Calcium Entry Channels Research Articles

Mitochondrial Calcium Transporters Mediate Sensitivity to Noise-Induced Losses of Hair Cells and Cochlear Synapses.

Mitochondria modulate cellular calcium homeostasis by the combined action of the mitochondrial calcium uniporter (MCU), a selective calcium entry channel, and the sodium calcium exchanger (NCLX), which extrudes calcium from mitochondria. In this study, we investigated MCU and NCLX in noise-induced hearing loss (NIHL) using adult CBA/J mice and noise-induced alterations of inner hair cell (IHC) synapses in MCU knockout mice. Following noise exposure, immunoreactivity of MCU increased in cochlear sensory hair cells of the basal turn, while immunoreactivity of NCLX decreased in a time- and exposure-dependent manner. Inhibition of MCU activity via MCU siRNA pretreatment or the specific pharmacological inhibitor Ru360 attenuated noise-induced loss of sensory hair cells and synaptic ribbons, wave I amplitudes, and NIHL in CBA/J mice. This protection was afforded, at least in part, through reduced cleavage of caspase 9 (CC9). Furthermore, MCU knockout mice on a hybrid genetic CD1 and C57/B6 background showed resistance to noise-induced seizures compared to wild-type littermates. Owing to the CD1 background, MCU knockouts and littermates suffer genetic high frequency hearing loss, but their IHCs remain intact. Noise-induced loss of IHC synaptic connections and reduction of auditory brainstem response (ABR) wave I amplitude were recovered in MCU knockout mice. These results suggest that cellular calcium influx during noise exposure leads to mitochondrial calcium overload via MCU and NCLX. Mitochondrial calcium overload, in turn, initiates cell death pathways and subsequent loss of hair cells and synaptic connections, resulting in NIHL.

The role of endothelial leak in pulmonary hypertension (2017 Grover Conference Series)

The canonical transient receptor potential 4 (TRPC4) protein contributes to the molecular make-up of endothelial store-operated calcium entry channels. Store-operated calcium entry is a prominent mode of calcium influx in endothelium. Store-operated calcium entry channels are activated by inflammatory mediators and growth factors, and in endothelium, this process induces inter-endothelial cell gaps that increase permeability. Pulmonary endothelium within extra-alveolar segments, including pulmonary arteries, is especially sensitive to the activation of store-operated calcium entry. Pulmonary arterial hypertension (PAH) is characterized by endothelial cell dysfunction in arteries. As one of the topics for the 2017 Grover Conference Series, we examined whether an endothelial cell permeability defect accompanies PAH and, if so, whether TRPC4 contributes to this defect. Through a series of studies conducted over the past five years, we find endothelial cell barrier dysfunction occurs early in the progression of experimental PAH. Endothelium within the arterial segment, and perhaps in other vascular segments, is highly susceptible to disruption secondary to both activation of store-operated calcium entry channels and high flow. This phenomenon partly depends upon TRPC4 channels. We discuss whether endothelial cell hyperpermeability is relevant to human disease, and more specifically, whether it is relevant to all groups of pulmonary hypertension.

