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Calcium is an important second messenger that is involved in almost all cellular processes. Disruptions in the regulation of intracellular Ca 2+ levels ([Ca 2+ ] i ) adversely impact normal physiological function and can contribute to various diseased conditions. STIM and Orai proteins play important roles in maintaining [Ca 2+ ] i through store-operated Ca 2+ entry (SOCE), with STIM being the primary regulatory protein that governs the function of Orai channels. STIM1 and STIM2 are single-pass ER-transmembrane proteins with their N- and C-termini located in the ER lumen and cytoplasm, respectively. The N-terminal EF-SAM domain of STIMs senses [Ca 2+ ] ER changes, while the C-terminus mediates clustering in ER-PM junctions and gating of Orai1. ER-Ca 2+ store depletion triggers activation of the STIM proteins, which involves their multimerization and clustering in ER-PM junctions, where they recruit and activate Orai1 channels. In this review, we will discuss the structure, organization, and function of EF-hand motifs and the SAM domain of STIM proteins in relation to those of other eukaryotic proteins.

Calcium (Ca 2+ ) is a secondary messenger that regulates various cellular processes. However, Ca 2+ mishandling could lead to pathological conditions. Orai1 is a Ca 2+ channel contributing to the store-operated calcium entry (SOCE) and plays a critical role in Ca 2+ homeostasis in several cell types. Dysregulation of Orai1 contributed to severe combined immune deficiency syndrome, some cancers, pulmonary arterial hypertension (PAH), and other cardiorespiratory diseases. During its activation process, Orai1 is mainly regulated by stromal interacting molecule (STIM) proteins, especially STIM1; however, many other regulatory partners have also been recently described. Increasing knowledge about these regulatory partners provides a better view of the downstream signalling pathways of SOCE and offers an excellent opportunity to decipher Orai1 dysregulation in these diseases. These proteins participate in other cellular functions, making them attractive therapeutic targets. This review mainly focuses on Orai1 regulatory partners in the physiological and pathological conditions of the pulmonary circulation and inflammation., Competing Interests: Declaration of competing interest DM has relationships with drug companies, including Actelion, Bayer, GSK, Novartis, and Pfizer. In addition to being investigators in trials involving these companies, other relationships include consultancy services and memberships to scientific advisory boards. The other authors have no conflicts of interest to declare., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The family of stromal interaction molecules (STIM) includes two widely expressed single-pass endoplasmic reticulum (ER) transmembrane proteins and additional splice variants that act as precise ER-luminal Ca 2+ sensors. STIM proteins mainly function as one of the two essential components of the so-called Ca 2+ release-activated Ca 2+ (CRAC) channel. The second CRAC channel component is constituted by pore-forming Orai proteins in the plasma membrane. STIM and Orai physically interact with each other to enable CRAC channel opening, which is a critical prerequisite for various downstream signalling pathways such as gene transcription or proliferation. Their activation commonly requires the emptying of the intracellular ER Ca 2+ store. Using their Ca 2+ sensing capabilities, STIM proteins confer this Ca 2+ content-dependent signal to Orai, thereby linking Ca 2+ store depletion to CRAC channel opening. Here we review the conformational dynamics occurring along the entire STIM protein upon store depletion, involving the transition from the quiescent, compactly folded structure into an active, extended state, modulation by a variety of accessory components in the cell as well as the impairment of individual steps of the STIM activation cascade associated with disease., (© 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Background: Stromal interaction molecule (STIM)/Orai calcium entry system appears to have a role in erectile dysfunction (ED) pathophysiology but its specific contribution to diabetic ED was not elucidated., Aim: To evaluate STIM/Orai inhibition on functional alterations associated with diabetic ED in rat and human penile tissues and on in vivo erectile responses in diabetic rats., Methods: Rat corpus cavernosum (RCC) strips from nondiabetic (No DM) and streptozotocin-induced diabetic (DM) rats and human penile resistance arteries (HPRA) and corpus cavernosum (HCC) from ED patients undergoing penile prosthesis insertion were functionally evaluated in organ chambers and wire myographs. Erectile function in vivo in rats was assessed by intracavernosal pressure (ICP) responses to cavernous nerve electrical stimulation (CNES). Expression of STIM/Orai elements in HCC was determined by immunofluorescence and immunoblot., Main Outcome Measures: Functional responses in RCC, HCC and HPRA and STIM/Orai protein expression in HCC. In vivo erectile responses to CNES., Results: Inhibition of Orai channels with YM-58483 (20 µM) significantly reduced adrenergic contractions in RCC but more effectively in DM. Thromboxane-induced and neurogenic contractions were reduced by STIM/Orai inhibition while defective endothelial, neurogenic and PDE5 inhibitor-induced relaxations were enhanced by YM-58483 (10 µM) in RCC from DM rats. In vivo, YM-58483 caused erections and attenuated diabetes-related impairment of erectile responses. YM-58483 potentiated the effects of PDE5 inhibition. In human tissues, STIM/Orai inhibition depressed adrenergic and thromboxane-induced contractions in ED patients more effectively in those with type 2 diabetes. Diabetes was associated with increased expression of Orai1 and Orai3 in ED patients., Clinical Translation: Targeting STIM/Orai to alleviate diabetes-related functional alterations of penile vascular tissue could improve erectile function and potentiate therapeutic effects of PDE5 inhibitors in diabetic ED., Strengths and Limitations: Improving effects of STIM/Orai inhibition on diabetes-related functional impairment was evidenced in vitro and in vivo in an animal model and validated in human tissues from ED patients. Functional findings were complemented with expression results. Main limitation was low numbers of human experiments due to limited human tissue availability., Conclusions: STIM/Orai inhibition alleviated alterations of functional responses in vitro and improved erectile responses in vivo in diabetic rats, potentiating the effects of PDE5 inhibition. STIM/Orai inhibition was validated as a target to modulate functional alterations of human penile vascular tissue in diabetic ED where Orai1 and Orai3 channels were upregulated. STIM/Orai inhibition could be a potential therapeutic strategy to overcome poor response to conventional ED therapy in diabetic patients. Sevilleja-Ortiz A, El Assar M, García-Gómez B, et al. STIM/Orai Inhibition as a Strategy for Alleviating Diabetic Erectile Dysfunction Through Modulation of Rat and Human Penile Tissue Contractility and in vivo Potentiation of Erectile Responses. J Sex Med 2022;19:1733-1749., (Copyright © 2022 International Society for Sexual Medicine. Published by Elsevier Inc. All rights reserved.)

