-exposed cells. 24h CSE exposure increased S-nitrosylation of ERα. Furthermore, 24h CSE-exposed bronchial rings showed increased bronchoconstrictor agonist responses that were not reduced as effectively by E2 compared to non-CSE controls. Conclusion: These data suggest that CS induces dysregulation of estrogen signaling in ASM, which could contribute to increased airway contractility in women exposed to CS.

Cameleons are sophisticated genetically encoded fluorescent probes that allow quantifying cellular Ca2+ signals. The probes are based on Förster resonance energy transfer (FRET) between terminally located fluorescent proteins (FPs), which move together upon binding of Ca2+ to the central calmodulin myosin light chain kinase M13 domain. Most of the available cameleons consist of cyan and yellow FPs (CFP and YFP) as the FRET pair. However, red-shifted versions with green and orange or red FPs (GFP, OFP, RFP) have some advantages such as less phototoxicity and minimal spectral overlay with autofluorescence of cells and fura-2, a prominent chemical Ca2+ indicator. While GFP/OFP- or GFP/RFP-based cameleons have been successfully used to study cytosolic and mitochondrial Ca2+ signals, red-shifted cameleons to visualize Ca2+ dynamics of the endoplasmic reticulum (ER) have not been developed so far. In this study, we generated and tested several ER targeted red-shifted cameleons. Our results show that GFP/OFP-based cameleons due to miss-targeting and their high Ca2+ binding affinity are inappropriate to record ER Ca2+ signals. However, ER targeted GFP/RFP-based probes were suitable to sense ER Ca2+ in a reliable manner. With this study we increased the palette of cameleons for visualizing Ca2+ dynamics within the main intracellular Ca2+ store.

Live yeast cell derivative (LYCD) is a medicinal yeast extract that has been used in the treatment of burns, wounds and hemorrhoids for over 70 years. It has been shown to enhance the closure of skin wounds in diabetic mice by increasing inflammation, angiogenesis, formation of granulation tissue and epithelial migration. An active fraction of LYCD has been identified as a mixture of peptides ranging in size from 5 kDA to 17 kDA. Despite its widespread use over many years, understanding of the mechanism by which LYCD acts to effect tissue repair responses is very limited. In this study, we have used a human monocyte-derived cell line, THP-1, as a representative of the inflammatory component of the wound healing process. We have identified two of the earliest responses to LYCD as an increase in cytoplasmic free calcium ([Ca2+]i) and the transcripts for c-fos. We have found that the increase in [Ca2+]i is both necessary and sufficient to account for the LYCD-induced elevation of c-fos. Furthermore, we have shown that the signaling pathway by which LYCD elevates [Ca2+]i involves both mobilization of Ca2+ from intracellular stores and influx of Ca2+ from the extracellular medium. Mobilization of store Ca2+ occurs first via activation of phospholipase C and this is followed by influx through activation of store operated calcium channels. These results constitute the first delineation of the early steps of the LYCD signaling pathway. [ABSTRACT FROM AUTHOR]

The endoplasmic reticulum (ER) stress sensor protein kinase RNA-like endoplasmic reticulum kinase (PERK) plays a major role during the unfolded protein response (UPR), mainly through eIF2α phosphorylation. We uncovered that PERK, by interacting with Filamin A, elicits F-actin remodeling required for ER-plasma membrane contact site formation after ER-Ca2+ depletion, through a UPR-independent mechanism.