The Role of S100A6 in the PP5C‐FKBP51‐Mediated Inhibition of Endothelial Isoc

BackgroundDuring inflammation, mediators increase calcium influx into endothelial cells through the store‐operated calcium entry (SOCE) channels. Increasing calcium influx through the SOCE current, Isoc, promotes endothelial barrier disruption through the formation of inter‐endothelial cell gaps. Inter‐endothelial cell gap formation leads to increased endothelial permeability, a vascular event contributing to pathophysiological states including inflammation and acute respiratory distress syndrome (ARDS). Identifying mechanisms of Isoc inhibition will aid in development of therapeutic strategies against endothelial permeability‐associated inflammation and ARDS. However, mechanisms of Isoc inhibition are poorly understood. We have shown that the immunophilin FKBP51 inhibits Isoc and contributes to the inhibition of calcium entry‐induced inter‐endothelial cell gap formation. We also determined that protein phosphatase 5 (PP5C) is required for the FKBP51‐mediated inhibition of Isoc and inter‐endothelial cell gap formation. However, PP5C has very low activity in the basal state. To exert its effect PP5C needs an activator. S100 proteins are potent activators of PP5C. S100 proteins are calcium‐dependent proteins and specifically, S100A6 can interact with tetratricopeptide repeat domain containing proteins such as PP5C and FKBP51. Further, S100A6 shows calcium‐induced translocation from cytosol to the plasma membrane where the ISOC channel is located. However, it is unknown whether S100A6 contributes to the PP5C‐FKBP51‐mediated inhibition of Isoc. The goal of this study was to determine whether S100A6 is critical in increasing the phosphatase activity of PP5C in a calcium‐dependent manner in endothelial cells to promote the PP5C‐FKBP51 axis‐mediated inhibition of Isoc.MethodsWe utilized quantitative RT‐PCR and immunoblot to determine whether S100 proteins are expressed in pulmonary endothelial cells. We performed co‐immunoprecipitation experiments to determine if S100A6 associates with the ISOC channel and PP5C protein in pulmonary microvascular endothelial cells (PMVECs) in presence and absence of calcium (2mM). To determine whether S100A6 activates PP5C in PMVECs we analyzed the phosphorylation status of tau protein with or without calcium (2mM), where decreased tau phosphorylation suggests increased PP5C activity. Whole cell patch clamp electrophysiology was used to measure Isoc in cells.ResultsWe showed that S100A6 is highly expressed in pulmonary endothelial cells. S100A6 co‐precipitated with the TRPC4 subunit of the ISOC channel as well as with PP5C in a calcium‐dependent manner. Upon S100A6 knock‐down in PMVECs, phosphorylation of tau increased suggesting that PP5C activity was deceased. In FKBP51 over‐expressed cells, Isoc was inhibited, but this effect was reduced by S100A6 knock‐down.ConclusionTogether these findings suggest that S100A6 activates PP5C leading to the FKBP51‐PP5C‐mediated inhibition of Isoc.Support or Funding InformationSupported by NIH R01HL107778.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of spatial heterogeneity and colocalization of eNOS and capacitative calcium entry channels on shear stress-induced NO production by endothelial cells: A modeling approach.

Colocalization of endothelial nitric oxide synthase (eNOS) and capacitative Ca2+ entry (CCE) channels in microdomains such as cavaeolae in endothelial cells (ECs) has been shown to significantly affect intracellular Ca2+ dynamics and NO production, but the effect has not been well quantified. We developed a two-dimensional continuum model of an EC integrating shear stress-mediated ATP production, intracellular Ca2+ mobilization, and eNOS activation to investigate the effects of spatial colocalization of plasma membrane eNOS and CCE channels on Ca2+ dynamics and NO production in response to flow-induced shear stress. Our model examines the hypothesis that subcellular colocalization of cellular components can be critical for optimal coupling of NO production to blood flow. Our simulations predict that heterogeneity of CCE can result in formation of microdomains with significantly higher Ca2+ compared to the average cytosolic Ca2+. Ca2+ buffers with lower or no mobility further enhanced Ca2+ gradients relative to mobile buffers. Colocalization of eNOS to CCE channels significantly increased NO production. Our results provide quantitative understanding for the role of spatial heterogeneity and the compartmentalization of signals in regulation of shear stress-induced NO production.

Skin Barrier and Calcium.

