Activation Of Store-operated Calcium Channels Research Articles

Pharmacological modulation of septins restores calcium homeostasis and is neuroprotective in models of Alzheimer's disease.

Abnormal calcium signaling is a central pathological component of Alzheimer's disease (AD). Here, we describe the identification of a class of compounds called ReS19-T, which are able to restore calcium homeostasis in cell-based models of tau pathology. Aberrant tau accumulation leads to uncontrolled activation of store-operated calcium channels (SOCCs) by remodeling septin filaments at the cell cortex. Binding of ReS19-T to septins restores filament assembly in the disease state and restrains calcium entry through SOCCs. In amyloid-β and tau-driven mouse models of disease, ReS19-T agents restored synaptic plasticity, normalized brain network activity, and attenuated the development of both amyloid-β and tau pathology. Our findings identify the septin cytoskeleton as a potential therapeutic target for the development of disease-modifying AD treatments.

SARS-CoV-2 spike protein receptor-binding domain perturbates intracellular calcium homeostasis and impairs pulmonary vascular endothelial cells

Exposure to the spike protein or receptor-binding domain (S-RBD) of SARS-CoV-2 significantly influences endothelial cells and induces pulmonary vascular endotheliopathy. In this study, angiotensin-converting enzyme 2 humanized inbred (hACE2 Tg) mice and cultured pulmonary vascular endothelial cells were used to investigate how spike protein/S-RBD impacts pulmonary vascular endothelium. Results show that S-RBD leads to acute-to-prolonged induction of the intracellular free calcium concentration ([Ca2+]i) via acute activation of TRPV4, and prolonged upregulation of mechanosensitive channel Piezo1 and store-operated calcium channel (SOCC) key component Orai1 in cultured human pulmonary arterial endothelial cells (PAECs). In mechanism, S-RBD interacts with ACE2 to induce formation of clusters involving Orai1, Piezo1 and TRPC1, facilitate the channel activation of Piezo1 and SOCC, and lead to elevated apoptosis. These effects are blocked by Kobophenol A, which inhibits the binding between S-RBD and ACE2, or intracellular calcium chelator, BAPTA-AM. Blockade of Piezo1 and SOCC by GsMTx4 effectively protects the S-RBD-induced pulmonary microvascular endothelial damage in hACE2 Tg mice via normalizing the elevated [Ca2+]i. Comparing to prototypic strain, Omicron variants (BA.5.2 and XBB) of S-RBD induces significantly less severe cell apoptosis. Transcriptomic analysis indicates that prototypic S-RBD confers more severe acute impacts than Delta or Lambda S-RBD. In summary, this study provides compelling evidence that S-RBD could induce persistent pulmonary vascular endothelial damage by binding to ACE2 and triggering [Ca2+]i through upregulation of Piezo1 and Orai1. Targeted inhibition of ACE2-Piezo1/SOCC-[Ca2+]i axis proves a powerful strategy to treat S-RBD-induced pulmonary vascular diseases.

A Genome-Wide Functional Screen Identifies Enhancer and Protective Genes for Amyloid Beta-Peptide Toxicity.

Alzheimer's disease (AD) is known to be caused by amyloid β-peptide (Aβ) misfolded into β-sheets, but this knowledge has not yet led to treatments to prevent AD. To identify novel molecular players in Aβ toxicity, we carried out a genome-wide screen in Saccharomyces cerevisiae, using a library of 5154 gene knock-out strains expressing Aβ1-42. We identified 81 mammalian orthologue genes that enhance Aβ1-42 toxicity, while 157 were protective. Next, we performed interactome and text-mining studies to increase the number of genes and to identify the main cellular functions affected by Aβ oligomers (oAβ). We found that the most affected cellular functions were calcium regulation, protein translation and mitochondrial activity. We focused on SURF4, a protein that regulates the store-operated calcium channel (SOCE). An in vitro analysis using human neuroblastoma cells showed that SURF4 silencing induced higher intracellular calcium levels, while its overexpression decreased calcium entry. Furthermore, SURF4 silencing produced a significant reduction in cell death when cells were challenged with oAβ1-42, whereas SURF4 overexpression induced Aβ1-42 cytotoxicity. In summary, we identified new enhancer and protective activities for Aβ toxicity and showed that SURF4 contributes to oAβ1-42 neurotoxicity by decreasing SOCE activity.

Genome-wide association study of the human brain functional connectome reveals strong vascular component underlying global network efficiency

Complex brain networks play a central role in integrating activity across the human brain, and such networks can be identified in the absence of any external stimulus. We performed 10 genome-wide association studies of resting state network measures of intrinsic brain activity in up to 36,150 participants of European ancestry in the UK Biobank. We found that the heritability of global network efficiency was largely explained by blood oxygen level-dependent (BOLD) resting state fluctuation amplitudes (RSFA), which are thought to reflect the vascular component of the BOLD signal. RSFA itself had a significant genetic component and we identified 24 genomic loci associated with RSFA, 157 genes whose predicted expression correlated with it, and 3 proteins in the dorsolateral prefrontal cortex and 4 in plasma. We observed correlations with cardiovascular traits, and single-cell RNA specificity analyses revealed enrichment of vascular related cells. Our analyses also revealed a potential role of lipid transport, store-operated calcium channel activity, and inositol 1,4,5-trisphosphate binding in resting-state BOLD fluctuations. We conclude that that the heritability of global network efficiency is largely explained by the vascular component of the BOLD response as ascertained by RSFA, which itself has a significant genetic component.

Substrate stiffness modulates migration and local intercellular membrane motion in pulmonary endothelial cell monolayers.