In 1986, J.W. Putney presented a model for capacitative calcium (Ca 2+ ) entry conveying that depletion of endoplasmic reticulum stored Ca 2+ levels leads to activation of plasma membrane Ca 2+ channels which mediate influx of Ca 2+ from the extracellular space into cells. Presently, the biomolecules regulating this process, more widely known as store operated Ca 2+ entry (SOCE) which is vital to myriad signaling pathways in health and disease, are known and the focus of intense structural biology research aiming to illuminate the atomic mechanisms of function. This brief review highlights the known structures with respect to the mechanisms of SOCE regulation and possible future directions in this field developed out of Putney’s conceptualization of this process. [ABSTRACT FROM AUTHOR]

The use of usnic acid as a weight loss agent is a safety concern due to reports of acute liver failure in humans. Previously we demonstrated that usnic acid induces apoptosis and cytotoxicity in hepatic HepG2 cells. We also demonstrated that usnic acid induces autophagy as a survival mechanism against its cytotoxicity. In this study, we investigated and characterized further molecular mechanisms underlying the toxicity of usnic acid in HepG2 cells. We found that usnic acid causes endoplasmic reticulum (ER) stress demonstrated by the increased expression of typical ER stress markers, including CHOP, ATF-4, p-eIF2a, and spliced XBP1. Usnic acid inhibited the secretion of Gaussia luciferase measured by an ER stress reporter assay. An ER stress inhibitor 4-phenylbutyrate attenuated usnic acid-induced apoptosis. Moreover, usnic acid significantly increased the cytosolic free Ca2+ concentration. Usnic acid increased the expression of calcium release-activated calcium channel protein 1 (CRAM1 or ORAI1) and stromal interaction molecule 1, two key components of store-operated calcium entry (SOCE), which is the major Ca2+ influx pathway in non-excitable cells, this finding was also confirmed in primary rat hepatocytes. Furthermore, knockdown of ORAI1 prevented ER stress and ATP depletion in response to usnic acid. In contrast, overexpression of ORAI1 increased ER stress and ATP depletion caused by usnic acid. Taken together, our results suggest that usnic acid disturbs calcium homeostasis, induces ER stress, and that usnic acid-induced cellular damage occurs at least partially via activation of the Ca2þ channel of SOCE. [ABSTRACT FROM AUTHOR]

Cameleons are sophisticated genetically encoded fluorescent probes that allow quantifying cellular Ca2+ signals. The probes are based on Förster resonance energy transfer (FRET) between terminally located fluorescent proteins (FPs), which move together upon binding of Ca2+ to the central calmodulin myosin light chain kinase M13 domain. Most of the available cameleons consist of cyan and yellow FPs (CFP and YFP) as the FRET pair. However, red-shifted versions with green and orange or red FPs (GFP, OFP, RFP) have some advantages such as less phototoxicity and minimal spectral overlay with autofluorescence of cells and fura-2, a prominent chemical Ca2+ indicator. While GFP/OFP- or GFP/RFP-based cameleons have been successfully used to study cytosolic and mitochondrial Ca2+ signals, red-shifted cameleons to visualize Ca2+ dynamics of the endoplasmic reticulum (ER) have not been developed so far. In this study, we generated and tested several ER targeted red-shifted cameleons. Our results show that GFP/OFP-based cameleons due to miss-targeting and their high Ca2+ binding affinity are inappropriate to record ER Ca2+ signals. However, ER targeted GFP/RFP-based probes were suitable to sense ER Ca2+ in a reliable manner. With this study we increased the palette of cameleons for visualizing Ca2+ dynamics within the main intracellular Ca2+ store. [ABSTRACT FROM AUTHOR]

Aims: Cigarette smoke (CS) in active smokers and second-hand smoke exposure exacerbate respiratory disorders such as asthma and chronic bronchitis. While women are known to experience a more asthmatic response to CS than emphysema in men, there is limited information on the mechanisms of CS-induced airway dysfunction. We hypothesize that CS interferes with a normal (protective) bronchodilatory role of estrogens, thus worsening airway contractility. Methods: We tested effects of cigarette smoke extract (CSE) on 17β-estradiol (E2) signaling in enzymatically-dissociated bronchial airway smooth muscle (ASM) obtained from lung samples of non-smoking female patients undergoing thoracic surgery. Results: In fura-2 loaded ASM cells, CSE increased intracellular calcium ([Ca2+].) responses to 10|JM histamine. Acute exposure to physiological concentrations of E2 decreased [Ca2+] responses. However, in 24h exposed CSE cells, although expression of estrogen receptors was increased, the effect of E2 on [Ca2+]i was blunted. Acute E2 exposure also decreased store-operated Ca2+ entry and inhibited stromal interaction molecule 1 (STIM1) phosphorylation: effects blunted by CSE. Acute exposure to E2 increased cAMP, but less so in 24h CSE-exposed cells. 24h CSE exposure increased S-nitrosylation of ERa. Furthermore, 24h CSE-exposed bronchial rings showed increased bronchoconstrictor agonist responses that were not reduced as effectively by E2 compared to non-CSE controls. Conclusion: These data suggest that CS induces dysregulation of estrogen signaling in ASM, which could contribute to increased airway contractility in women exposed to CS. [ABSTRACT FROM AUTHOR]

