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The single-pass transmembrane protein Stromal Interaction Molecule 1 (STIM1), located in the endoplasmic reticulum (ER) membrane, possesses two main functions: It senses the ER-Ca2+ concentration and directly binds to the store-operated Ca2+ channel Orai1 for its activation when Ca2+ recedes. At high resting ER-Ca2+ concentration, the ER-luminal STIM1 domain is kept monomeric but undergoes di/multimerization once stores are depleted. Luminal STIM1 multimerization is essential to unleash the STIM C-terminal binding site for Orai1 channels. However, structural basis of the luminal association sites has so far been elusive. Here, we employed molecular dynamics (MD) simulations and identified two essential di/multimerization segments, the a7 and the adjacent region near the a9-helix in the sterile alpha motif (SAM) domain. Based on MD results, we targeted the two STIM1 SAM domains by engineering point mutations. These mutations interfered with higher-order multimerization of ER-luminal fragments in biochemical assays and puncta formation in live-cell experiments upon Ca2+ store depletion. The STIM1 multimerization impeded mutants significantly reduced Ca2+ entry via Orai1, decreasing the Ca2+ oscillation frequency as well as store-operated Ca2+ entry. Combination of the ER-luminal STIM1 multimerization mutations with gain of function mutations and coexpression of Orai1 partially ameliorated functional defects. Our data point to a hydrophobicity-driven binding within the ER-luminal STIM1 multimer that needs to switch between resting monomeric and activated multimeric state. Altogether, these data reveal that interactions between SAM domains of STIM1 monomers are critical for multimerization and activation of the protein. [ABSTRACT FROM AUTHOR]

Key components of endoplasmic reticulum (ER) Ca2+ release and store‐operated Ca2+ entry (SOCE) are likely expressed in all metazoan cells. Due to the complexity of canonical Ca2+ entry mechanisms in neurons, the functional significance of ER‐Ca2+ release and SOCE has been difficult to identify and establish. In this review we present evidence of how these two related mechanisms of Ca2+ signalling impact multiple aspects of neuronal physiology and discuss their interaction with the better understood classes of ion channels that are gated by either voltage changes or extracellular ligands in neurons. Given how a small imbalance in Ca2+ homeostasis can have strongly detrimental effects on neurons, leading to cell death, it is essential that neuronal SOCE is carefully regulated. We go on to discuss some mechanisms of SOCE regulation that have been identified in Drosophila and mammalian neurons. These include specific splice variants of stromal interaction molecules, different classes of membrane‐interacting proteins and an ER–Ca2+ channel. So far these appear distinct from the mechanisms of SOCE regulation identified in non‐excitable cells. Finally, we touch upon the significance of these studies in the context of certain human neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

The concentration of free cytosolic Ca2+ is a critical second messenger in almost every cell type, with the signal often being carried by the period of oscillations, or spikes, in the cytosolic Ca2+ concentration. We have previously studied how Ca2+ influx across the plasma membrane affects the period and shape of Ca2+ oscillations in HEK293 cells. However, our theoretical work was unable to explain how the shape of Ca2+ oscillations could change qualitatively, from thin spikes to broad oscillations, during the course of a single time series. Such qualitative changes in oscillation shape are a common feature of HEK293 cells in which STIM1 and 2 have been knocked out. Here, we present an extended version of our earlier model that suggests that such time-dependent qualitative changes in oscillation shape might be the result of balanced positive and negative feedback from Ca2+ to the production and degradation of inositol trisphosphate. [ABSTRACT FROM AUTHOR]

The skin is a complex organ that acts as a protective layer against the external environment. It protects the internal tissues from harmful agents, dehydration, ultraviolet radiation and physical injury as well as conferring thermoregulatory control, sensation, immunological surveillance and various biochemical functions. The diverse cell types that make up the skin include 1) keratinocytes, which form the bulk of the protective outer layer; 2) melanocytes, which protect the body from ultraviolet radiation by secreting the pigment melanin; and 3) cells that form the secretory appendages: eccrine and apocrine sweat glands, and the sebaceous gland. Emerging evidence suggests that store-operated Ca2+ entry (SOCE), whereby depletion of intracellular Ca2+ stores triggers Ca2+ influx across the plasma membrane, is central to the normal physiology of these cells and thus skin function. Numerous skin pathologies including dermatitis, anhidrotic ectodermal dysplasia, hyperhidrosis, hair loss and cancer are now linked to dysfunction in SOCE proteins. Principal amongst these are the stromal interaction molecules (STIMs) that sense Ca2+ depletion and Orai channels that mediate Ca2+ influx. In this review, the roles of STIM, Orai and other store-operated channels are discussed in the context of keratinocyte differentiation, melanogenesis, and eccrine sweat secretion. We explore not only STIM1-Orai1 as drivers of SOCE, but also independent actions of STIM, and emerging signal cascades stemming from their activities. Roles are discussed for the elusive transient receptor potential canonical channel (TRPC) complex in keratinocytes, Orai channels in Ca2+-cyclic AMP signal crosstalk in melanocytes, and Orai isoforms in eccrine sweat gland secretion.

The endoplasmic reticulum (ER) is the major intracellular calcium (Ca2+) storage compartment in eukaryotic cells. In most instances, the mobilization of Ca2+ from this store is followed by a delayed and sustained uptake of Ca2+ through Ca2+-permeable channels of the cell surface named store-operated Ca2+ channels (SOCCs). This gives rise to a store-operated Ca2+ entry (SOCE) that has been thoroughly investigated in electrically non-excitable cells where it is the principal regulated Ca2+ entry pathway. The existence of this Ca2+ route in neurons has long been a matter of debate. However, a growing body of experimental evidence indicates that the recruitment of Ca2+ from neuronal ER Ca2+ stores generates a SOCE. The present review summarizes the main studies supporting the presence of a depletion-dependent Ca2+ entry in neurons. It also addresses the question of the molecular composition of neuronal SOCCs, their expression, pharmacological properties, as well as their physiological relevance. [ABSTRACT FROM AUTHOR]

Loss of endoplasmic reticular (ER) Ca2+ activates store-operated Ca2+ entry (SOCE) by causing the ER localized Ca2+ sensor STIM to unfurl domains that activate Orai channels in the plasma membrane at membrane contact sites (MCS). Here, we demonstrate a novel mechanism by which the inositol 1,4,5 trisphosphate receptor (IP3R), an ER-localized IP3-gated Ca2+ channel, regulates neuronal SOCE. In human neurons, SOCE evoked by pharmacological depletion of ER-Ca2+ is attenuated by loss of IP3Rs, and restored by expression of IP3Rs even when they cannot release Ca2+, but only if the IP3Rs can bind IP3. Imaging studies demonstrate that IP3Rs enhance association of STIM1 with Orai1 in neuronal cells with empty stores; this requires an IP3-binding site, but not a pore. Convergent regulation by IP3Rs, may tune neuronal SOCE to respond selectively to receptors that generate IP3.