Epidermal barrier formation and the maintenance of barrier homeostasis are essential to protect us from the external environments and organisms. Moreover, impaired keratinocytes differentiation and dysfunctional skin barrier can be the primary causes or aggravating factors for many inflammatory skin diseases including atopic dermatitis and psoriasis. Therefore, understanding the regulation mechanisms of keratinocytes differentiation and skin barrier homeostasis is important to understand many skin diseases and establish an effective treatment strategy. Calcium ions (Ca2+) and their concentration gradient in the epidermis are essential in regulating many skin functions, including keratinocyte differentiation, skin barrier formation, and permeability barrier homeostasis. Recent studies have suggested that the intracellular Ca2+ stores such as the endoplasmic reticulum (ER) are the major components that form the epidermal calcium gradient and the ER calcium homeostasis is crucial for regulating keratinocytes differentiation, intercellular junction formation, antimicrobial barrier, and permeability barrier homeostasis. Thus, both Ca2+ release from intracellular stores, such as the ER and Ca2+ influx mechanisms are important in skin barrier. In addition, growing evidences identified the functional existence and the role of many types of calcium channels which mediate calcium flux in keratinocytes. In this review, the origin of epidermal calcium gradient and their role in the formation and regulation of skin barrier are focused. We also focus on the role of ER calcium homeostasis in skin barrier. Furthermore, the distribution and role of epidermal calcium channels, including transient receptor potential channels, store-operated calcium entry channel Orai1, and voltage-gated calcium channels in skin barrier are discussed.

Quantitative and qualitative analysis of calcium oscillation considering store-operated calcium entry channel

As an important second messenger in cell, calcium ions can regulate various cellular processes by the way of oscillation. Variety of information or signals in cells are encoded by frequency and amplitude of calcium oscillation. In present paper, we focus on an influx channel, called store-operated calcium entry channel (SOCE for short), and establish a new model to describe the calcium exchanges and interactions between different stores. By employing bifurcation analysis on the model, change laws of the amplitude and period of oscillation, with varying the maximum capacity of SOCE, is obtained. Then we select other four important parameters to perform simulation separately, and discuss the influences on the amplitude and period of oscillation. Based on intensive quantitative and qualitative analysis, we conclude that SOCE plays a key role in calcium oscillation system.

248 - TRPV4 Has a Role as a Ca2+–Permeable Channel in the Differentiation of Human Ventricular Fibroblasts Into Myofibroblasts

Introduction: Cardiac fibroblasts (CFs) are the predominant cell types that reside in the interstitial space of the heart. In response to pathological stimuli, CFs are stimulated to undergo phenotypic modulation and become myofibroblasts that are hypersecretory and highly contractile and which deposit excessive extracellular matrix proteins. Ca2 + signals are essential to a diversity of cellular functions, including differentiation, gene expression, cell proliferation, and growth. CaV1.2-mediated L-type Ca2+ current (ICa,L) is a fundamental myocardial calcium entry channel, but CFs do not typically express the voltage-gated Ca2+ channel. Transient receptor potential (TRP) channels are nonselective calcium entry channels, and their function has been studied in many cell types. TRP is expressed in a cell-specific manner, and its role has not been evaluated in human ventricular fibroblasts. Methods: Here, we evaluate the role of the TRP channel in the differentiation of human ventricular fibroblasts into myofibroblasts. Results: Concordant with previous studies, Ca2+ was found in the current study to be an essential component in fibroblast differentiation. Intracellular chelation inhibited p-ERK expression and fibroblast differentiation. TRPV4 mRNA was expressed in human ventricular fibroblasts and increased on account of TGF-β1 (2 ng/mL). TRPV4 agonist (GSK1016790A) treatment induced the robust differentiation of fibroblasts into myofibroblasts. TRPV4 antagonist (RN-9893) significantly inhibited TGF-β1-induced p–ERK expression and the differentiation of CFs. Using ratio Ca2+ imaging measurements, we found a robust increase in Ca2+ influx after fibroblasts were treated with TRPV4 agonist (GSK1016790A). Conclusions: These results demonstrate that TRPV4 has a role as a Ca2+–permeable channel in the differentiation of human ventricular fibroblasts into myofibroblasts, through the ERK signaling pathway.

Detection of TRPV4 channel current-like activity in Fawn Hooded hypertensive (FHH) rat cerebral arterial muscle cells.