Stromal Interaction Molecule1 (STIM1) is an endoplasmic reticulum membrane-localized calcium (Ca 2+ ) sensor that plays a critical role in the store-operated Ca 2+ entry (SOCE) pathway. STIM1 regulates a variety of physiological processes and contributes to a plethora of pathophysiological conditions. Several disease states and enhanced biological phenomena are associated with increased STIM1 levels and activity. However, molecular mechanisms driving STIM1 expression remain largely unappreciated. We recently reported that STIM1 expression augments during pigmentation. Nonetheless, the molecular choreography regulating STIM1 expression in melanocytes is completely unexplored. Here, we characterized the molecular events that regulate STIM1 expression during pigmentation. We demonstrate that physiological melanogenic stimuli α-melanocyte stimulating hormone (αMSH) increases STIM1 mRNA and protein levels. Further, αMSH stimulates STIM1 promoter-driven luciferase activity, thereby suggesting transcriptional upregulation of STIM1. We show that downstream of αMSH, microphthalmia-associated transcription factor (MITF) drives STIM1 expression. By performing knockdown and overexpression studies, we corroborated that MITF regulates STIM1 expression and SOCE. Next, we conducted extensive bioinformatics analysis and identified MITF-binding sites on the STIM1 promoter. We validated significance of the MITF-binding sites in controlling STIM1 expression by performing ChIP and luciferase assays with truncated STIM1 promoters. Moreover, we confirmed MITF's role in regulating STIM1 expression and SOCE in primary human melanocytes. Importantly, analysis of publicly available datasets substantiates a positive correlation between STIM1 and MITF expression in sun-exposed tanned human skin, thereby highlighting physiological relevance of this regulation. Taken together, we have identified a novel physiologically relevant molecular pathway that transcriptionally enhances STIM1 expression., Competing Interests: Conflict of interest The authors declare that they do not have any conflict of interest with the content of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

• CaV1.3 is upregulated in prostate cancer during androgen deprivation therapy • Expression of short 170kDa isoform determines CaV1.3 function in PCa • Store operated calcium entry is enhanced through CaV1.3 under androgen deprivation • Androgen independent proliferation and survival is linked to CaV1.3 calcium entry • CaV1.3 represents a novel oncochannel linked to castrate resistant prostate cancer Androgen deprivation therapy (ADT) is the main treatment for advanced prostate cancer (PCa) but resistance results in progression to terminal castrate resistant PCa (CRPC), where there is an unmet therapeutic need. Aberrant intracellular calcium (Ca i 2+) is known to promote neoplastic transformation and treatment resistance. There is growing evidence that voltage gated calcium channel (VGCC) expression is increased in cancer, particularly CACNA1D /CaV1.3 in CRPC. The aim of this study was to investigate if increased CaV1.3 drives resistance to ADT and determine its associated impact on Ca i 2+ and cancer biology. Bioinformatic analysis revealed that CACNA1D gene expression is increased in ADT treated PCa patients. This was corroborated in both in vivo LNCaP xenograft mouse and in vitro PCa cell line models, which demonstrated a significant increase in CaV1.3 protein expression following ADT with bicalutamide. Expression was found to be of a shortened 170kDa CaV1.3 isoform associated with plasma and intracellular membranes, which failed to induce calcium influx following membrane depolarisation. Instead, under ADT CaV1.3 mediated a rise in basal cytosolic calcium and an increase in store operated calcium entry (SOCE). This mechanism was found to promote the proliferation and survival of ADT resistant CRPC cells. Overall, this study demonstrates for the first time in PCa that under ADT specific CaV1.3 isoforms promote an upregulation of SOCE which contributes to treatment resistance and CRPC biology. Thus, this novel oncochannel represents a target for therapeutic development to improve PCa patient outcomes. Graphical Abstract [Display omitted]. [ABSTRACT FROM AUTHOR]