Alzheimer's disease (AD) is a progressive neurological disease that slowly causing memory impairments with no effective treatment. We have recently reported that kisspeptin-13 (KP-13) ameliorates Aβ toxicity-induced memory deficit in rats. Here, the possible cellular impact of kisspeptin receptor activation in a rat model of the early stage AD was assessed using whole-cell patch-clamp recording from CA1 pyramidal neurons and molecular approaches. Compared to neurons from the control group, cells from the Aβ-treated group displayed spontaneous and evoked hyperexcitability with lower spike frequency adaptation. These cells had also a lower sag ratio in response to hyperpolarizing prepulse current delivered before a depolarizing current injection. Neurons from the Aβ-treated group exhibited short spike onset latency, lower rheobase and short utilization time compared with those in the control group. Furthermore, phase plot analysis of action potential showed that Aβ treatment affected the action potential features. These electrophysiological changes induced by Aβ were associated with increased expression of stromal interaction molecules (STIMs), particularly (STIM2) and decreased pCREB/CREB ratio. Treatment with KP-13 following Aβ injection into the entorhinal cortex, however, prevented the excitatory effect of Aβ on spontaneous and evoked neuronal activity, increased the latency of onset, enhanced the sag ratio, increased the rheobase and utilization time, and prevented the changes induced Aβ on spike parameters. In addition, the KP-13 application after Aβ treatment reduced the expression of STIMs and increased the pCREB/CREB ratio compared to those receiving Aβ treatment alone. In summary, these results provide evidence that activation of kisspeptin receptor may be effective against pathology of Aβ., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Sustained and intermittent hypoxia produce vasoconstriction, arterial remodeling, and hypertension in the lung. Stromal interaction molecule (STIM)-activated transient receptor potential channels (TRPC) and calcium release-activated calcium channel protein (ORAI) channels (STOC) play key roles in the progression of pulmonary hypertension in pre-clinical models of animals subjected to sustained and intermittent hypoxia. The available evidence supports the theory that oxidative stress and hypoxic inducible factors upregulate and activate STIM-activated TRPC-ORAI Ca 2+ channels, contributing to the pulmonary remodeling and hypertension induced by sustained hypoxia. However, less is known about the effects of oxidative stress and hypoxic inducible factors on the modulation of STIM-activated TRPC-ORAI channels following chronic intermittent hypoxia. In this review, we examined the emerging evidence supporting the theory that oxidative stress and hypoxic inducible factors induced by intermittent hypoxia upregulate and activate STIM-activated TRPC-ORAI Ca 2+ channels. In addition, we used bioinformatics tools to search public databases for the genes involved in the upregulation of STIMactivated TRPC-ORAI Ca 2+ channels and compare the differential gene expression and biological processes induced by intermittent and sustained hypoxia in lung cells., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Cardiac fibrotization is a well-known process characteristic of many cardiac pathological conditions. The key element is excessive activation of cardiac fibroblasts, their transdifferentiation into myofibroblasts, increased production, and accumulation of extracellular matrix proteins, resulting in cardiac stiffness. The exact cellular mechanisms and molecular components involved in the process are not fully elucidated, but the SOCE mechanism could play an important role. Its key molecules are the molecular sensor of calcium in ER/SR - STIM and the highly selective calcium channels Orai located in the plasma membrane. This study aims to evaluate selected SOCE-associated genes in the activation of HCF cell culture by several known substances (phenylephrine, isoprenaline) that represent cardiovascular overload. After cell cultivation, cell medium was collected to measure the soluble collagen content. From the harvested cells, qRT-PCR was performed to determine the mRNA levels of the corresponding genes. The activation of cells was based on changes in the relative expression of collagen genes as well as the collagen content in the medium of the cell culture. We detected an increase in the expression of the Orai2 isoform, a change in the Orai1/Orai3 ratio and also an increase in the expression of the STIM2 isoform. These results suggest an increased activation of the SOCE mechanism under stress conditions of fibroblasts, which supports the hypothesis of fibroblast activation in pathological processes by altering calcium homeostasis through the SOCE mechanism.