The transient receptor potential vallinoid type 4 (TRPV4) is a calcium entry channel known to modulate vascular function by mediating endothelium–dependent vasodilation. The present study investigated if isolated cerebral arterial myocytes of the Fawn Hooded hypertensive (FHH) rat, known to display exaggerated KCa channel current activity and impaired myogenic tone, express TRPV4 channels at the transcript and protein level and exhibit TRPV4-like single-channel cationic current activity. Reverse transcription polymerase chain reaction (RT-PCR), Western blot, and immunostaining analysis detected the expression of mRNA transcript and translated protein of TRPV4 channel in FHH rat cerebral arterial myocytes. Patch clamp recording of single-channel current activity identified the presence of a single-channel cationic current with unitary conductance of ~85 pS and ~96 pS at hyperpolarizing and depolarizing potentials, respectively, that was inhibited by the TRPV4 channel antagonist RN 1734 or HC 067074 and activated by the potent TRPV4 channel agonist GSK1016790A. Application of negative pressure via the interior of the patch pipette increased the NPo of the TRPV4-like single-channel cationic current recorded in cell-attached patches at a patch potential of 60 mV that was inhibited by prior application of the TRPV4 channel antagonist RN 1734 or HC 067047. Treatment with the TRPV4 channel agonist GSK1016790A caused concentration-dependent increase in the NPo of KCa single-channel current recorded in cell-attached patches of cerebral arterial myocytes at a patch potential of 40 mV, which was not influenced by pretreatment with the voltage-gated L-type Ca2+ channel blocker nifedipine or the T-type Ca2+ channel blocker Ni2+. These findings demonstrate that FHH rat cerebral arterial myocytes express mRNA transcript and translated protein for TRPV4 channel and display TRPV4-like single-channel cationic current activity that was stretch-sensitive and activation of which increased the open state probability of KCa single-channel current in these arterial myocytes.

P2Y6 Receptor Antagonist MRS2578 Inhibits Neutrophil Activation and Aggregated Neutrophil Extracellular Trap Formation Induced by Gout-Associated Monosodium Urate Crystals.

Human neutrophils (polymorphonuclear leukocytes [PMNs]) generate inflammatory responses within the joints of gout patients upon encountering monosodium urate (MSU) crystals. Neutrophil extracellular traps (NETs) are found abundantly in the synovial fluid of gout patients. The detailed mechanism of MSU crystal-induced NET formation remains unknown. Our goal was to shed light on possible roles of purinergic signaling and neutrophil migration in mediating NET formation induced by MSU crystals. Interaction of human neutrophils with MSU crystals was evaluated by high-throughput live imaging using confocal microscopy. We quantitated NET levels in gout synovial fluid supernatants and detected enzymatically active neutrophil primary granule enzymes, myeloperoxidase, and human neutrophil elastase. Suramin and PPADS, general P2Y receptor blockers, and MRS2578, an inhibitor of the purinergic P2Y6 receptor, blocked NET formation triggered by MSU crystals. AR-C25118925XX (P2Y2 antagonist) did not inhibit MSU crystal-stimulated NET release. Live imaging of PMNs showed that MRS2578 represses neutrophil migration and blocked characteristic formation of MSU crystal-NET aggregates called aggregated NETs. Interestingly, the store-operated calcium entry channel inhibitor (SK&F96365) also reduced MSU crystal-induced NET release. Our results indicate that the P2Y6/store-operated calcium entry/IL-8 axis is involved in MSU crystal-induced aggregated NET formation, but MRS2578 could have additional effects affecting PMN migration. The work presented in the present study could lead to a better understanding of gouty joint inflammation and help improve the treatment and care of gout patients.

Neurotransmitter Release Can Be Stabilized by a Mechanism That Prevents Voltage Changes Near the End of Action Potentials from Affecting Calcium Currents.

The shape of presynaptic action potentials (APs), particularly the falling phase, affects calcium entry and small changes in calcium influx can produce large changes in postsynaptic responses. We hypothesized that afterpotentials, which often follow APs, affect calcium entry and neurotransmitter release. We tested this in calyx of Held nerve terminals, which allow simultaneous recording of presynaptic calcium currents and postsynaptic responses. Surprisingly, presynaptic afterpotentials did not alter calcium current or neurotransmitter release. We show that the AP falling phase causes afterpotential-induced changes in electrical driving force and calcium channel gating to cancel each other out. This mechanism regulates calcium entry at the end of APs and therefore stabilizes synaptic transmission. This also stabilizes responses when the presynaptic resting potential changes.