Store-operated Ca2+ entry (SOCE) is activated in response to the inositol-1,4,5-trisphosphate (InsP3)-dependent depletion of the endoplasmic reticulum (ER) Ca2+ store and represents a ubiquitous mode of Ca2+ influx. In vascular endothelial cells, SOCE regulates a plethora of functions that maintain cardiovascular homeostasis, such as angiogenesis, vascular tone, vascular permeability, platelet aggregation, and monocyte adhesion. The molecular mechanisms responsible for SOCE activation in vascular endothelial cells have engendered a long-lasting controversy. Traditionally, it has been assumed that the endothelial SOCE is mediated by two distinct ion channel signalplexes, i.e., STIM1/Orai1 and STIM1/Transient Receptor Potential Canonical 1(TRPC1)/TRPC4. However, recent evidence has shown that Orai1 can assemble with TRPC1 and TRPC4 to form a non-selective cation channel with intermediate electrophysiological features. Herein, we aim at bringing order to the distinct mechanisms that mediate endothelial SOCE in the vascular tree from multiple species (e.g., human, mouse, rat, and bovine). We propose that three distinct currents can mediate SOCE in vascular endothelial cells: (1) the Ca2+-selective Ca2+-release activated Ca2+ current (ICRAC), which is mediated by STIM1 and Orai1; (2) the store-operated non-selective current (ISOC), which is mediated by STIM1, TRPC1, and TRPC4; and (3) the moderately Ca2+-selective, ICRAC-like current, which is mediated by STIM1, TRPC1, TRPC4, and Orai1. [ABSTRACT FROM AUTHOR]

The changes in intracellular free calcium (Ca2+) levels are one of the most widely regulators of cell function; therefore, calcium as a universal intracellular mediator is involved in very important human diseases and disorders. In many cells, Ca2+ inflow is mediated by store-operated calcium channels, and it is recognized that the store-operated calcium entry (SOCE) is mediated by the two partners: the pore-forming proteins Orai (Orai1-3) and the calcium store sensor, stromal interaction molecule (STIM1-2). Importantly, the Orai/STIM channels are involved in crucial cell signalling processes such as growth factors, neurotransmitters, and cytokines via interaction with protein tyrosine kinase coupled receptors and G protein-coupled receptors. Therefore, in recent years, the issue of Orai/STIM channels as a drug target in human diseases has received considerable attention. This review summarizes and highlights our current knowledge of the Orai/STIM channels in human diseases and disorders, including immunodeficiency, myopathy, tubular aggregate, Stormorken syndrome, York platelet syndrome, cardiovascular and metabolic disorders, and cancers, as well as suggesting these channels as drug targets for pharmacological therapeutic intervention. Moreover, this work will also focus on the pharmacological modulators of Orai/STIM channel complexes. Together, our thoughtful of the biology and physiology of the Orai/STIM channels have grown remarkably during the past three decades, and the next important milestone in the field of store-operated calcium entry will be to identify potent and selective small molecules as a therapeutic agent with the purpose to target human diseases and disorders for patient benefit. [ABSTRACT FROM AUTHOR]

Moderate coolness is sensed by TRPM8 ion channels in peripheral sensory nerves, but the mechanism by which noxious cold is detected remains elusive. Here, we show that somatosensory and sympathetic neurons express two distinct mechanisms to detect noxious cold. In the first, inhibition by cold of a background outward current causes membrane depolarization that activates an inward current through voltage‐dependent calcium (CaV) channels. A second cold‐activated mechanism is independent of membrane voltage, is inhibited by blockers of ORAI ion channels and by downregulation of STIM1, and is recapitulated in HEK293 cells by co‐expression of ORAI1 and STIM1. Using total internal reflection fluorescence microscopy we found that cold causes STIM1 to aggregate with and activate ORAI1 ion channels, in a mechanism similar to that underlying store‐operated calcium entry (SOCE), but directly activated by cold and not by emptying of calcium stores. This novel mechanism may explain the phenomenon of cold‐induced vasodilation (CIVD), in which extreme cold increases blood flow in order to preserve the integrity of peripheral tissues. Synopsis: Extreme cold is not sensed via TRPM8 ion channels but by unclear alternative mechanisms. Here, we show that extreme cold causes STIM1‐mediated aggregation and activation of ORAI calcium channels in somatosensory and sympathetic neurons. Extreme cold (mean threshold 9–10°C) causes the ER‐resident membrane protein STIM1 to aggregate into puncta.STIM1 aggregation triggers aggregation of surface‐membrane calcium‐selective ORAI1 channels.The subsequent opening of ORAI1 channels causes a calcium influx without detectable membrane depolarization.Operation of this mechanism in sympathetic neurons may explain the phenomenon of cold‐induced vasodilation that increases blood flow in order to protect exposed tissues from frostbite. [ABSTRACT FROM AUTHOR]

Polymer membranes are conventionally prepared using high-energy particles from radioactive decay or by the bombardment of hundreds of MeVs energy ions. In both circumstances, tracks of damage are produced by particles/ions passing through the polymer, and successively, the damaged material is removed by chemical etching to create narrow pores. This process ensures nanosized pore diameter but with random placement, leading to non-uniform local pore density and low membrane porosity, which is necessary to reduce the risk of their overlapping. The present study is focused on the use of polyethylene terephthalate (PET) foils irradiated by 10.0 MeV carbon ions, easily achievable with ordinary ion accelerators. The ion irradiation conditions and the chemical etching conditions were monitored to obtain customized pore locations without pore overlapping in PET. The quality, shape, and size of the pores generated in the micromembranes can have a large impact on their applicability. In this view, the Scanning Transmission Ion Microscopy coupled with a computer code created in our laboratory was implemented to acquire new visual and quantitative insights on fabricated membranes. [ABSTRACT FROM AUTHOR]