The Ca sensor stromal interacting molecule 1 (STIM1) and the Ca channel Orai1 mediate the ubiquitous store-operated Ca entry (SOCE) pathway activated by depletion of internal Ca stores and mediated through the highly Ca-selective, Ca release-activated Ca (CRAC) current. Furthermore, STIM1 and Orai1, along with Orai3, encode store-independent Ca currents regulated by either arachidonate or its metabolite, leukotriene C. Orai channels are emerging as important contributors to numerous cell functions, including proliferation, migration, differentiation, and apoptosis. Recent studies suggest critical involvement of STIM/Orai proteins in controlling the development of several cancers, including malignancies of the breast, prostate, and cervix. Here, we quantitatively compared the magnitude of SOCE and the expression levels of STIM1 and Orai1 in non-malignant human primary astrocytes (HPA) and in primary human cell lines established from surgical samples of the brain tumor glioblastoma multiforme (GBM). Using Ca imaging, patch-clamp electrophysiology, pharmacological reagents, and gene silencing, we established that in GBM cells, SOCE and CRAC are mediated by STIM1 and Orai1. We further found that GBM cells show upregulation of SOCE and increased Orai1 levels compared to HPA. The functional significance of SOCE was evaluated by studying the effects of STIM1 and Orai1 knockdown on cell proliferation and invasion. Utilizing Matrigel assays, we demonstrated that in GBM, but not in HPA, downregulation of STIM1 and Orai1 caused a dramatic decrease in cell invasion. In contrast, the effects of STIM1 and Orai1 knockdown on GBM cell proliferation were marginal. Overall, these results demonstrate that STIM1 and Orai1 encode SOCE and CRAC currents and control invasion of GBM cells. Our work further supports the potential use of channels contributed by Orai isoforms as therapeutic targets in cancer. [ABSTRACT FROM AUTHOR]

Abstract: Store operated Ca2+ entry (SOCE) replenishes intracellular Ca2+ stores and activates a number of intracellular signalling pathways. Whilst several molecular components forming store operated Ca2+ channels (SOCC) have been identified, their modulation in neurons remains poorly understood. Here, we extend on our previous findings and show that neuronal SOCE is modulated by tyrosine phosphorylation. Cyclopiazonic acid induced SOCE was characterised in hippocampal cultures derived from forebrain specific protein tyrosine phosphatase 1B knockout (PTP1B KO) mice and wild type (WT) litter mates using Fura-2 Ca2+ imaging. PTP1B KO cultures expressed elevated SOCE relative to WT cultures without changes in cytoplasmic Ca2+ homeostasis or depolarisation-induced Ca2+ influx. WT and PTP1B KO cultures displayed similar pharmacological sensitivities towards the SOCE inhibitors gadolinium and 2-aminoethoxydiphenyl borate, as well as the tyrosine kinase inhibitor Ag126 indicating an augmentation of native SOCCs by PTP1B. Following store depletion WT culture homogenates showed heightened phospho-tyrosine levels, an increase in Src tyrosine kinase activation and two minor PTP1B species. These data suggest tyrosine phosphorylation gating SOCE, and implicate PTP1B as a key regulatory enzyme. The involvement of PTP1B in SOCE and its relation to SOCC components and mechanism of regulation are discussed. [Copyright &y& Elsevier]