The role of the store-operated calcium entry channel Orai1 in cultured rat hippocampal synapse formation and plasticity.

The role of non-synaptic calcium entry in the formation and functions of dendritic spines was studied in dissociated cultured rat hippocampal neurons. Orai1, a store-operated calcium channel, is found in dendritic spines. Orai1 co-localizes in dendritic spines with STIM2 under conditions of lower [Ca2+ ]o. Orai1 channels are associated with the formation of new dendritic spines in response to elevated [Ca2+ ]o. Lack of Orai1, either by transfection with a dominant negative construct or with small interfering RNA to Orai1, results in retarded dendritic spines, an increase in density of filopodia, lower synaptic connectivity and the ability to undergo plastic changes. These results highlight a novel role for Orai1 in synapse formation, maturation and plasticity. The possible role of store operated calcium entry (SOCE) through the Orai1 channel in the formation and functions of dendritic spines was studied in cultured hippocampal neurons. In calcium store-depleted neurons, a transient elevation of extracellular calcium concentration ([Ca2+ ]o ) caused a rise in [Ca2+ ]i that was mediated by activation of the SOCE. The store depletion resulted in an increase in stromal interacting molecule 2 (an endoplasmic calcium sensor) association with Orai1 in dendritic spines. The response to the rise in [Ca2+ ]o was larger in spines endowed with a cluster of Orai1 molecules than in spines devoid of Orai1. Transfection of neurons with a dominant negative Orai1 resulted in retarded maturation of dendritic spines, a reduction in synaptic connectivity with afferent neurons and a reduction in the ability to undergo morphological changes following induction of chemical long-term potentiation. Similarly, small interfering RNA (siRNA)-treated neurons had fewer mature dendritic spines, and lower rates of mEPSCs compared to scrambled control siRNA-treated neurons. Thus, influx of calcium through Orai1 channels facilitates the maturation of dendritic spines and the formation of functional synapses in central neurons.

Tumor necrosis factor regulates NMDA receptor-mediated airway smooth muscle contractile function and airway responsiveness

We have shown that N-methyl-d-aspartate receptors (NMDA-Rs) are receptor-operated calcium entry channels in human airway smooth muscle (HASM) during contraction. Tumor necrosis factor (TNF) augments smooth muscle contractility by influencing pathways that regulate intracellular calcium flux and can alter NMDA-R expression and activity in cortical neurons and glial cells. We hypothesized that NMDA-R-mediated Ca(2+) and contractile responses of ASM can be altered by inflammatory mediators, including TNF. In cultured HASM cells, we assessed TNF (10 ng/ml, 48 h) effect on NMDA-R subunit abundance by quantitative PCR, confocal imaging, and immunoblotting. We observed dose- and time-dependent changes in NMDA-R composition: increased obligatory NR1 subunit expression and altered regulatory NR2 and inhibitory NR3 subunits. Measuring intracellular Ca(2+) flux in Fura-2-loaded HASM cultures, we observed that TNF exposure enhanced cytosolic Ca(2+) mobilization and changed the temporal pattern of Ca(2+) flux in individual myocytes induced by NMDA, an NMDA-R selective analog of glutamate. We measured airway responses to NMDA in murine thin-cut lung slices (TCLS) from allergen-naive animals and observed significant airway contraction. However, NMDA acted as a bronchodilator in TCLS from house dust mice-challenged mice and in allergen-naive TCLS subjected to TNF exposure. All contractile or bronchodilator responses were blocked by a selective NMDA-R antagonist, (2R)-amino-5-phosphonopentanoate, and bronchodilator responses were prevented by N(G)-nitro-l-arginine methyl ester (nitric oxide synthase inhibitor) or indomethacin (cyclooxygenase inhibitor). Collectively, we show that TNF augments NMDA-R-mediated Ca(2+) mobilization in HASM cells, whereas in multicellular TCLSs allergic inflammation and TNF exposure leads to NMDA-R-mediated bronchodilation. These findings reveal the unique contribution of ionotrophic NMDA-R to airway hyperreactivity.