Calcium is a major intracellular signaling messenger in innate immune cells. Similar to other immune cell subsets, the majority of calcium entry into innate immune cells is induced by cell surface receptors that stimulate store-operated calcium entry through calcium-release activated calcium (CRAC) channels. Since the molecular description of the STIM family of calcium sensors and the ORAI family of CRAC channel proteins, the majority of studies support a dominant role for these proteins in calcium signaling in innate cells. In reviewing the literature on CRAC channel function in innate cells, several general themes emerge. All innate cells express multiple members of the STIM and ORAI family members, however the ratio and relative contribution of individual isoforms changes depending on the cell type and activation state of the cell. It is evident that study of functional roles for STIM molecules is clearly ahead of studies of specific ORAI family members in all innate cell types, and that studies of CRAC channels in innate cells are not nearly as advanced as studies in lymphocytes. However, taken together, evidence from both STIM calcium sensors and ORAI channels in innate cells indicates that deficiency of STIM and ORAI proteins tends not to affect the development of any innate cell lineage, but certainly affects their function, in particular activation of the neutrophil oxidase and mast cell activation via IgE receptors. Furthermore, there are clearly hints that therapeutic targeting of CRAC channels in innate cells offers a new approach to various inflammatory and allergic diseases.

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Routinely, in membrane technology, the decay from radioactive particles or the bombardment of ions with MeV energy per nucleon have been employed for the production of narrow and long pores in membranes. Presently, the ion lithography is proposed to make the fabrication cost more affordable. It is prospective for the use of medium capacity accelerators making more feasible the fabrication of customized membranes. Thin polyethylene terephthalate foils have been patterned using 12 MeV O5+ ions and then processed to obtain good aspect ratio ion track pores in membranes. Pores of micrometric diameter with the following profiles were fabricated in the membranes: truncated cone, double conical, ideal cone, and cylindrical. Monitoring of the shape and size of pores has been attempted with a combination of Scanning Transmission Ion Microscope and a newly designed simulation program. This study is focused on the use of low-energy ions, accomplished in all laboratories, for the fabrication of membranes where the pores are not randomly traced and exhibit higher surface density and negligible overlapping than in membranes commonly manufactured. The good reproducibility and the ordered pore locations can be potentially utilized in applications such as microfluidics and organ-on-chip microsystems, where cells growing over porous substrates are used in simulation of biological barriers and transport processes. [ABSTRACT FROM AUTHOR]

The skin is a complex organ that acts as a protective layer against the external environment. It protects the internal tissues from harmful agents, dehydration, ultraviolet radiation and physical injury as well as conferring thermoregulatory control, sensation, immunological surveillance and various biochemical functions. The diverse cell types that make up the skin include 1) keratinocytes, which form the bulk of the protective outer layer; 2) melanocytes, which protect the body from ultraviolet radiation by secreting the pigment melanin; and 3) cells that form the secretory appendages: eccrine and apocrine sweat glands, and the sebaceous gland. Emerging evidence suggests that store-operated Ca2+ entry (SOCE), whereby depletion of intracellular Ca2+ stores triggers Ca2+ influx across the plasma membrane, is central to the normal physiology of these cells and thus skin function. Numerous skin pathologies including dermatitis, anhidrotic ectodermal dysplasia, hyperhidrosis, hair loss and cancer are now linked to dysfunction in SOCE proteins. Principal amongst these are the stromal interaction molecules (STIMs) that sense Ca2+ depletion and Orai channels that mediate Ca2+ influx. In this review, the roles of STIM, Orai and other store-operated channels are discussed in the context of keratinocyte differentiation, melanogenesis, and eccrine sweat secretion. We explore not only STIM1-Orai1 as drivers of SOCE, but also independent actions of STIM, and emerging signal cascades stemming from their activities. Roles are discussed for the elusive transient receptor potential canonical channel (TRPC) complex in keratinocytes, Orai channels in Ca2+-cyclic AMP signal crosstalk in melanocytes, and Orai isoforms in eccrine sweat gland secretion.

ER-PM junctions are membrane contact sites formed by the endoplasmic reticulum (ER) and plasma membrane (PM) in close apposition together. The formation and stability of these junctions are dependent on constitutive and dynamic enrichment of proteins, which either contribute to junctional stability or modulate the lipid levels of both ER and plasma membranes. The ER-PM junctions have come under much scrutiny recently as they serve as hubs for assembling the Ca2+ signaling complexes. This review summarizes: (1) key findings that underlie the abilities of STIM proteins to accumulate in ER-PM junctions; (2) the modulation of Orai/STIM complexes by other components found within the same junction; and (3) how Orai1 channel activation is coordinated and coupled with downstream signaling pathways. [ABSTRACT FROM AUTHOR]

Neurons are highly specialised cells that need to relay information over long distances and integrate signals from thousands of synaptic inputs. The complexity of neuronal function is evident in the morphology of their plasma membrane (PM), by far the most intricate of all cell types. Yet, within the neuron lies an organelle whose architecture adds another level to this morphological sophistication - the endoplasmic reticulum (ER). Neuronal ER is abundant in the cell body and extends to distant axonal terminals and postsynaptic dendritic spines. It also adopts specialised structures like the spine apparatus in the postsynapse and the cisternal organelle in the axon initial segment. At membrane contact sites (MCSs) between the ER and the PM, the two membranes come in close proximity to create hubs of lipid exchange and Ca2+ signalling called ER-PM junctions. The development of electron and light microscopy techniques extended our knowledge on the physiological relevance of ER-PM MCSs. Equally important was the identification of ER and PM partners that interact in these junctions, most notably the STIM-ORAI and VAP-Kv2.1 pairs. The physiological functions of ER-PM junctions in neurons are being increasingly explored, but their molecular composition and the role in the dynamics of Ca2+ signalling are less clear. This review aims to outline the current state of research on the topic of neuronal ER-PM contacts. Specifically, we will summarise the involvement of different classes of Ca2+ channels in these junctions, discuss their role in neuronal development and neuropathology and propose directions for further research., (© 2024 The Author(s).)