The amyloid-beta (Aβ) fibrillation process seems to execute a principal role in the neuropathology of Alzheimer's disease (AD). Accordingly, novel therapeutic plans have concentrated on the inhibition or degradation of Aβ oligomers and fibrils. Biocompatible nanoparticles (NPs), e.g., gold and iron oxide NPs, take a unique capacity in redirecting Aβ fibrillation kinetics; nevertheless, their impacts on AD-related memory impairment have not been adequately evaluated in vivo. Here, we examined the effect of commercial PEGylated superparamagnetic iron oxide nanoparticles (SPIONs) on the learning and memory of an AD-animal model. The outcomes demonstrated the dose-dependent effect of SPIONs on Aβ fibrillation and learning and memory processes. In vitro and in vivo findings revealed that Low doses of SPIONs inhibited Aβ aggregation and ameliorated learning and memory deficit in the AD model, respectively. Enhanced level of hippocampal proteins, including brain-derived neurotrophic factor, BDNF, phosphorylated-cAMP response element-binding protein, p-CREB, and stromal interaction molecules, e.g., STIM1 and STIM2, were also observed. However, at high doses, SPIONs did not improve the detrimental impacts of Aβ fibrillation on spatial memory and hippocampal proteins expression. Overall, we revealed the potential capacity of SPIONs on retrieval of behavioral and molecular manifestations of AD in vivo, which needs further investigations to determine the mechanistic effect of SPIONs in the AD conundrum., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In eukaryotic cells, ultimate specificity in activation and action-for example, by means of second messengers-of the myriad of signaling cascades is primordial. In fact, versatile and ubiquitous second messengers, such as calcium (Ca 2+ ) and cyclic adenosine monophosphate (cAMP), regulate multiple-sometimes opposite-cellular functions in a specific spatiotemporal manner. Cells achieve this through segregation of the initiators and modulators to specific plasma membrane (PM) subdomains, such as lipid rafts and caveolae, as well as by dynamic close contacts between the endoplasmic reticulum (ER) membrane and other intracellular organelles, including the PM. Especially, these membrane contact sites (MCSs) are currently receiving a lot of attention as their large influence on cell signaling regulation and cell physiology is increasingly appreciated. Depletion of ER Ca 2+ stores activates ER membrane STIM proteins, which activate PM-residing Orai and TRPC Ca 2+ channels at ER-PM contact sites. Within the MCS, Ca 2+ fluxes relay to cAMP signaling through highly interconnected networks. However, the precise mechanisms of MCS formation and the influence of their dynamic lipid environment on their functional maintenance are not completely understood. The current review aims to provide an overview of our current understanding and to identify open questions of the field.