NET formation induced by gout-associated monosodium urate crystals requires IL-8-mediated neutrophil migration and is inhibited by the P2Y6 receptor antagonist MRS2578.

Abstract Human neutrophils generate inflammatory responses within the joints of gout patients upon encountering Monosodium Urate (MSU) crystals. Studies (including ours) show that neutrophil extracellular traps (NETs) are found abundantly in the synovial fluid (SF) of gout patients and are crucial for the onset of the autoinflammatory cascade. Our goal is to shed light on possible roles of purinergic signaling and neutrophil migration in mediating in vitro NET formation induced by MSU crystals. Our data show that gout SF supernatants have significantly higher levels of the neutrophil granule markers, myeloperoxidase (MPO) and human neutrophil elastase (HNE), and NETs than SF specimens from MSU-negative osteoarthritis patients. MSU crystal-triggered NETs in human neutrophils contain active DNA-bound MPO and HNE. MRS2578, a purinergic P2Y6 receptor antagonist, reduces the release of extracellular DNA, MPO and HNE in MSU crystal-stimulated neutrophils. Releases of MPO, HNE and NETs were measured by commercial ELISA and our proprietary NET-DNA ELISA assays. We found that MRS2578 reduces MSU crystal-induced IL-8 release, neutrophil chemotaxis and characteristic formation of DNA aggregates. The store operated calcium entry (SOCE) channel inhibitor (SK96365) blocked MSU crystal-induced NET release. Our results indicating that the P2Y6-SOCE-IL-8 axis is involved in MSU crystal-induced NET formation provide a novel mechanism potentially relevant in gout inflammation. Our work on the role of the P2Y6-SOCE-IL-8 axis in MSU crystal-triggered NET formation could potentially lead to a better understanding of the mechanism of gout joint inflammation and, thereby, help in improving the care of gout patients.

Expression of calcium-buffering proteins in rat intrinsic laryngeal muscles.

Intrinsic laryngeal muscles (ILM) are highly specialized muscles involved in phonation and airway protection, with unique properties that allow them to perform extremely rapid contractions and to escape from damage in muscle dystrophy. Due to that, they may differ from limb muscles in several physiological aspects. Because a better ability to handle intracellular calcium has been suggested to explain ILM unique properties, we hypothesized that the profile of the proteins that regulate calcium levels in ILM is different from that in a limb muscle. Calcium-related proteins were analyzed in the ILM, cricothyroid (CT), and tibialis anterior (TA) muscles from male Sprague–Dawley rats (8 weeks of age) using quantitative PCR and western blotting. Higher expression of key Ca2+ regulatory proteins was detected in ILM compared to TA, such as the sarcoplasmic reticulum (SR) Ca2+-reuptake proteins (Sercas 1 and 2), the Na+/Ca2+ exchanger, phospholamban, and the Ca2+-binding protein calsequestrin. Parvalbumin, calmodulin and the ATPase, Ca2+-transporting, and plasma membrane 1 were also expressed at higher levels in ILM compared to TA. The store-operated calcium entry channel molecule was decreased in ILM compared to the limb muscle and the voltage-dependent L-type and ryanodine receptor were expressed at similar levels in ILM and TA. These results show that ILM have a calcium regulation system profile suggestive of a better ability to handle calcium changes in comparison to limb muscles, and this may provide a mechanistic insight for their unique pathophysiological properties.

Noggin inhibits hypoxia-induced proliferation by targeting store-operated calcium entry and transient receptor potential cation channels.