All human life starts with a calcium (Ca2+) wave. This ion regulates a plethora of cellular functions ranging from fertilisation and birth to development and cell death. A sophisticated system is responsible for maintaining the essential, tight concentration of calcium within cells. Intricate components of this Ca2+ network are store-operated calcium channels in the cells' membrane. The best-characterised store-operated channel is the Ca2+ release-activated Ca2+ (CRAC) channel. Currents through CRAC channels are critically dependent on the correct function of two proteins: STIM1 and Orai1. A disruption of the precise mechanism of Ca2+ entry through CRAC channels can lead to defects and in turn to severe impacts on our health. Mutations in either STIM1 or Orai1 proteins can have consequences on our immune cells, the cardiac and nervous system, the hormonal balance, muscle function, and many more. There is solid evidence that altered Ca2+ signalling through CRAC channels is involved in the hallmarks of cancer development: uncontrolled cell growth, resistance to cell death, migration, invasion, and metastasis. In this work we highlight the importance of Ca2+ and its role in human health and disease with focus on CRAC channels. [ABSTRACT FROM AUTHOR]

• Microglia respond to chemotactic signals from the CNS, exhibiting high dynamism. • iPSC-derived microglia (iMGL) show distance- and concentration-dependent migration towards purinergic cues. • ER- and SOCE Ca2+ signals are dispensable for iMGL directed migration upon purinergic stimulation. • iMGL chemotaxis to purinergic signals primarily relies on cyclic AMP. The central nervous system (CNS) is constantly surveilled by microglia, highly motile and dynamic cells deputed to act as the first line of immune defense in the brain and spinal cord. Alterations in the homeostasis of the CNS are detected by microglia that respond by extending their processes or – following major injuries – by migrating toward the affected area. Understanding the mechanisms controlling directed cell migration of microglia is crucial to dissect their responses to neuroinflammation and injury. We used a combination of pharmacological and genetic approaches to explore the involvement of calcium (Ca2+) signaling in the directed migration of human induced pluripotent stem cell (iPSC)-derived microglia challenged with a purinergic stimulus. This approach mimics cues originating from injury of the CNS. Unexpectedly, simultaneous imaging of microglia migration and intracellular Ca2+ changes revealed that this phenomenon does not require Ca2+ signals generated from the endoplasmic reticulum (ER) and store-operated Ca2+ entry (SOCE) pathways. Instead, we find evidence that human microglial chemotaxis to purinergic signals is mediated by cyclic AMP in a Ca2+-independent manner. These results challenge prevailing notions, with important implications in neurological conditions characterized by perturbation in Ca2+ homeostasis. [Display omitted] [ABSTRACT FROM AUTHOR]

Tight spatiotemporal regulation of intracellular Ca2+ plays a critical role in regulating diverse cellular functions including cell survival, metabolism, and transcription. As a result, eukaryotic cells have developed a wide variety of mechanisms for controlling Ca2+ influx and efflux across the plasma membrane as well as Ca2+ release and uptake from intracellular stores. The STIM and Orai protein families comprising of STIM1, STIM2, Orai1, Orai2, and Orai3, are evolutionarily highly conserved proteins that are core components of all mammalian Ca2+ signaling systems. STIM1 and Orai1 are considered key players in the regulation of Store Operated Calcium Entry (SOCE), where release of Ca2+ from intracellular stores such as the Endoplasmic/Sarcoplasmic reticulum (ER/SR) triggers Ca2+ influx across the plasma membrane. SOCE, which has been widely characterized in non-excitable cells, plays a central role in Ca2+-dependent transcriptional regulation. In addition to their role in Ca2+ signaling, STIM1 and Orai1 have been shown to contribute to the regulation of metabolism and mitochondrial function. STIM and Orai proteins are also subject to redox modifications, which influence their activities. Considering their ubiquitous expression, there has been increasing interest in the roles of STIM and Orai proteins in excitable cells such as neurons and myocytes. While controversy remains as to the importance of SOCE in excitable cells, STIM1 and Orai1 are essential for cellular homeostasis and their disruption is linked to various diseases associated with aging such as cardiovascular disease and neurodegeneration. The recent identification of splice variants for most STIM and Orai isoforms while complicating our understanding of their function, may also provide insight into some of the current contradictions on their roles. Therefore, the goal of this review is to describe our current understanding of the molecular regulation of STIM and Orai proteins and their roles in normal physiology and diseases of aging, with a particular focus on heart disease and neurodegeneration.

Store-operated Ca2+ entry (SOCE) is activated in response to the inositol-1,4,5-trisphosphate (InsP3)-dependent depletion of the endoplasmic reticulum (ER) Ca2+ store and represents a ubiquitous mode of Ca2+ influx. In vascular endothelial cells, SOCE regulates a plethora of functions that maintain cardiovascular homeostasis, such as angiogenesis, vascular tone, vascular permeability, platelet aggregation, and monocyte adhesion. The molecular mechanisms responsible for SOCE activation in vascular endothelial cells have engendered a long-lasting controversy. Traditionally, it has been assumed that the endothelial SOCE is mediated by two distinct ion channel signalplexes, i.e., STIM1/Orai1 and STIM1/Transient Receptor Potential Canonical 1(TRPC1)/TRPC4. However, recent evidence has shown that Orai1 can assemble with TRPC1 and TRPC4 to form a non-selective cation channel with intermediate electrophysiological features. Herein, we aim at bringing order to the distinct mechanisms that mediate endothelial SOCE in the vascular tree from multiple species (e.g., human, mouse, rat, and bovine). We propose that three distinct currents can mediate SOCE in vascular endothelial cells: (1) the Ca2+-selective Ca2+-release activated Ca2+ current (ICRAC), which is mediated by STIM1 and Orai1; (2) the store-operated non-selective current (ISOC), which is mediated by STIM1, TRPC1, and TRPC4; and (3) the moderately Ca2+-selective, ICRAC-like current, which is mediated by STIM1, TRPC1, TRPC4, and Orai1.

