Arriving at the next level of complexity in IP3R and SOCE signaling.

The endoplasmic reticulum (ER) has long been recognized as the master regulator of cellular Ca2+ signaling. In this context, IP3R channels may be envisioned as this conductor's baton, which enables virtuous orchestration of cellular Ca2+ signaling tunes. IP3Rs serve the generation of spatiotemporally defined Ca2+ changes and are key for the ER´s function as an autonomous Ca2+ signaling unit, which is able to govern its own refilling from the extracellular Ca2+ pool. As yet, IP3R signaling has been primarily attributed to its precisely-tunable Ca2+ channel function and IP3-mediated control over Ca2+ levels within signaling domains. A recent report from the Hasan laboratory [1] provides evidence for an as yet overlooked function of IP3R1 in terms of supporting STIM/Orai-mediated SOCE in neurons. IP3R1 is demonstrated to remarkably facilitate productive STIM-Orai interactions and SOCE by a process that is triggered by IP3 but independent of the receptors' function as an ER Ca2+ channel.

Excitatory cholinergic responses in mouse primary bronchial smooth muscle require both Ca2+ entry via l-type Ca2+ channels and store operated Ca2+ entry via Orai channels.

Malfunctions in airway smooth muscle Ca2+-signalling leads to airway hyperresponsiveness in asthma and chronic obstructive pulmonary disease. Ca2+-release from intracellular stores is important in mediating agonist-induced contractions, but the role of influx via l-type Ca2+ channels is controversial. We re-examined roles of the sarcoplasmic reticulum Ca2+ store, refilling of this store via store-operated Ca2+ entry (SOCE) and l-type Ca2+ channel pathways on carbachol (CCh, 0.1-10 µM)-induced contractions of mouse bronchial rings and intracellular Ca2+ signals of mouse bronchial myocytes. In tension experiments, the ryanodine receptor (RyR) blocker dantrolene (100 µM) reduced CCh-responses at all concentrations, with greater effects on sustained rather than initial components of contraction. 2-Aminoethoxydiphenyl borate (2-APB, 100 µM), in the presence of dantrolene, abolished CCh-responses, suggesting the sarcoplasmic reticulum Ca2+ store is essential for contraction. The SOCE blocker GSK-7975A (10 µM) reduced CCh-contractions, with greater effects at higher (e.g. 3 and 10 µM) CCh concentrations. Nifedipine (1 µM), abolished remaining contractions in GSK-7975A (10 µM). A similar pattern was observed on intracellular Ca2+-responses to 0.3 µM CCh, where GSK-7975A (10 µM) substantially reduced Ca2+ transients induced by CCh, and nifedipine (1 µM) abolished remaining responses. When nifedipine (1 µM) was applied alone it had less effect, reducing tension responses at all CCh concentrations by 25% - 50%, with greater effects at lower (e.g. 0.1 and 0.3 µM) CCh concentrations. When nifedipine (1 µM) was examined on the intracellular Ca2+-response to 0.3 µM CCh, it only modestly reduced Ca2+ signals, while GSK-7975A (10 µM) abolished remaining responses. In conclusion, Ca2+-influx from both SOCE and l-type Ca2+ channels contribute to excitatory cholinergic responses in mouse bronchi. The contribution of l-type Ca2+ channels was especially pronounced at lower doses of CCh, or when SOCE was blocked. This suggests l-type Ca2+ channels might be a potential target for bronchoconstriction under certain circumstances.

[Mechanism of total flavonoids of Rhododendra simsii in alleviating ischemic brain injury].