Spermatozoa must translate information from their environment and the egg to achieve fertilization in sexually reproducing animals. These tasks require decoding a variety of signals in the form of intracellular Ca changes. As TRP channels constitute a large family of versatile multi-signal transducers, they are interesting subjects in which to explore their possible participation in sperm function. Here, we review the evidence for their presence and involvement in sperm motility, maturation, and the acrosome reaction, an exocytotic process required for sperm-egg fusion. Since store-operated Ca entry (SOCE) has been proposed to play an important role in these three functions, the main proteins responsible for this transport (STIM and ORAI) and their interaction with TRPs are also discussed. Improving our tools to solve infertility, improve animal breeding, and preserve biodiversity requires a better understanding of how Ca is regulated in spermatozoa. [ABSTRACT FROM AUTHOR]

Abstract: Reactive oxygen species (ROS) are increasingly recognized as second messengers in many cellular processes. While high concentrations of oxidants damage proteins, lipids and DNA, ultimately resulting in cell death, selective and reversible oxidation of key residues in proteins is a physiological mechanism that can transiently alter their activity and function. Defects in ROS producing enzymes cause disturbed immune response and disease. Changes in the intracellular free Ca2+ concentration are key triggers for diverse cellular functions. Ca2+ homeostasis thus needs to be precisely tuned by channels, pumps, transporters and cellular buffering systems. Alterations of these key regulatory proteins by reversible or irreversible oxidation alter the physiological outcome following cell stimulation. It is therefore necessary to understand which proteins are regulated and if this regulation is relevant in a physiological- and/or pathophysiological context. Because ROS are inherently difficult to identify and to measure, we first review basic oxygen redox chemistry and methods of ROS detection with special emphasis on electron paramagnetic resonance (EPR) spectroscopy. We then focus on the present knowledge of redox regulation of Ca2+ permeable ion channels such as voltage-gated (CaV) Ca2+ channels, transient receptor potential (TRP) channels and Orai channels. [Copyright &y& Elsevier]

Abstract: The purpose of this research was to analyse experimental data concerning cytosolic calcium concentration in view of the mechanisms involved in calcium fluxes in human platelets. The parameters of model curves are related to the properties of the entities responsible for control or maintenance of cytosolic calcium concentration. It has been shown that: (a) biphasicity of increase in cytosolic calcium concentration caused by inhibition of SERCAs either by TBHQ and TG or by TG alone is related to fast and slow discharge of acidic calcium stores and DTS; (b) biphasicity of decline in cytosolic calcium concentration after its rise caused by stimulation of platelets by the agonists is related to non-synchronous extrusion of calcium by PMCA and NCX; (c) NCX is active only in calcium containing medium: calcium ion(s) are necessary to be bound to the site(s) located on the medium-facing side of the (macro)molecule; (d) PMCA is likely to be activated either by binding calcium ion(s) to the site(s) located on its cytosol-facing side or by unbinding identical ion(s) from the site(s) on its medium-facing side. [Copyright &y& Elsevier]

Abstract: In neutrophils, increases in intracellular calcium [Ca2+]i provide a crucial link between inflammatory mediators and inflammatory responses. The modulation of [Ca2+]i fluxes in non-excitable cells such as neutrophils has been studied for more than 25years yet remains to be resolved. In these cells, the Ca2+ influx can occur through at least two mechanisms, as follows: one dependent on the state of filling of the endoplasmic reticulum Ca2+ stores, termed store operated calcium entry (SOCE), and the other less studied mechanism in neutrophils which is not dependent on the state of the Ca2+ stores but is regulated by receptor occupation, termed receptor operated calcium entry (ROCE). Over the past tenyears, the molecular components of SOCE have been extensively characterized, but in neutrophils, the molecular components of ROCE have only recently been explored. In this review, we discuss recent research findings that have demonstrated an important role for ROCE in human neutrophils. In addition, an overview of pharmacological approaches used to discriminate between ROCE and SOCE will be discussed. The elucidation of the molecular components of ROCE may well provide important pharmacological targets for the development of novel anti-inflammatory drugs. [ABSTRACT FROM AUTHOR]