Polymer membranes are conventionally prepared using high-energy particles from radioactive decay or by the bombardment of hundreds of MeVs energy ions. In both circumstances, tracks of damage are produced by particles/ions passing through the polymer, and successively, the damaged material is removed by chemical etching to create narrow pores. This process ensures nanosized pore diameter but with random placement, leading to non-uniform local pore density and low membrane porosity, which is necessary to reduce the risk of their overlapping. The present study is focused on the use of polyethylene terephthalate (PET) foils irradiated by 10.0 MeV carbon ions, easily achievable with ordinary ion accelerators. The ion irradiation conditions and the chemical etching conditions were monitored to obtain customized pore locations without pore overlapping in PET. The quality, shape, and size of the pores generated in the micromembranes can have a large impact on their applicability. In this view, the Scanning Transmission Ion Microscopy coupled with a computer code created in our laboratory was implemented to acquire new visual and quantitative insights on fabricated membranes.

The changes in intracellular free calcium (Ca2+) levels are one of the most widely regulators of cell function; therefore, calcium as a universal intracellular mediator is involved in very important human diseases and disorders. In many cells, Ca2+ inflow is mediated by store-operated calcium channels, and it is recognized that the store-operated calcium entry (SOCE) is mediated by the two partners: the pore-forming proteins Orai (Orai1-3) and the calcium store sensor, stromal interaction molecule (STIM1-2). Importantly, the Orai/STIM channels are involved in crucial cell signalling processes such as growth factors, neurotransmitters, and cytokines via interaction with protein tyrosine kinase coupled receptors and G protein-coupled receptors. Therefore, in recent years, the issue of Orai/STIM channels as a drug target in human diseases has received considerable attention. This review summarizes and highlights our current knowledge of the Orai/STIM channels in human diseases and disorders, including immunodeficiency, myopathy, tubular aggregate, Stormorken syndrome, York platelet syndrome, cardiovascular and metabolic disorders, and cancers, as well as suggesting these channels as drug targets for pharmacological therapeutic intervention. Moreover, this work will also focus on the pharmacological modulators of Orai/STIM channel complexes. Together, our thoughtful of the biology and physiology of the Orai/STIM channels have grown remarkably during the past three decades, and the next important milestone in the field of store-operated calcium entry will be to identify potent and selective small molecules as a therapeutic agent with the purpose to target human diseases and disorders for patient benefit.

Store-operated calcium entry through calcium release-activated calcium (CRAC) channels replenishes intracellular calcium stores and plays a critical role in cellular calcium signaling. CRAC channels are activated by tightly regulated interaction between the endoplasmic reticulum (ER) calcium sensor STIM proteins and plasma membrane (PM) Orai channels. Our current understanding of the role of STIM-Orai-dependent calcium signals under physiologically relevant conditions remains limited in part due to a lack of spatiotemporally precise methods for direct manipulation of endogenous CRAC channels. Here, we report the synthesis and characterization of azoboronate light-operated CRAC channel inhibitors (LOCIs) that allow for a dynamic and fully reversible remote modulation of the function of native CRAC channels using ultraviolet (UV) and visible light. We demonstrate the use of LOCI-1 to modulate gene expression in T lymphocytes, cancer cell seeding at metastatic sites, and pain-related behavior. [ABSTRACT FROM AUTHOR]

Store-operated Ca2+ entry (SOCE) is a ubiquitous Ca2+ signaling pathway that is evolutionarily conserved across eukaryotes. SOCE is triggered physiologically when the endoplasmic reticulum (ER) Ca2+ stores are emptied through activation of inositol 1,4,5-trisphosphate receptors. SOCE is mediated by the Ca2+ release-activated Ca2+ (CRAC) channels, which are highly Ca2+ selective. Upon store depletion, the ER Ca2+-sensing STIM proteins aggregate and gain extended conformations spanning the ER–plasma membrane junctional space to bind and activate Orai, the pore-forming proteins of hexameric CRAC channels. In recent years, studies on STIM and Orai tissue-specific knockout mice and gain- and loss-of-function mutations in humans have shed light on the physiological functions of SOCE in various tissues. Here, we describe recent findings on the composition of native CRAC channels and their physiological functions in immune, muscle, secretory, and neuronal systems to draw lessons from transgenic mice and human diseases caused by altered CRAC channel activity. [ABSTRACT FROM AUTHOR]

Heart failure is often preceded by pathological cardiac hypertrophy, a thickening of the heart musculature driven by complex gene regulatory and signaling processes. The Drosophila heart has great potential as a genetic model for deciphering the underlying mechanisms of cardiac hypertrophy. However, current methods for evaluating hypertrophy of the Drosophila heart are laborious and difficult to carry out reproducibly. Here, we demonstrate that microcomputerized tomography (microCT) is an accessible, highly reproducible method for nondestructive, quantitative analysis of Drosophila heart morphology and size. To validate our microCT approach for analyzing Drosophila cardiac hypertrophy, we show that expression of constitutively active Ras (Ras85DV12), previously shown to cause hypertrophy of the fly heart, results in significant thickening of both adult and larval heart walls when measured from microCT images. We then show using microCT analysis that genetic upregulation of store-operated Ca2+ entry (SOCE) driven by expression of constitutively active Stim (StimCA) or Orai (OraiCA) proteins also results in significant hypertrophy of the Drosophila heart, through a process that specifically depends on Orai Ca2+ influx channels. Intravital imaging of heart contractility revealed significantly reduced end-diastolic and end-systolic dimensions in StimCA- and OraiCA-expressing hearts, consistent with the hypertrophic phenotype. These results demonstrate that increased SOCE activity is an important driver of hypertrophic cardiomyocyte growth, and demonstrate how microCT analysis combined with tractable genetic tools in Drosophila can be used to delineate molecular signaling processes that underlie cardiac hypertrophy and heart failure. NEW & NOTEWORTHY Genetic analysis of Drosophila cardiac hypertrophy holds immense potential for the discovery of new therapeutic targets to prevent and treat heart failure. This potential has been hindered by a lack of rapid and effective methods for analyzing heart size in flies. Here, we demonstrate that analysis of the Drosophila heart with microcomputerized tomography yields accurate and highly reproducible heart size measurements that can be used to analyze heart growth and cardiac hypertrophy in Drosophila. [ABSTRACT FROM AUTHOR]