This study explores the effect of total flavonoids of Rhododendra simsii(TFR) on middle cerebral artery occlusion(MCAO)-induced cerebral injury in rats and oxygen-glucose deprivation/reoxygenation(OGD/R) injury in PC12 cells and the underlying mechanism. The MCAO method was used to induce focal ischemic cerebral injury in rats. Male SD rats were randomized into sham group, model group, and TFR group. After MCAO, TFR(60 mg·kg~(-1)) was administered for 3 days. The content of tumor necrosis factor-α(TNF-α), interleukin-1(IL-1), and interleukin-6(IL-6) in serum was detected by enzyme-linked immunosorbent assay(ELISA). The pathological changes of brain tissue and cerebral infarction were observed based on hematoxylin and eosin(HE) staining and 2,3,5-triphenyltetrazolium chloride(TTC) staining. RT-qPCR and Western blot were used to detect the mRNA and protein levels of calcium release-activated calcium channel modulator 1(ORAI1), stromal interaction molecule 1(STIM1), stromal intera-ction molecule 2(STIM2), protein kinase B(PKB), and cysteinyl aspartate specific proteinase 3(caspase-3) in brain tissues. The OGD/R method was employed to induce injury in PC12 cells. Cells were randomized into the normal group, model group, gene silencing group, TFR(30 µg·mL~(-1)) group, and TFR(30 µg·mL~(-1))+gene overexpression plasmid group. Intracellular Ca~(2+) concentration and apoptosis rate of PC12 cells were measured by laser scanning confocal microscopy and flow cytometry. The effect of STIM-ORAI-regulated store-operated calcium entry(SOCE) pathway on TFR was explored based on gene silencing and gene overexpression techniques. The results showed that TFR significantly alleviated the histopathological damage of brains in MCAO rats after 3 days of admini-stration, reduced the contents of TNF-α, IL-1, and IL-6 in the serum, down-regulated the expression of ORAI1, STIM1, STIM2, and caspase-3 genes, and up-regulated the expression of PKB gene in brain tissues of MCAO rats. TFR significantly decreased OGD/R induced Ca~(2+) overload and apoptosis in PC12 cells. However, it induced TFR-like effect by ORAI1, STIM1 and STIM2 genes silencing. However, overexpression of these genes significantly blocked the effect of TFR in reducing Ca~(2+) overload and apoptosis in PC12 cells. In summary, in the early stage of focal cerebral ischemia-reperfusion injury and OGD/R-induced injury in PC12 cells TFR attenuates ischemic brain injury by inhibiting the STIM-ORAI-regulated SOCE pathway and reducing Ca~(2+) overload and inflammatory factor expression, and apoptosis.

Endoplasmic reticulum-Phagosome contact sites from the cradle to the grave.

Phagocytosis is a key component of the innate immune system used to ingest apoptotic cells and microorganisms for their destruction and recycling of macromolecules and the presentation of antigens to adaptive immune system cells. The newly formed vacuole or nascent phagosome undergoes a maturation process reminiscent of the classical endocytic maturation process, reaching a highly degradative phagolysosome stage before its tubulovesicular breakdown into lysosomes. The process is highly regulated and can be disrupted by various pathogenic organisms. The exchange of proteins, lipids, and other metabolites between organelles, including maturing phagosomes, is enabled by two processes, vesicular and non-vesicular transport at membrane contact sites (MCS). For decades the specific role(s) of the endoplasmic reticulum (ER) in phagocytosis has been the subject of much debate. In parallel, the last two decades have seen a burst in research on the numerous roles of ER contact sites and resident proteins in all aspects of organelle biology. Here, in this minireview, we describe ER-phagosome contact sites' functions from the early stages of particle engulfment to the phagolysosome dissolution into lysosomes. We also discuss several aspects of ER-phagosome contact sites that remain to be explored.

Effects of Inhalation of STIM-Orai Antagonist SKF 96365 on Ovalbumin-Induced Airway Remodeling in Guinea Pigs.

Airway remodeling (AR) consists of wall thickening and hyperreactivity. STIM (stromal interaction molecule) and Orai protein pathways mediate extracellular Ca2+ signals involved in AR. This study aims to define the effects on AR of the STIM-Orai antagonist SKF 96365 given by inhalation in three increasing doses in ovalbumin-induced AR. In the control group, the antiasthmatic budesonide and salbutamol were given in the same model. The airway structure was evaluated by histological and immunohistochemistry and reactivity by specific airway resistance, contraction strength of isolated airway smooth muscles, and mucociliary clearance expressed by ciliary beating frequency. The immuno-biochemical markers of chronic inflammation were evaluated by BioPlex and ELISA assays. The AR was mediated by inflammatory cytokines and growth factors. The findings show significant anti-remodeling effects of SKF 96365, which were associated with a decrease in airway hyperreactivity. The anti-remodeling effect of SKF 96365 was mediated via the suppression of IL-4, IL-5, and IL-13 synthesis, and IL-12-INF-γ-TGF-ß pathway. The budesonide-related AR suppression had to do with a decrease in proinflammatory cytokines and an increase in the anti-inflammatory IL-10, with negligible influence on growth factors synthesis and mucous glands activity.

Lessons from the Endoplasmic Reticulum Ca2+ Transporters-A Cancer Connection.

Ca2+ is an integral mediator of intracellular signaling, impacting almost every aspect of cellular life. The Ca2+-conducting transporters located on the endoplasmic reticulum (ER) membrane shoulder the responsibility of constructing the global Ca2+ signaling landscape. These transporters gate the ER Ca2+ release and uptake, sculpt signaling duration and intensity, and compose the Ca2+ signaling rhythm to accommodate a plethora of biological activities. In this review, we explore the mechanisms of activation and functional regulation of ER Ca2+ transporters in the establishment of Ca2+ homeostasis. We also contextualize the aberrant alterations of these transporters in carcinogenesis, presenting Ca2+-based therapeutic interventions as a means to tackle malignancies.