Spatiotemporally discrete cytoplasmic Ca2+ fluctuations are fundamental eukaryotic signals in myriad physiological and pathophysiological functions. Store-operated Ca2+ entry is the process whereby a decrease in endoplasmic reticulum (ER) luminal Ca2+ levels activates Ca2+ release activated calcium (CRAC) channels on the plasma membrane (PM), providing a sustained Ca2+ elevation to the cytoplasm and ultimately replenishing the ER lumen Ca2+ supply. Stromal interaction molecules (STIMs) are the Ca2+ sensors of the ER lumen, which macromolecularly couple depleted ER Ca2+ to the assembly and opening of PM CRAC channels. The considerable stability difference caused by Ca2+ loading and depletion within the luminal portion of STIMs modulates intramolecular cytoplasmic domain interactions essential to the assembly of PM CRAC channels. Thus, the action of the entire complex is tightly regulated through the Ca2+ sensitivity of luminal STIM domains. Recent structural and biochemical studies suggest that partial unfolding - coupled oligomerization of STIMs is a crucial step in CRAC channel activation. Based on these and other published data, this minireview discusses what is currently known about the molecular mechanism of ER Ca2+ sensing by STIMs. Les fluctuations spatio-temporelles du Ca2+ intracellulaire constituent des signaux fondamentaux dans une myriade de fonctions physiologiques et pathophysiologiques chez les eucaryotes. L’influx capacitif du Ca2+ (« store-operated calcium entry ») est un processus par lequel une diminution des niveaux de Ca2+ dans la lumière du réticulum endoplasmique (RE) active les canaux CRAC (« Ca2+release activated calcium ») de la membrane plasmique, résultant en une élévation prolongée de Ca2+ dans le cytoplasme et ultimement, en une reconstitution des réserves de Ca2+ dans la lumière du RE. Les STIM (« stromal interaction molecules ») sont des senseurs de Ca2+ de la lumière du RE qui couplent de façon macromoléculaire le RE épuisé en Ca2+ à l’assemblage et à l’ouverture des canaux CRAC. La différence de stabilité considérable causée par le chargement et la déplétion de Ca2+ à l’intérieur de la partie luminale des STIM module les interactions intramoléculaires des domaines cytoplasmiques essentielles à l’assemblage des canaux CRAC de la membrane plasmique. Ainsi, l’action du complexe entier est intimement modulée par la sensibilité au Ca2+ des domaines luminaux des STIM. Des études structurales et biochimiques récentes suggèrent que l’oligomérisation des STIM couplée à un dépliement partiel est une étape clé de l’activation des canaux CRAC. Cette mini-revue présente des éléments connus du mécanisme moléculaire de la détection de Ca2+du RE par les STIM, à partir de ces travaux et d’autres données publiées. [ABSTRACT FROM AUTHOR]

TRPC and Orai proteins have both been proposed to form Ca2+- selective, store-operated calcium entry (SOCE) channels that are activated by store-depletion with Ca2+ chelators or calcium pump inhibitors. In contrast, only TRPC proteins have been proposed to form nonselective receptor-operated calcium entry (ROCE) cation channels that are activated by Gq/Gi-PLCβ signaling, which is the physiological stimulus for store depletion. We reported previously that a dominant negative Orai1 mutant, R91W. inhibits Ca2+ entry through both SOCE and ROCE channels, implicating Oral participation in both channel complexes. However, the argument for Orai participating in ROCE independently of store depletion is tenuous because store depletion is an integral component of the ROCE response, which includes formation of lP3, a store-depleting agent. Here we show that the R91W mutant also blocks diacylglycerol (DAG)-activated Ca2+ entry into cells that stably, or transiently, express DAG-responsive TRPC proteins. This strongly suggests that Oral and TRPC proteins form complexes that participate in Ca2+ entry with or without activation of store depletion. To integrate these results with recent data linking SOCE with recruitment of Orai and TRPCs to lipid rafts by STIM, we develop the hypothesis that Orai:TRPC complexes recruited to lipid rafts mediate SOCE, whereas the same complexes mediate ROCE when they are outside of lipid rafts. It remains to be determined whether the molecules forming the permeation pathway are the same when Orai:TRPC complexes mediate ROCE or SOCE. [ABSTRACT FROM AUTHOR]