Introduction. Functional gastrointestinal disorders (FGID) occupy one of the leading positions among intestinal pathologies. At present, scientific data indicate the lack of efficiency of existing methods of FGID treatment and the need for further research. This study is devoted to the assessment of the impact of STIM Lax and STIM treatment on clinical manifestations in patients with FC and FD.Aim of the study. Evaluation of the effectiveness of biologically active additives (BAA) STIM and STIM Laks in 39 patients with functional gastrointestinal disorders. Objectives of the study: to evaluate the effect of the drug on clinical symptoms according to an individual diary and using specialized questionnaires with a score of symptoms before and after treatment.Materials of the research. 39 patients divided into two groups were admitted to the research. Of these, 20 patients were with FC (4 men and 16 women; mean age – 40.3 ± 8.9 years). Monotherapy with STIM Laks was conducted (1 tablet 3 times a day for 30 days), and 19 FD patients (10 men and 9 women; mean age – 36.9 ± 14.1 years). Before and after the course of treatment a carbolene sample was conducted, stool form and frequency, intensity of flatulence, purring, transfusion, abdominal pain were clinically evaluated.Results. Therapy of FС and FD with STIM and STIM Laks effectively reduces the frequency and intensity of clinical symptoms, normalizes the transit time of the carbolene sample, the frequency of stool. Increase of flatulence was observed in 7 (17.9%) patients, its resolution within 1–3 days led to a decrease in the dose of the drug, without affecting the end result.Conclusions. This study indicates a high efficacy and good tolerability of treatment with STIM Laks and STIM in patients with FGID.

The central nervous system (CNS) is constantly surveilled by microglia, highly motile and dynamic cells deputed to act as the first line of immune defense in the brain and spinal cord. Alterations in the homeostasis of the CNS are detected by microglia that respond by extending their processes or - following major injuries - by migrating toward the affected area. Understanding the mechanisms controlling directed cell migration of microglia is crucial to dissect their responses to neuroinflammation and injury. We used a combination of pharmacological and genetic approaches to explore the involvement of calcium (Ca 2+ ) signaling in the directed migration of human induced pluripotent stem cell (iPSC)-derived microglia challenged with a purinergic stimulus. This approach mimics cues originating from injury of the CNS. Unexpectedly, simultaneous imaging of microglia migration and intracellular Ca 2+ changes revealed that this phenomenon does not require Ca 2+ signals generated from the endoplasmic reticulum (ER) and store-operated Ca 2+ entry (SOCE) pathways. Instead, we find evidence that human microglial chemotaxis to purinergic signals is mediated by cyclic AMP in a Ca 2+ -independent manner. These results challenge prevailing notions, with important implications in neurological conditions characterized by perturbation in Ca 2+ homeostasis., Competing Interests: Declaration of competing interest M.B.-J. is a co-founder of NovoGlia Inc. I.F.S. is a consultant for, and holds stock options in NeuroQure, Inc. All other authors declare no conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Orai1 comprises the pore-forming subunit of the Ca(2+) release-activated Ca(2+) (CRAC) channel. When bound and activated by stromal interacting molecule 1 (STIM1), an endoplasmic reticulum (ER)-resident calcium sensor, Orai1 channels possess high selectivity for calcium but extremely small conductance that has precluded direct recording of single-channel currents. We have developed an approach to visualize Orai1 activity by fusing Orai1 to fluorescent, genetically encoded calcium indicators (GECIs). The GECI-Orai1 probes reveal local Ca(2+) influx at STIM1-Orai1 puncta. By whole cell recording, these fusions are fully functional as CRAC channels. When GECI-Orai1 and the CRAC-activating domain (CAD) of STIM1 were coexpressed at low levels and imaged using a total internal reflectance fluorescence microscope, cells exhibited sporadic fluorescence transients the size of diffraction-limited spots and the brightness of a few activated GECI proteins. Transients typically rose rapidly and fell into two classes according to duration: briefer "flickers" lasting only a few hundred milliseconds, and longer "pulses" lasting one to several seconds. The size, intensity, trace shape, frequency, distribution, physiological characteristics, and association with CAD binding together demonstrate that GECI-Orai1 fluorescence transients correspond to single-channel Orai1 responses. Single Orai1 channels gated by CAD, and small Orai1 puncta gated by STIM1, exhibit repetitive fluctuations in single-channel output. CAD binding supports a role in open state maintenance and reveals a second phase of CAD/STIM1 binding after channel opening. These first recordings of single-channel Orai1 currents reveal unexpected dynamics, and when paired with CAD association, support multiple single-channel states.

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 SOCEassociated 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. [ABSTRACT FROM AUTHOR]

Ryanodine receptors (RyRs) are intracellular Ca2+ release channels ubiquitously expressed in various cell types. RyRs were extensively studied in striated muscle cells due to their crucial role in muscle contraction. In contrast, the role of RyRs in Ca2+ signalling and functions in non‐excitable cells, such as T lymphocytes, remains poorly understood. Expression of different isoforms of RyRs was shown in primary T cells and T cell lines. In T cells, RyRs co‐localize with the plasmalemmal store‐operated Ca2+ channels of the Orai family and endoplasmic reticulum Ca2+ sensing Stim family proteins and are activated by store‐operated Ca2+ entry and pyridine nucleotide metabolites, the intracellular second messengers generated upon stimulation of T cell receptors. Experimental data indicate that together with d‐myo‐inositol 1,4,5‐trisphosphate receptors, RyRs regulate intercellular Ca2+ dynamics by controlling Ca2+ concentration within the lumen of the endoplasmic reticulum and, consequently, store‐operated Ca2+ entry. Gain‐of‐function mutations, genetic deletion or pharmacological inhibition of RyRs alters T cell Ca2+ signalling and effector functions. The picture emerging from the collective data shows that RyRs are the essential regulators of T cell Ca2+ signalling and can be potentially used as molecular targets for immunomodulation or T cell‐based diagnostics of the disorders associated with RyRs dysregulation. [ABSTRACT FROM AUTHOR]