Stromal interaction molecules (STIM) are a distinct class of ubiquitously expressed single-pass transmembrane proteins in the endoplasmic reticulum (ER) membrane. Together with Orai ion channels in the plasma membrane (PM), they form the molecular basis of the calcium release-activated calcium (CRAC) channel. An intracellular signaling pathway known as store-operated calcium entry (SOCE) is critically dependent on the CRAC channel. The SOCE pathway is activated by the ligand-induced depletion of the ER calcium store. STIM proteins, acting as calcium sensors, subsequently sense this depletion and activate Orai ion channels via direct physical interaction to allow the influx of calcium ions for store refilling and downstream signaling processes. This review article is dedicated to the latest advances in the field of STIM proteins. New results of ongoing investigations based on the recently published functional data as well as structural data from nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations are reported and complemented with a discussion of the latest developments in the research of STIM protein isoforms and their differential functions in regulating SOCE.

Voltage-gated L-type Ca 2+ channel (Ca v 1.2) blockers (LCCBs) are major drugs for treating hypertension, the preeminent risk factor for heart failure. Vascular smooth muscle cell (VSMC) remodeling is a pathological hallmark of chronic hypertension. VSMC remodeling is characterized by molecular rewiring of the cellular Ca 2+ signaling machinery, including down-regulation of Ca v 1.2 channels and up-regulation of the endoplasmic reticulum (ER) stromal-interacting molecule (STIM) Ca 2+ sensor proteins and the plasma membrane ORAI Ca 2+ channels. STIM/ORAI proteins mediate store-operated Ca 2+ entry (SOCE) and drive fibro-proliferative gene programs during cardiovascular remodeling. SOCE is activated by agonists that induce depletion of ER Ca 2+ , causing STIM to activate ORAI. Here, we show that the three major classes of LCCBs activate STIM/ORAI-mediated Ca 2+ entry in VSMCs. LCCBs act on the STIM N terminus to cause STIM relocalization to junctions and subsequent ORAI activation in a Ca v 1.2-independent and store depletion-independent manner. LCCB-induced promotion of VSMC remodeling requires STIM1, which is up-regulated in VSMCs from hypertensive rats. Epidemiology showed that LCCBs are more associated with heart failure than other antihypertensive drugs in patients. Our findings unravel a mechanism of LCCBs action on Ca 2+ signaling and demonstrate that LCCBs promote vascular remodeling through STIM-mediated activation of ORAI. Our data indicate caution against the use of LCCBs in elderly patients or patients with advanced hypertension and/or onset of cardiovascular remodeling, where levels of STIM and ORAI are elevated., Competing Interests: The authors declare no competing interest.

VGF gene encodes for a neuropeptide precursor of 68 kDa composed by 615 (human) and 617 (rat, mice) residues, expressed prevalently in the central nervous system (CNS), but also in the peripheral nervous system (PNS) and in various endocrine cells. This precursor undergoes proteolytic cleavage, generating a family of peptides different in length and biological activity. Among them, TLQP-21, a peptide of 21 amino acids, has been widely investigated for its relevant endocrine and extraendocrine activities. The complement complement C3a receptor-1 (C3aR1) has been suggested as the TLQP-21 receptor and, in different cell lines, its activation by TLQP-21 induces an increase of intracellular Ca 2+ . This effect relies both on Ca 2+ release from the endoplasmic reticulum (ER) and extracellular Ca 2+ entry. The latter depends on stromal interaction molecules (STIM)-Orai1 interaction or transient receptor potential channel (TRPC) involvement. After Ca 2+ entry, the activation of outward K + -Ca 2+ -dependent currents, mainly the K Ca3.1 currents, provides a membrane polarizing influence which offset the depolarizing action of Ca 2+ elevation and indirectly maintains the driving force for optimal Ca 2+ increase in the cytosol. In this review, we address the main endocrine and extraendocrine actions displayed by TLQP-21, highlighting recent findings on its mechanism of action and its potential in different pathological conditions.