Insights and perspectives on calcium channel functions in the cockpit of cancerous space invaders.

Development of metastasis causes the most serious clinical consequences of cancer and is responsible for over 90 % of cancer-related deaths. Hence, a better understanding of the mechanisms that drive metastasis formation appears critical for drug development designed to prevent the spread of cancer and related mortality. Metastasis dissemination is a multistep process supported by the increased motility and invasiveness capacities of tumor cells. To succeed in overcoming the mechanical constraints imposed by the basement membrane and surrounding tissues, cancer cells reorganize their focal adhesions or extend acto-adhesive cellular protrusions, called invadosomes, that can both contact the extracellular matrix and tune its degradation through metalloprotease activity. Over the last decade, accumulating evidence has demonstrated that altered Ca2+ channel activities and/or expression promote tumor cell-specific phenotypic changes, such as exacerbated migration and invasion capacities, leading to metastasis formation. While several studies have addressed the molecular basis of Ca2+ channel-dependent cancer cell migration, we are still far from having a comprehensive vision of the Ca2+ channel-regulated mechanisms of migration/invasion. This is especially true regarding the specific context of invadosome-driven invasion. This review aims to provide an overview of the current evidence supporting a central role for Ca2+ channel-dependent signaling in the regulation of these dynamic degradative structures. It will present available data on the few Ca2+ channels that have been studied in that specific context and discuss some potential interesting actors that have not been fully explored yet.

Review: Structure and Activation Mechanisms of CRAC Channels.

Ca2+ release activated Ca2+ (CRAC) channels represent a primary pathway for Ca2+ to enter non-excitable cells. The two key players in this process are the stromal interaction molecule (STIM), a Ca2+ sensor embedded in the membrane of the endoplasmic reticulum, and Orai, a highly Ca2+ selective ion channel located in the plasma membrane. Upon depletion of the internal Ca2+ stores, STIM is activated, oligomerizes, couples to and activates Orai. This review provides an overview of novel findings about the CRAC channel activation mechanisms, structure and gating. In addition, it highlights, among diverse STIM and Orai mutants, also the disease-related mutants and their implications.

Dynamic compartmentalization of calcium channel signalling in neurons.

Calcium fluxes through the neuronal membrane are strictly limited in time due to biophysical properties of voltage-gated and ligand-activated ion channels and receptors. Being embedded into the crowded dynamic environment of biological membranes, Ca2+-permeable receptors and channels undergo perpetual spatial rearrangement, which enables their temporary association and formation of transient signalling complexes. Thus, efficient calcium-mediated signal transduction requires mechanisms to support very precise spatiotemporal alignment of the calcium source and Ca2+-binding lipids and proteins in a highly dynamic environment. The mobility of calcium channels and calcium-sensing proteins themselves can be considered as a physiologically meaningful variable that affects calcium-mediated signalling in neurons. In this review, we will focus on voltage-gated calcium channels (VGCCs) and activity-induced relocation of stromal interaction molecules (STIMs) in the endoplasmic reticulum (ER) to show that particularly in time ranges between milliseconds to minutes, dynamic rearrangement of calcium conducting channels and sensor molecules is of physiological relevance. This article is part of the special issue entitled 'Mobility and trafficking of neuronal membrane proteins'.

Critical parameters maintaining authentic CRAC channel hallmarks.

Ca2+ ions represent versatile second messengers that regulate a huge diversity of processes throughout the cell's life. One prominent Ca2+ entry pathway into the cell is the Ca2+ release-activated Ca2+ (CRAC) ion channel. It is fully reconstituted by the two molecular key players: the stromal interaction molecule (STIM1) and Orai. STIM1 is a Ca2+ sensor located in the membrane of the endoplasmic reticulum, and Orai, a highly Ca2+ selective ion channel embedded in the plasma membrane. Ca2+ store-depletion leads initially to the activation of STIM1 which subsequently activates Orai channels via direct binding. Authentic CRAC channel hallmarks and biophysical characteristics include high Ca2+ selectivity with a reversal potential in the range of + 50 mV, small unitary conductance, fast Ca2+-dependent inactivation and enhancements in currents upon the switch from a Na+-containing divalent-free to a Ca2+-containing solution. This review provides an overview on the critical determinants and structures within the STIM1 and Orai proteins that establish these prominent CRAC channel characteristics.