Exposure of neurones in culture to excitotoxic levels of glutamate results in an initial transient spike in [Ca2+]i followed by a delayed, irreversible [Ca2+]i rise governed by rapid kinetics, with Ca2+ originating from the extracellular medium. The molecular mechanism responsible for the secondary Ca2+ rise is unknown. Here, we report that the delayed Ca2+ entry in cortical neurones is diminished by 2-aminoethoxydiphenyl borate (2-APB: IC50 = 62 ± 9 μM) and La3+ (IC50 = 7.2 ± 3 μM), both known to inhibit transient receptor potential (TRP) and store-operated Ca2+ (SOC) channels. Application of thapsigargin, however, failed to exacerbate the delayed Ca2+ deregulation, arguing against a store depletion event as the stimulus for induction of the secondary [Ca2+]i rise. In addition, these neurones did not exhibit SOC entry. Unexpectedly, application of ryanodine or caffeine significantly inhibited glutamate-induced delayed Ca2+ deregulation. In basal Ca2+ entry experiments, La3+ and 2-APB modulated the rapid rise in [Ca2+]i caused by exposure of neurones to Ca2+ after pre-incubating in a calcium-free medium. This basal Ca2+ influx was mitigated by extracellular Mg2+ but not aggravated by thapsigargin, ryanodine or caffeine. These results indicate that 2-APB and La3+ influence non-store-operated Ca2+ influx in cortical neurones and that this route of Ca2+ entry is involved in glutamate-induced delayed Ca2+ deregulation. [ABSTRACT FROM AUTHOR]

Simple Summary: Pancreatic cancer (PC) is one of the most lethal forms of cancers with 5-year mean survival rate of less than 10%. Most of the PC associated deaths are due to metastasis to secondary sites. Calcium (Ca2+) signaling plays a critical role in regulating hallmarks of cancer progression including cell proliferation, migration and apoptotic resistance. Here, we demonstrate that a highly Ca2+ selective plasma membrane channel Orai3 is overexpressed in PC and is associated with poor prognosis in PC patients. Our data demonstrate that Orai3 modulates PC cell proliferation, apoptosis and migration. We further reveal that Orai3 regulates PC metastasis in immune-compromised mice. Collectively, our study establishes Orai3 as an attractive therapeutic target for managing PC metastasis, which may lead to better prognosis. Store operated Ca2+ entry (SOCE) mediated by Orai1/2/3 channels is a highly regulated and ubiquitous Ca2+ influx pathway. Although the role of Orai1 channels is well studied, the significance of Orai2/3 channels is still emerging in nature. In this study, we performed extensive bioinformatic analysis of publicly available datasets and observed that Orai3 expression is inversely associated with the mean survival time of PC patients. Orai3 expression analysis in a battery of PC cell lines corroborated its differential expression profile. We then carried out thorough Ca2+ imaging experiments in six PC cell lines and found that Orai3 forms a functional SOCE channel in PC cells. Our in vitro functional assays show that Orai3 regulates PC cell cycle progression, apoptosis and migration. Most importantly, our in vivo xenograft studies demonstrate a critical role of Orai3 in PC tumor growth and secondary metastasis. Mechanistically, Orai3 controls G1 phase progression, matrix metalloproteinase expression and epithelial-mesenchymal transition in PC cells. Taken together, this study for the first-time reports that Orai3 drives aggressive phenotypes of PC cells, i.e., migration in vitro and metastasis in vivo. Considering that Orai3 overexpression leads to poor prognosis in PC patients, it appears to be a highly attractive therapeutic target. [ABSTRACT FROM AUTHOR]