Store-operated calcium entry (SOCE) is one of regulatory mechanisms which regulates Ca2+ cycling in the heart. SOCE alterations in pathological conditions contribute to progression of heart failure and cardiac hypertrophy by multiple signaling pathways such as Cn/NFAT and CaMKII/MEF2. Several components mediating SOCE have been identified, such as STIM and Orai. Different isoforms of both Orai and STIM have been detected in animal studies, exhibiting distinct functional properties. This study is focused on the analysis of STIM and Orai isoforms expression in the end-stage human failing myocardium. Left ventricle samples isolated from 43 explanted hearts from patients undergoing heart transplant and from 5 healthy donor hearts were used to determine the mRNA levels of Orai1, Orai2 and Orai3, STIM1, STIM2 and STIM2.1 by qRT-PCR. The expression was further analyzed for connection with gender, related co-morbidities, pathoetiology, clinical data and biochemical parameters. We show that Orai1 expression is decreased by 30 % in failing myocardium, even though we detected no significant changes in expression of Orai2 or Orai3. Interestingly, this decrease in Orai1 was gender-specific and was present only in men, with no change in women. The ratio Orai1/Orai3 was significantly lower in males as well. The novel STIM2.1 isoform was detected both in healthy and failing human myocardium. In the end-stage heart failure, the expression of STIM2.1 was significantly decreased. The lower ratio of STIM2.1/STIM2 in failing hearts indicates a switch from SOCE-inhibiting STIM2.1 isoform to stimulatory STIM2.2. STIM1 mRNA levels were not significantly changed. These observed alterations in Orai and STIM expression were independent of functional heart parameters, clinical or biochemical patient characteristics. These results provide detailed insight into the alterations of SOCE regulation in human failing myocardium. Gender-specific change in Orai1 expression might represent a possible mechanism of cardioprotective effects of estrogens. The switch from STIM2.1 to STIM2.2 indicates an amplification of SOCE and could contribute to the hypertrophy development in the filing heart.

Effective therapy of hypertension represents a key strategy for reducing the burden of cardiovascular disease and its associated mortality. The significance of voltage dependent L-type Ca²⁺ channels to Ca²⁺ influx, and of their regulatory mechanisms in the development of heart disease, is well established. A wide variety of L-type Ca²⁺ channel inhibitors and Ca²⁺ antagonists have been found to be beneficial not only in the treatment of hypertension, but also in myocardial infarction and heart failure. Over the past two decades, another class of Ca²⁺ channel - the voltage independent store-operated Ca²⁺ channel - has been implicated in the regulation and fine tuning of Ca²⁺ entry in both cardiac and smooth muscle cells. Store-operated Ca²⁺ channels are activated by the depletion of Ca²⁺ stores within the endoplasmic/sarcoplasmic reticulum, or by low levels of cytosolic Ca²⁺, thereby facilitating agonist-induced Ca²⁺ influx. Store-operated Ca²⁺ entry through this pivotal pathway involves both stromal interaction molecule (STIM) and Orai channels. Different degrees of changes in these proteins are considered to promote Ca²⁺ entry and hence contribute to the pathogenesis of cardiovascular dysfunction. Several blockers of store-operated Ca²⁺ channels acting at the level of both STIM and Orai channels have been shown to depress Ca²⁺ influx and lower blood pressure. However, their specificity, safety, and clinical significance remain to be established. Thus, there is an ongoing challenge in the development of selective inhibitors of store-operated Ca²⁺ channels that act in vascular smooth muscles for the improved treatment of hypertension., Competing Interests: The authors declare no conflict of interest., (©2019 Bhullar et al. Published by IMR press. All rights reserved.)