Abstract Background Openers of mitochondrial adenosine triphosphate-dependent potassium (mKATP) channels like diazoxide increase reactive oxygen species (ROS) production in cardiac cells and reduce Ca2+ elevations produced by ischemia–reperfusion, protecting the heart from damage. In this study we tested the hypothesis that opening mKATP channels regulates expression of the major components of store-operated Ca2+ entry (SOCE) STIM1 and Orai1. Results Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blot experiments showed that diazoxide increased expression of STIM1 and Orai1 at the mRNA and protein levels, respectively, in adult rat cardiomyocytes. Immunofluorescence analyses revealed that diazoxide also disrupted the striated distribution pattern of STIM1. These effects were prevented by the ROS scavenger N-acetyl cysteine (NAC), the mKATP channel antagonist 5-hydroxydecanoate (5-HD), or the protein synthesis inhibitor cycloheximide (CHX). Confocal microscopy revealed that diazoxide also led to nuclear translocation of the transcription factors c-Fos and NFκB, which was also blocked by NAC or 5-HD. Finally, the MAPK pathway inhibitor UO126 attenuated diazoxide-induced upregulation of STIM1 and Orai1 expression. Conclusions Our results suggest that opening mitochondrial potassium ATP channels with diazoxide upregulates the expression of STIM1 and Orai1 by de novo synthesis by a mechanism that involves NFkB, c-Fos, and ROS via MAPK/ERK signaling.

The solute carrier family 10 member SLC10A7 is a negative regulator of intracellular calcium signaling (RCAS). In cell culture, SLC10A7 expression is negatively correlated with store-operated calcium entry (SOCE) via the plasma membrane. SLC10A7-deficient cells have significantly increased calcium influx after treatment with thapsigargin for depletion of ER calcium stores, whereas SLC10A7/RCAS overexpression limits calcium influx. Genetic variants in the human SLC10A7 gene are associated with skeletal dysplasia and amelogenesis imperfecta and reveal loss of function on cellular calcium influx. More recently, an additional disease-related genetic variant (P303L) as well as some novel genetic variants (V235F, T221M, I136M, L210F, P285L, and G146S) have been identified. In the present study, these variants were expressed in HEK293 cells to study their subcellular localization and their effect on cellular calcium influx. All variants were properly sorted to the ER compartment and closely co-localized with the STIM protein, a functional component of SOCE. The variants P303L and L210F showed significantly reduced effects on cellular calcium influx compared to the wild type but still maintained some degree of residual activity. This might explain the milder phenotype of patients bearing the P303L variant and might indicate disease potential for the newly identified L210F variant. In contrast, all other variants behaved like the wild type. In conclusion, the occurrence of variants in the SLC10A7 gene should be considered in patients with skeletal dysplasia and amelogenesis imperfecta. In addition to the already established variants, the present study identifies another potential disease-related SLC10A7/RCAS variant, namely, L210F, which seems to be most frequent in South Asian populations.

Routinely, in membrane technology, the decay from radioactive particles or the bombardment of ions with MeV energy per nucleon have been employed for the production of narrow and long pores in membranes. Presently, the ion lithography is proposed to make the fabrication cost more affordable. It is prospective for the use of medium capacity accelerators making more feasible the fabrication of customized membranes. Thin polyethylene terephthalate foils have been patterned using 12 MeV O5+ ions and then processed to obtain good aspect ratio ion track pores in membranes. Pores of micrometric diameter with the following profiles were fabricated in the membranes: truncated cone, double conical, ideal cone, and cylindrical. Monitoring of the shape and size of pores has been attempted with a combination of Scanning Transmission Ion Microscope and a newly designed simulation program. This study is focused on the use of low-energy ions, accomplished in all laboratories, for the fabrication of membranes where the pores are not randomly traced and exhibit higher surface density and negligible overlapping than in membranes commonly manufactured. The good reproducibility and the ordered pore locations can be potentially utilized in applications such as microfluidics and organ-on-chip microsystems, where cells growing over porous substrates are used in simulation of biological barriers and transport processes.

Tumor metastasis is the primary cause of treatment failure and cancer-related deaths. Store-operated Ca2+ entry (SOCE), which is mediated by stromal interaction molecules (STIM) and ORAI proteins, has been implicated in the tumor invasion-metastasis cascade. Epithelial-mesenchymal transition (EMT) is a cellular program that enables tumor cells to acquire the capacities needed for migration and invasion and the formation of distal metastases. Tumor-associated angiogenesis contributes to metastasis because aberrantly developed vessels offer a path for tumor cell dissemination as well as supply sufficient nutrients for the metastatic colony to develop into metastasis. Recently, increasing evidence has indicated that SOCE alterations actively participate in the multi-step process of tumor metastasis. In addition, the dysregulated expression of STIM/ORAI has been reported to be a predictor of poor prognosis. Herein, we review the latest advances about the critical role of SOCE in the tumor metastasis cascade and the underlying regulatory mechanisms. We emphasize the contributions of SOCE to the EMT program, tumor cell migration and invasion, and angiogenesis. We further discuss the possibility of modulating SOCE or intervening in the downstream signaling pathways as a feasible targeting therapy for cancer treatment. [ABSTRACT FROM AUTHOR]

Ion channels and transporters mediate the transport of charged ions across hydrophobic lipid membranes. In immune cells, divalent cations such as calcium, magnesium, and zinc have important roles as second messengers to regulate intracellular signaling pathways. By contrast, monovalent cations such as sodium and potassium mainly regulate the membrane potential, which indirectly controls the influx of calcium and immune cell signaling. Studies investigating human patients with mutations in ion channels and transporters, analysis of gene-targeted mice, or pharmacological experiments with ion channel inhibitors have revealed important roles of ionic signals in lymphocyte development and in innate and adaptive immune responses. We here review the mechanisms underlying the function of ion channels and transporters in lymphocytes and innate immune cells and discuss their roles in lymphocyte development, adaptive and innate immune responses, and autoimmunity, as well as recent efforts to develop pharmacological inhibitors of ion channels for immunomodulatory therapy.