Abnormally elevated bone morphogenetic protein 4 (BMP4) expression and mediated signaling play a critical role in the pathogenesis of chronic hypoxia-induced pulmonary hypertension (CHPH). In this study, we investigated the expression level and functional significance of four reported naturally occurring BMP4 antagonists, noggin, follistatin, gremlin1, and matrix gla protein (MGP), in the lung and distal pulmonary arterial smooth muscle cell (PASMC). A 21-day chronic hypoxic (10% O2) exposure rat model was utilized, which has been previously shown to successfully establish experimental CHPH. Among the four antagonists, noggin, but not the other three, was selectively downregulated by hypoxic exposure in both the lung tissue and PASMC, in correlation with markedly elevated BMP4 expression, suggesting that the loss of noggin might account for the hypoxia-triggered BMP4 signaling transduction. Then, by using treatment of extrogenous recombinant noggin protein, we further found that noggin significantly normalized 1) BMP4-induced phosphorylation of cellular p38 and ERK1/2; 2) BMP4-induced phosphorylation of cellular JAK2 and STAT3; 3) hypoxia-induced PASMC proliferation; 4) hypoxia-induced store-operated calcium entry (SOCE), and 5) hypoxia-increased expression of transient receptor potential cation channels (TRPC1 and TRPC6) in PASMC. In combination, these data strongly indicated that the hypoxia-suppressed noggin accounts, at least partially, for hypoxia-induced excessive PASMC proliferation, while restoration of noggin may be an effective way to inhibit cell proliferation by suppressing SOCE and TRPC expression.

Role of Calcium Signaling in B Cell Activation and Biology.

Increase in intracellular levels of calcium ions (Ca2+) is one of the key triggering signals for the development of B cell response to the antigen. The diverse Ca2+ signals finely controlled by multiple factors participate in the regulation of gene expression, B cell development, and effector functions. B cell receptor (BCR)-initiated Ca2+ mobilization is sourced from two pathways: one is the release of Ca2+ from the intracellular stores, endoplasmic reticulum (ER), and other is the prolonged influx of extracellular Ca2+ induced by depleting the stores via store-operated calcium entry (SOCE) and calcium release-activated calcium (CRAC) channels. The identification of stromal interaction molecule 1(STIM1), the ER Ca2+ sensor, and Orai1, a key subunit of the CRAC channel pore, has now provided the tools to understand the mode of Ca2+ influx regulation and physiological relevance. Herein, we discuss our current understanding of the molecular mechanisms underlying BCR-triggered Ca2+ signaling as well as its contribution to the B cell biological processes and diseases.

Sodium entry through endothelial store-operated calcium entry channels: regulation by Orai1.

Orai1 interacts with transient receptor potential protein of the canonical subfamily (TRPC4) and contributes to calcium selectivity of the endothelial cell store-operated calcium entry current (ISOC). Orai1 silencing increases sodium permeability and decreases membrane-associated calcium, although it is not known whether Orai1 is an important determinant of cytosolic sodium transitions. We test the hypothesis that, upon activation of store-operated calcium entry channels, Orai1 is a critical determinant of cytosolic sodium transitions. Activation of store-operated calcium entry channels transiently increased cytosolic calcium and sodium, characteristic of release from an intracellular store. The sodium response occurred more abruptly and returned to baseline more rapidly than did the transient calcium rise. Extracellular choline substitution for sodium did not inhibit the response, although 2-aminoethoxydiphenyl borate and YM-58483 reduced it by ∼50%. After this transient response, cytosolic sodium continued to increase due to influx through activated store-operated calcium entry channels. The magnitude of this sustained increase in cytosolic sodium was greater when experiments were conducted in low extracellular calcium and when Orai1 expression was silenced; these two interventions were not additive, suggesting a common mechanism. 2-Aminoethoxydiphenyl borate and YM-58483 inhibited the sustained increase in cytosolic sodium, only in the presence of Orai1. These studies demonstrate that sodium permeates activated store-operated calcium entry channels, resulting in an increase in cytosolic sodium; the magnitude of this response is determined by Orai1.

Synaptopodin regulates spine plasticity: mediation by calcium stores.