Calcium (Ca 2+ ) is an important element for many physiological functions of the uterus, including embryo implantation. Here, we investigated the possible involvement of altered intracellular Ca 2+ levels in decidualization in human endometrial stromal cells (hEMSCs). hEMSCs showed high levels of mesenchymal stem cell marker expression (CD73, CD90, and CD105) and did not express markers of hematopoietic progenitor cells (CD31, CD34, CD45, and HLA-DR). Decidualization is a process of ovarian steroid-induced endometrial stromal cell proliferation and differentiation. Several types of ion channels, which are regulated by the ovarian hormones progesterone and estradiol, as well as growth factors, are important for endometrial receptivity and embryo implantation. The combined application of progesterone (1 μM medroxyprogesterone acetate) and cyclic AMP (0.5 mM) for 6 days not only elevated inositol 1,4,5-triphosphate receptor (IP 3 R)-mediated Ca 2+ release and IP 3 R expression, it also promoted ORAI and STIM expression as well as cyclopiazonic acid-induced Ca 2+ release. Finally, intracellular Ca 2+ levels and ion channel gene expression influenced hEMSC proliferation. These results suggest that cytosolic Ca 2+ dynamics, mediated by specific ion channels, serve as an important step in the decidualization of hEMSCs., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Inositol 1,4,5-trisphosphate receptors (IP 3 R) are the most widely expressed intracellular Ca 2+ release channels. Their activation by IP 3 and Ca 2+ allows Ca 2+ to pass rapidly from the ER lumen to the cytosol. The resulting increase in cytosolic [Ca 2+ ] may directly regulate cytosolic effectors or fuel Ca 2+ uptake by other organelles, while the decrease in ER luminal [Ca 2+ ] stimulates store-operated Ca 2+ entry (SOCE). We are close to understanding the structural basis of both IP 3 R activation, and the interactions between the ER Ca 2+ -sensor, STIM, and the plasma membrane Ca 2+ channel, Orai, that lead to SOCE. IP 3 Rs are the usual means through which extracellular stimuli, through ER Ca 2+ release, stimulate SOCE. Here, we review evidence that the IP 3 Rs most likely to respond to IP 3 are optimally placed to allow regulation of SOCE. We also consider evidence that IP 3 Rs may regulate SOCE downstream of their ability to deplete ER Ca 2+ stores. Finally, we review evidence that IP 3 Rs in the plasma membrane can also directly mediate Ca 2+ entry in some cells., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Skeletal muscle contracts or relaxes to maintain the body position and locomotion. For the contraction and relaxation of skeletal muscle, Ca2＋ in the cytosol of skeletal muscle fibers acts as a switch to turn on and off a series of contractile proteins. The cytosolic Ca2＋ level in skeletal muscle fibers is governed mainly by movements of Ca2＋ between the cytosol and the sarcoplasmic reticulum (SR). Store-operated Ca2＋ entry (SOCE), a Ca2＋ entryway from the extracellular space to the cytosol, has gained a significant amount of attention from muscle physiologists. Orai1 and stromal interaction molecule 1 (STIM1) are the main protein identities of SOCE. This mini-review focuses on the roles of STIM proteins and SOCE in the physiological and pathophysiological functions of skeletal muscle and in their correlations with recently identified proteins, as well as historical proteins that are known to mediate skeletal muscle function. [BMB Reports 2018; 51(8): 378-387].

Electrical pacemaker activity generates phasic contractions and motility patterns such as segmentation and peristalsis in the gastrointestinal tract. Pacemaker currents are generated in interstitial cells of Cajal (ICC), which release Ca 2+ from intracellular stores that stimulates Ca 2+ -activated Cl - channels (CaCCs) in the plasma membrane. Thus, Ca 2+ stores must be maintained to sustain pacemaker activity. Store-operated Ca 2+ entry (SOCE) facilitates the refilling of Ca 2+ stores by a mechanism dependent upon interactions between STIM and Orai proteins. We investigated the role of SOCE in ICC pacemaker activity. Reintroduction of extracellular Ca 2+ in store-depleted ICC resulted in CaCC activation. Blocking CaCCs revealed an inwardly rectifying current with properties of a Ca 2+ release-activated current ( I CRAC ). An inhibitory peptide that interfered with the STIM-Orai interaction blocked I CRAC in HEK 293 cells expressing STIM1 and Orai1 and blocked spontaneous transient inward currents (STICs) and slow wave currents in ICC. STICs, which are fundamental pacemaker events in ICC, were blocked by an Orai antagonist. Imaging of Ca 2+ transients linked to pacemaker activity in ICC in intact muscles showed that the Orai antagonist blocked Ca 2+ transients in ICC. These data suggest that Ca 2+ recovery through STIM-Orai interactions is necessary to maintain ICC pacemaker activity., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The expression of chimeras that consist of a fluorescent protein (FP) conjugated with a protein of interest provides the ability to visualize, track, and quantify the subcellular localization and dynamics of specific proteins in biological samples. Array confocal laser scanning microscopy is an eminently suitable technique for live-cell imaging of FP-tagged fusion proteins. Here, we describe real-time monitoring of the subcellular dynamics of the stromal-interacting molecule 1 (STIM1) and Orai1, the key protagonists of store-operated Ca 2+ entry (SOCE) under resting conditions, and upon Ca 2+ mobilization from the endoplasmic reticulum (ER).

The characterization of protein-protein interactions through methods such as co-immunoprecipitation, followed by Western blot analysis, is a crucial step in the understanding of protein functions and the biology of the cell. Since the discovery of ORAI and STIM proteins as component of store-operated channel (SOC), overexpressing systems have been used to demonstrate how ORAI and STIM can associate with physiological and pathological conditions. Here we describe a protocol allowing endogenous studies.