ER-PM junctions are membrane contact sites formed by the endoplasmic reticulum (ER) and plasma membrane (PM) in close apposition together. The formation and stability of these junctions are dependent on constitutive and dynamic enrichment of proteins, which either contribute to junctional stability or modulate the lipid levels of both ER and plasma membranes. The ER-PM junctions have come under much scrutiny recently as they serve as hubs for assembling the Ca2+ signaling complexes. This review summarizes: (1) key findings that underlie the abilities of STIM proteins to accumulate in ER-PM junctions; (2) the modulation of Orai/STIM complexes by other components found within the same junction; and (3) how Orai1 channel activation is coordinated and coupled with downstream signaling pathways.

What is known and objective: RP3128, a novel, orally available modulator of calcium released activated calcium (CRAC) channel, is being developed for the potential treatment of autoimmune and inflammatory diseases. RP3128 showed nano‐molar potency and activity in a range of in vitro and in vivo models of inflammation. We report a first‐in‐human study investigating the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of RP3128 in healthy subjects. Methods: A randomized, double‐blind, placebo‐controlled trial of single (25, 50, 100, 200 and 400 mg) and multiple (7 days: 25, 100 and 400 mg once daily) doses of RP3128 were performed. Thirty‐two and 24 subjects were randomized in the single ascending dose (SAD) and multiple ascending dose (MAD) parts, respectively. Results and discussion: RP3128 was well tolerated, with no dose‐limiting toxicity at single and multiple doses. Incidence of treatment emergent adverse events (TEAEs) did not increase with ascending RP3128 doses. No changes were seen in cognitive function and ECG parameters. RP3128 was rapidly absorbed. Elimination was slow with a half‐life of more than 80 h. Exposures increased with increasing doses. Accumulation was seen on repeated dosing. PD response, as evidenced by lower plasma levels of tumour necrosis factor‐alfa (TNFα) and interleukin‐4 (IL‐4), was seen when compared to pre‐dose values or placebo. What is new and conclusion: The safety, tolerability and PK/PD profile of RP3128 demonstrates its potential to be developed in inflammatory disorders and support further clinical development (ClinicalTrials.gov number: NCT02958982). [ABSTRACT FROM AUTHOR]

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All human life starts with a calcium (Ca2+) wave. This ion regulates a plethora of cellular functions ranging from fertilisation and birth to development and cell death. A sophisticated system is responsible for maintaining the essential, tight concentration of calcium within cells. Intricate components of this Ca2+ network are store-operated calcium channels in the cells’ membrane. The best-characterised store-operated channel is the Ca2+ release-activated Ca2+ (CRAC) channel. Currents through CRAC channels are critically dependent on the correct function of two proteins: STIM1 and Orai1. A disruption of the precise mechanism of Ca2+ entry through CRAC channels can lead to defects and in turn to severe impacts on our health. Mutations in either STIM1 or Orai1 proteins can have consequences on our immune cells, the cardiac and nervous system, the hormonal balance, muscle function, and many more. There is solid evidence that altered Ca2+ signalling through CRAC channels is involved in the hallmarks of cancer development: uncontrolled cell growth, resistance to cell death, migration, invasion, and metastasis. In this work we highlight the importance of Ca2+ and its role in human health and disease with focus on CRAC channels.

L’intégration de disciplines scolaires telles que les sciences et la mathématique (S-M) permet de favoriser l’intérêt des élèves et le sentiment d’efficacité personnelle (SEP). Cependant, peu de questionnaires semblent avoir été développés pour mesurer ces variables perceptuelles dans ce type de contexte. Il ne semble pas non plus en exister qui soient adaptés pour les élèves du primaire au sein d’écoles défavorisées et multiculturelles. Cet article rapporte les résultats du processus de validation d’un tel questionnaire comprenant six échelles auprès de 1553 élèves de 5e ou 6e année dans des écoles de la région de Montréal, au Canada. Dans sa version finale, le Questionnaire sur l’intégration de la mathématique et des sciences (QIMS) permet de mesurer six variables perceptuelles : le SEP en sciences, le SEP en mathématique, l’intérêt pour les sciences, l’intérêt pour la mathématique, l’intérêt pour les activités intégrées S-M ainsi que la perception du lien entre sciences et mathématique. À notre connaissance, les deux dernières variables n’ont jamais été mesurées et les échelles proposées pour les mesurer sont inédites.

The integration of academic disciplines such as science and mathematics (M&S) can help foster students’ interest and sense of self-efficacy (S-E). However, few questionnaires appear to have been developed to measure these perceptual variables in this type of context. Nor do any appear to exist that are suitable for elementary students from low-income and multicultural schools. This article reports the results of the validation process of such a questionnaire comprising six scales conducted among 1,553 Grade 5 or 6 students from schools in Canada’s Montréal area. In its final version, the Science and Mathematics Integration Questionnaire (SMIQ) measures six perceptual variables: S-E in science, S-E in mathematics, interest in science, interest in mathematics, interest in integrated M&S activities and perception of the links between science and mathematics. To our knowledge, the last two variables have never been measured and the proposed scales to measure them are new.

There are growing indications for the involvement of calcium stores in the plastic properties of neurons and particularly in dendritic spines of central neurons. The store-operated calcium entry (SOCE) channels are assumed to be activated by the calcium sensor stromal interaction molecule (STIM)which leads to activation of its associated Orai channel. There are two STIM species, and the differential role of the two in SOCE is not entirely clear. In the present study, we were able to distinguish between transfected STIM1, which is more mobile primarily in young neurons, and STIM2 which is less mobile and more prominent in older neurons in culture. STIM1 mobility is associated with spontaneous calcium sparks, local transient rise in cytosolic [Ca2+]i, and in the formation and elongation of dendritic filopodia/spines. In contrast, STIM2 is associated with older neurons, where it is mobile and moves into dendritic spines primarily when cytosolic [Ca2+]i levels are reduced, apparently to activate resident Orai channels. These results highlight a role for STIM1 in the regulation of [Ca2+]i fluctuations associated with the formation of dendritic spines or filopodia in the developing neuron, whereas STIM2 is associated with the maintenance of calcium entry into stores in the adult neuron.