Ca 2+ signalling plays an essential role in T cell activation, which is a key step to start an adaptive immune response. During the transition from a quiescent to a fully activated state, Ca 2+ microdomains of reduced spatial and temporal extents develop in the junctions between the plasma membrane and the endoplasmic reticulum (ER). These microdomains rely on Ca 2+ entry from the extracellular medium, via the ORAI1/STIM1/STIM2 system that mediates store operated Ca 2+ entry Store operated calcium entry. The mechanism leading to local store depletion and subsequent Ca 2+ entry depends on the activation state of the cells. The initial, smaller microdomains are triggered by D- myo -inositol 1,4,5-trisphosphate (IP 3 ) signalling in response to T cell adhesion. T cell receptor (TCR)/CD3 stimulation then initiates nicotinic acid adenine dinucleotide phosphate signalling, which activates ryanodine receptors (RYR). We have recently developed a mathematical model to elucidate the spatiotemporal Ca 2+ dynamics of the microdomains triggered by IP 3 signalling in response to T cell adhesion (Gil et al., 2021). This reaction-diffusion model describes the evolution of the cytosolic and endoplasmic reticulum Ca 2+ concentrations in a three-dimensional ER-PM junction and was solved using COMSOL Multiphysics. Modelling predicted that adhesion-dependent microdomains result from the concerted activity of IP 3 receptors and pre-formed ORAI1-STIM2 complexes. In the present study, we extend this model to include the role of RYRs rapidly after TCR/CD3 stimulation. The involvement of STIM1, which has a lower K D for Ca 2+ than STIM2, is also considered. Detailed 3D spatio-temporal simulations show that these Ca 2+ microdomains rely on the concerted opening of ∼7 RYRs that are simultaneously active in response to the increase in NAADP induced by T cell stimulation. Opening of these RYRs provoke a local depletion of ER Ca 2+ that triggers Ca 2+ flux through the ORAI1 channels. Simulations predict that RYRs are most probably located around the junction and that the increase in junctional Ca 2+ concentration results from the combination between diffusion of Ca 2+ released through the RYRs and Ca 2+ entry through ORAI1 in the junction. The computational model moreover provides a tool allowing to investigate how Ca 2+ microdomains occur, extend and interact in various states of T cell activation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gil, Diercks, Guse and Dupont.)

Store-operated calcium entry (SOCE) is activated by depletion of Ca2+ from the endoplasmic reticulum (ER) and mediated by stromal interaction molecule (STIM) proteins. Here, we show that in rat and mouse hippocampal neurons, acute ER Ca2+ depletion increases presynaptic Ca2+ levels and glutamate release through a pathway dependent on STIM2 and the synaptic Ca2+ sensor synaptotagmin-7 (syt7). In contrast, synaptotagmin-1 (syt1) can suppress SOCE-mediated spontaneous release, and STIM2 is required for the increase in spontaneous release seen during syt1 loss of function. We also demonstrate that chronic ER stress activates the same pathway leading to syt7-dependent potentiation of spontaneous glutamate release. During ER stress, inhibition of SOCE or syt7-driven fusion partially restored basal neurotransmission and decreased expression of pro-apoptotic markers, indicating that these processes participate in the amplification of ER-stress-related damage. Taken together, we propose that presynaptic SOCE links ER stress and augmented spontaneous neurotransmission, which may, in turn, facilitate neurodegeneration. [Display omitted] • SOCE can potentiate excitatory spontaneous neurotransmission • SOCE is mediated by STIM2 and drives neurotransmitter release through syt7 • Syt1 clamps SOCE-mediated spontaneous release • The SOCE/syt7 pathway participates in propagation of ER-stress-related neuronal damage Chanaday et al. uncover the underlying molecular mechanism coupling store-operated Ca2+ entry to neurotransmitter release at excitatory presynaptic terminals. Activation of this mechanism during chronic ER stress may exacerbate and help propagate the neuronal damage via increased glutamate release. [ABSTRACT FROM AUTHOR]