Calcium influx through store-operated Ca 2+ entry (SOCE), mediated by STIM-operated Orai channels, is crucial for many cellular functions. To dissect the molecular mechanisms underlying the process of STIM-Orai activation and identify regulators that modify this process, ratiometric imaging of SOCE responses in HEK cells overexpressing STIM and Orai is a routinely used method. Here we describe one commonly used procedure of monitoring SOCE activity with a ratiometric membrane-permeable dye fura-2-AM. Other ratiometric indicators suitable for SOCE measurements are also discussed.

Calcium (Ca 2+ ) is a ubiquitous second messenger that performs significant physiological task such as neurosecretion, exocytosis, neuronal growth/differentiation, and the development and/or maintenance of neural circuits. An important regulatory aspect of neuronal Ca 2+ homeostasis is store-operated Ca 2+ entry (SOCE) which, in recent years, has gained much attention for influencing a variety of nerve cell responses. Essentially, activation of SOCE ensues following the activation of the plasma membrane (PM) store-operated Ca 2+ channels (SOCC) triggered by the depletion of endoplasmic reticulum (ER) Ca 2+ stores. In addition to the TRPC (transient receptor potential canonical) and the Orai family of ion channels, STIM (stromal interacting molecule) proteins have been baptized as key molecular regulators of SOCE. Functional significance of the TRPC channels in neurons has been elaborately studied; however, information on Orai and STIM components of SOCE, although seems imminent, is currently limited. Importantly, perturbations in SOCE have been implicated in a spectrum of neuropathological conditions. Hence, understanding the precise involvement of SOCC in neurodegeneration would presumably unveil avenues for plausible therapeutic interventions. We thus review the role of SOCE-regulated neuronal Ca 2+ signaling in selecting neurodegenerative conditions.

Store-operated Ca 2+ entry (SOCE) mediated by STIM and Orai proteins is a highly regulated and ubiquitous signaling pathway that plays an important role in various cellular and physiological functions. Endoplasmic reticulum (ER) serves as the major site for intracellular Ca 2+ storage. Stromal Interaction Molecule 1/2 (STIM1/2) sense decrease in ER Ca 2+ levels and transmits the message to plasma membrane Ca 2+ channels constituted by Orai family members (Orai1/2/3) resulting in Ca 2+ influx into the cells. This increase in cytosolic Ca 2+ in turn activates a variety of signaling cascades to regulate a plethora of cellular functions. Evidence from the literature suggests that SOCE dysregulation is associated with several pathophysiologies, including vascular disorders. Interestingly, recent studies have suggested that STIM proteins may also regulate vascular functions independent of their contribution to SOCE. In this updated book chapter, we will focus on the physiological role of STIM and Orai proteins in the vasculature (endothelial cells and vascular smooth muscle cells). We will further retrospect the literature implicating a critical role for these proteins in vascular disease.

Cysteines are among the least abundant amino acids found in proteins. Due to their unique nucleophilic thiol group, they are able to undergo a broad range of chemical modifications besides their known role in disulfide formation, such as S-sulfenylation (-SOH), S-sulfinylation (-SO(2)H), S-sufonylation (-SO(3)H), S-glutathionylation (-SSG), and S-sulfhydration (-SSH), among others. These posttranslational modifications can be irreversible and act as transitional modifiers or as reversible on-off switches for the function of proteins. Disturbances of the redox homeostasis, for example, in situations of increased oxidative stress, can contribute to a range of diseases. Because Ca 2+ signaling mediated by store-operated calcium entry (SOCE) is involved in a plethora of cellular responses, the cross-talk between reactive oxygen species (ROS) and Ca 2+ is critical for homeostatic control. Identification of calcium regulatory protein targets of thiol redox modifications is needed to understand their role in biology and disease.

Ca 2+ signals regulate a plethora of cellular functions that include muscle contraction, heart beating, hormone secretion, lymphocyte activation, gene expression, and metabolism. To study the impact of Ca 2+ signals on biological processes, pharmacological tools and caged compounds have been commonly applied to induce fluctuations of intracellular Ca 2+ concentrations. These conventional approaches, nonetheless, lack rapid reversibility and high spatiotemporal resolution. To overcome these disadvantages, we and others have devised a series of photoactivatable genetically encoded Ca 2+ actuators (GECAs) by installing light sensitivities into a bona fide highly selective Ca 2+ channel, the Ca 2+ release-activated Ca 2+ (CRAC) channel. Store-operated CRAC channel serves as a major route for Ca 2+ entry in many cell types. These GECAs enable remote and precise manipulation of Ca 2+ signaling in both excitable and non-excitable cells. When combined with nanotechnology, it becomes feasible to wirelessly photo-modulate Ca 2+ -dependent activities in vivo. In this chapter, we briefly review most recent advances in engineering CRAC channels to achieve optical control over Ca 2+ signaling, outline their design principles and kinetic features, and present exemplary applications of GECAs engineered from CRAC channels.