The role of synaptopodin (SP), an actin-binding protein residing in dendritic spines, in synaptic plasticity was studied in dissociated cultures of hippocampus taken from control and SP knock-out (SPKO) mice. Unlike controls, SPKO cultures were unable to express changes in network activity or morphological plasticity after intense activation of their NMDA receptors. SPKO neurons were transfected with SP-GFP, such that the only SP resident in these neurons is the fluorescent species. The localization and intensity of the transfected SP were similar to that of the native one. Because less than half of the spines in the transfected neurons contained SP, comparisons were made between SP-containing (SP(+)) and SP lacking (SP(-)) spines in the same dendritic segments. Synaptic plasticity was induced either in the entire network by facilitation of the activation of the NMDA receptor, or specifically by local flash photolysis of caged glutamate. After activation, spines that were endowed with SP puncta were much more likely to expand than SP(-) spines. The spine expansion was suppressed by thapsigargin, which disables calcium stores. The mechanism through which SP may promote plasticity is indicated by the observations that STIM-1, the sensor of calcium concentration in stores, and Orai-1, the calcium-induced calcium entry channel, are colocalized with SP, in the same dendritic spines. The structural basis of SP is likely to be the spine apparatus, found in control but not in SPKO cells. These results indicate that SP has an essential, calcium store-related role in regulating synaptic plasticity in cultured hippocampal neurons.

STIM and Orai isoform expression in pregnant human myometrium: a potential role in calcium signaling during pregnancy.

Store-operated calcium (Ca2+) entry (SOCE) can be mediated by two novel proteins, STIM/Orai. We have previously demonstrated that members of the TRPC family, putative basal and store operated calcium entry channels, are present in human myometrium and regulated by labor associated stimuli IL-1β and mechanical stretch. Although STIM and Orai isoforms (1-3) have been reported in other smooth muscle cell types, there is little known about the expression or gestational regulation of STIM and Orai expression in human myometrium. Total RNA was isolated from lower segment human myometrial biopsies obtained at Cesarean section from women at the time of preterm no labor (PTNL), preterm labor (PTL), term non-labor (TNL), and term with labor (TL); primary cultured human uterine smooth muscle cells, and a human myometrial cell line (hTERT-HM). STIM1-2, and Orai1-3 mRNA expression was assessed by quantitative real-time PCR. All five genes were expressed in myometrial tissue and cultured cells. STIM1-2 and Orai2-3 expression was significantly lower in cultured cells compared tissue. This has implications with regard investigation of the contribution of these proteins in cultured cells. Orai2 was the most abundant Orai isoform in human myometrium. Expression of STIM1-2/Orai1-3 did not alter with the onset of labor. Orai1 mRNA expression in cultured cells was enhanced by IL-1β treatment. This novel report of STIM1-2 and Orai1-3 mRNA expression in pregnant human myometrium and Orai1 regulation by IL-1β indicates a potential role for these proteins in calcium signaling in human myometrium during pregnancy.

Vascular endothelial growth factor A evokes distinct calcium entry by promoting surface accumulation of Orai3 (1057.5)

Vascular endothelial growth factor A (VEGF‐A) regulates endothelial cell function and angiogenesis via activation of vascular endothelial growth factor receptor 2 (VEGFR2). One consequence of VEGFR2‐activation is calcium entry into endothelial cells. The responsible pathways are, however, not fully understood. Here we show that the VEGF‐A‐evoked calcium response in human umbilical vein endothelial cells (HUVEC) is dependent on Orai3, a membrane protein reported to form calcium entry channels. VEGFR2 activation and subsequent arachidonic acid release from the plasma membrane caused forward trafficking of Orai3 to the cell surface and association with STIM1. Knockdown of Orai3 via siRNA caused inhibition of endothelial tube formation in vitro and in vivo, indicating a potential role in angiogenesis. Our findings provide evidence for a previously unrecognised Orai3 mechanism that contributes importantly to VEGF‐A signalling.Grant Funding Source: Supported by the Medical Research Council.