The STIM-Orai Pathway: The Interactions Between STIM and Orai.

A primary Ca2+ entry pathway in non-excitable cells is established by the Ca2+ release-activated Ca2+ channels. Their two limiting molecular components include the Ca2+-sensor protein STIM1 located in the endoplasmic reticulum and the Orai channel in the plasma membrane. STIM1 senses the luminal Ca2+ content, and store depletion induces its oligomerization into puncta-like structures, thereby triggering coupling to as well as activation of Orai channels. A C-terminal STIM1 domain is assumed to couple to both C- and N-terminal, cytosolic strands of Orai, accomplishing gating of the channel. Here we highlight the inter- and intramolecular steps of the STIM1-Orai signaling cascade together with critical sites of the pore structure that accomplishes Ca2+ permeation.

Cardiovascular and Hemostatic Disorders: SOCE in Cardiovascular Cells: Emerging Targets for Therapeutic Intervention.

The discovery of the store-operated Ca2+ entry (SOCE) phenomenon is tightly associated with its recognition as a pathway of high (patho)physiological significance in the cardiovascular system. Early on, SOCE has been investigated primarily in non-excitable cell types, and the vascular endothelium received particular attention, while a role of SOCE in excitable cells, specifically cardiac myocytes and pacemakers, was initially ignored and remains largely enigmatic even to date. With the recent gain in knowledge on the molecular components of SOCE as well as their cellular organization within nanodomains, potential tissue/cell type-dependent heterogeneity of the SOCE machinery along with high specificity of linkage to downstream signaling pathways emerged for cardiovascular cells. The basis of precise decoding of cellular Ca2+ signals was recently uncovered to involve correct spatiotemporal organization of signaling components, and even minor disturbances in these assemblies trigger cardiovascular pathologies. With this chapter, we wish to provide an overview on current concepts of cellular organization of SOCE signaling complexes in cardiovascular cells with particular focus on the spatiotemporal aspects of coupling to downstream signaling and the potential disturbance of these mechanisms by pathogenic factors. The significance of these mechanistic concepts for the development of novel therapeutic strategies will be discussed.

Chloroquine inhibits Ca2+ permeable ion channels-mediated Ca2+ signaling in primary B lymphocytes.

BACKGROUND: Chloroquine, a bitter tastant, inhibits Ca2+ signaling, resulting in suppression of B cell activation; however, the inhibitory mechanism remains unclear. RESULTS: In this study, thapsigargin (TG), but not caffeine, induced sustained intracellular Ca2+ increases in mouse splenic primary B lymphocytes, which were markedly inhibited by chloroquine. Under Ca2+-free conditions, TG elicited transient Ca2+ increases, which additionally elevated upon the restoration of 2 mM Ca2+. The former were from release of intracellular Ca2+ store and the latter from Ca2+ influx. TG-induced release was inhibited by 2-APB (an inhibitor of inositol-3-phosphate receptors, IP3Rs) and chloroquine, and TG-caused influx was inhibited by pyrazole (Pyr3, an inhibitor of transient receptor potential C3 (TRPC3) and stromal interaction molecule (STIM)/Orai channels) and chloroquine. Moreover, chloroquine also blocked Ca2+ increases induced by the engagement of B cell receptor (BCR) with anti-IgM. CONCLUSIONS: These results indicate that chloroquine inhibits Ca2+ elevations in splenic B cells through inhibiting Ca2+ permeable IP3R and TRPC3 and/or STIM/Orai channels. These findings suggest that chloroquine would be a potent immunosuppressant.

Molecular mechanisms of STIM/Orai communication.

Ca(2+)entry into the cell via store-operated Ca(2+)release-activated Ca(2+)(CRAC) channels triggers diverse signaling cascades that affect cellular processes like cell growth, gene regulation, secretion, and cell death. These store-operated Ca(2+)channels open after depletion of intracellular Ca(2+)stores, and their main features are fully reconstituted by the two molecular key players: the stromal interaction molecule (STIM) and Orai. STIM represents an endoplasmic reticulum-located Ca(2+)sensor, while Orai forms a highly Ca(2+)-selective ion channel in the plasma membrane. Functional as well as mutagenesis studies together with structural insights about STIM and Orai proteins provide a molecular picture of the interplay of these two key players in the CRAC signaling cascade. This review focuses on the main experimental advances in the understanding of the STIM1-Orai choreography, thereby establishing a portrait of key mechanistic steps in the CRAC channel signaling cascade. The focus is on the activation of the STIM proteins, the subsequent coupling of STIM1 to Orai1, and the consequent structural rearrangements that gate the Orai channels into the open state to allow Ca(2+)permeation into the cell.