The basic paradigm of a mechanism for calcium influx triggered after a reduction on calcium store content implies a sensor of calcium concentration on the endoplasmic reticulum (the stores) and a calcium channel immersed on the plasma membrane. These two basic components are STIM and Orai, the most fundamental and minimal molecular constituents of the store-operated calcium entry mechanism. However, even when minimal components can be reduced to these two proteins, the intricate process involved in approximating two cellular membranes (endoplasmic reticulum, ER and plasma membrane, PM) require the participation of several other components, many of which remain unidentified to this date. Here we review several of the proteins identified as constituents of the so-called store-operated calcium influx complex (SOCIC) and discuss their role in modulating this complex phenomenon.

This chapter focuses on the Orai proteins, Orai1-Orai3, with special emphasis on Orai1, in humans and other mammals, and on the definitive evidence that Orai is the pore subunit of the CRAC channel. It begins by reviewing briefly the defining characteristics of the CRAC channel, then discusses the studies that implicated Orai as part of the store-operated Ca 2+ entry pathway and as the CRAC channel pore subunit, and finally examines ongoing work that is providing insights into CRAC channel structure and gating.

Engagement of T cell receptors (TCRs) with cognate antigens triggers cascades of signaling pathways in helper T cells. TCR signaling is essential for the effector function of helper T cells including proliferation, differentiation, and cytokine production. It also modulates effector T cell fate by inducing cell death, anergy (nonresponsiveness), exhaustion, and generation of regulatory T cells. One of the main axes of TCR signaling is the Ca 2+ -calcineurin-nuclear factor of activated T cells (NFAT) signaling pathway. Stimulation of TCRs triggers depletion of intracellular Ca 2+ store and, in turn, activates store-operated Ca 2+ entry (SOCE) to raise the intracellular Ca 2+ concentration. SOCE in T cells is mediated by the Ca 2+ release-activated Ca 2+ (CRAC) channels, which have been very well characterized in terms of their electrophysiological properties. Identification of STIM1 as a sensor to detect depletion of the endoplasmic reticulum (ER) Ca 2+ store and Orai1 as the pore subunit of CRAC channels has dramatically advanced our understanding of the regulatory mechanism of Ca 2+ signaling in T cells. In this review, we discuss our current understanding of Ca 2+ signaling in T cells with specific focus on the mechanism of CRAC channel activation and regulation via protein interactions. In addition, we will discuss the role of CRAC channels in effector T cells, based on the analyses of genetically modified animal models.

Calcium (Ca 2+ ) is a key regulator of cardiomyocyte contraction. The Ca 2+ channels, pumps, and exchangers responsible for the cyclical cytosolic Ca 2+ signals that underlie contraction are well known. In addition to those Ca 2+ signaling components responsible for contraction, it has been proposed that cardiomyocytes express channels that promote the influx of Ca 2+ from the extracellular milieu to the cytosol in response to depletion of intracellular Ca 2+ stores. With non-excitable cells, this store-operated Ca 2+ entry (SOCE) is usually easily demonstrated and is essential for prolonging cellular Ca 2+ signaling and for refilling depleted Ca 2+ stores. The role of SOCE in cardiomyocytes, however, is rather more elusive. While there is published evidence for increased Ca 2+ influx into cardiomyocytes following Ca 2+ store depletion, it has not been universally observed. Moreover, SOCE appears to be prominent in embryonic cardiomyocytes but declines with postnatal development. In contrast, there is overwhelming evidence that the molecular components of SOCE (e.g., STIM, Orai, and TRPC proteins) are expressed in cardiomyocytes from embryo to adult. Moreover, these proteins have been shown to contribute to disease conditions such as pathological hypertrophy, and reducing their expression can attenuate hypertrophic growth. It is plausible that SOCE might underlie Ca 2+ influx into cardiomyocytes and may have important signaling functions perhaps by activating local Ca 2+ -sensitive processes. However, the STIM, Orai, and TRPC proteins appear to cooperate with multiple protein partners in signaling complexes. It is therefore possible that some of their signaling activities are not mediated by Ca 2+ influx signals, but by protein-protein interactions.