The pulmonary artery endothelium forms a semipermeable barrier that limits macromolecular flux through intercellular junctions. This barrier is maintained by an intrinsic forward protrusion of the interacting membranes between adjacent cells. However, the dynamic interactions of these membranes have been incompletely quantified. Here, we present a novel technique to quantify the motion of the peripheral membrane of the cells, called paracellular morphological fluctuations (PMFs), and to assess the impact of substrate stiffness on PMFs. Substrate stiffness impacted large-length scale morphological changes such as cell size and motion. Cell size was larger on stiffer substrates, whereas the speed of cell movement was decreased on hydrogels with stiffness either larger or smaller than 1.25 kPa, consistent with cells approaching a jammed state. Pulmonary artery endothelial cells moved fastest on 1.25 kPa hydrogel, a stiffness consistent with a healthy pulmonary artery. Unlike these large-length scale morphological changes, the baseline of PMFs was largely insensitive to the substrate stiffness on which the cells were cultured. Activation of store-operated calcium channels using thapsigargin treatment triggered a transient increase in PMFs beyond the control treatment. However, in hypocalcemic conditions, such an increase in PMFs was absent on 1.25 kPa hydrogel but was present on 30 kPa hydrogel-a stiffness consistent with that of a hypertensive pulmonary artery. These findings indicate that 1) PMFs occur in cultured endothelial cell clusters, irrespective of the substrate stiffness; 2) PMFs increase in response to calcium influx through store-operated calcium entry channels; and 3) stiffer substrate promotes PMFs through a mechanism that does not require calcium influx.

Cationic Mechanosensitive Channels Mediate Trabecular Meshwork Responses to Cyclic Mechanical Stretch.

The trabecular meshwork (TM) is responsible for intraocular pressure (IOP) homeostasis in the eye. The tissue senses IOP fluctuations and dynamically adapts to the mechanical changes to either increase or decrease aqueous humor outflow. Cationic mechanosensitive channels (CMCs) have been reported to play critical roles in mediating the TM responses to mechanical forces. However, how CMCs influence TM cellular function affect aqueous humor drainage is still elusive. In this study, human TM (HTM) cells were collected from a Chinese donor and subjected to cyclically equiaxial stretching with an amplitude of 20% at 1 Hz GsMTx4, a non-selective inhibitor for CMCs, was added to investigate the proteomic changes induced by CMCs in response to mechanical stretch of HTM. Gene ontology enrichment analysis demonstrated that inhibition of CMCs significantly influenced several biochemical pathways, including store-operated calcium channel activity, microtubule cytoskeleton polarity, toll-like receptor signaling pathway, and neuron cell fate specification. Through heatmap analysis, we grouped 148 differentially expressed proteins (DEPs) into 21 clusters and focused on four specific patterns associated with Ca2+ homeostasis, autophagy, cell cycle, and cell fate. Our results indicated that they might be the critical downstream signals of CMCs adapting to mechanical forces and mediating AH outflow.

Peripheral myelin protein 22 modulates store-operated calcium channel activity, providing insights into Charcot-Marie-Tooth disease etiology

Charcot-Marie-Tooth (CMT) disease is a peripheral neuropathy associated with gene duplication and point mutations in the peripheral myelin protein 22 (PMP22) gene. However, the role of PMP22 in Schwann cell physiology and the mechanisms by which PMP22 mutations cause CMT are not well-understood. On the basis of homology between PMP22 and proteins associated with modulation of ion channels, we hypothesized that PMP22 alters ion channel activity. Using whole-cell electrophysiology, we show here that heterologous PMP22 expression increases the amplitude of currents similar to those ascribed to store-operated calcium (SOC) channels, particularly those involving transient receptor canonical channel 1 (TrpC1). These channels help replenish Ca2+ in the endoplasmic reticulum (ER) following stimulus-induced depletion. Currents with similar properties were recorded in WT but not pmp22 -/- mouse Schwann cells. Heterologous expression of the CMT-associated PMP22_L16P variant, which fails to reach the plasma membrane and localizes to the ER, led to larger currents than WT PMP22. Similarly, Schwann cells isolated from Trembler J (TrJ; PMP22_L16P) mice had larger currents than WT littermates. Calcium imaging in live nerves and cultured Schwann cells revealed elevated intracellular Ca2+ in TrJ mice compared with WT. Moreover, we found that PMP22 co-immunoprecipitated with stromal interaction molecule 1 (STIM1), the Ca2+ sensor SOC channel subunit in the ER. These results suggest that in the ER, PMP22 interacts with STIM1 and increases Ca2+ influx through SOC channels. Excess or mutant PMP22 in the ER may elevate intracellular Ca2+ levels, which could contribute to CMT pathology.

STIMs and Orai1 regulate cytokine production in spinal astrocytes.

BackgroundOur previous study demonstrated that a store-operated calcium channel (SOCC) inhibitor (YM-58483) has central analgesic effects. However, the cellular and molecular mechanisms of such effects remain to be determined. It is well-known that glial cells play important roles in central sensitization. SOC entry (SOCE) has been implicated in many cell types including cortical astrocytes. However, the role of the SOCC family in the function of astrocytes has not been determined. Here, we thoroughly investigated the expression and the functional significance of SOCCs in spinal astrocytes.MethodsPrimary cultured astrocytes were prepared from neonatal (P2–P3) CD1 mice. Expressions of mRNAs and proteins were respectively assessed by real-time PCR and Western blot analysis. SOCE was measured using a calcium imaging system. Live-cell STIM1 translocation was detected using a confocal microscope. Cytokine levels were measured by the enzyme-linked immunosorbent assay.ResultsWe found that the SOCC family is expressed in spinal astrocytes and that depletion of calcium stores from the endoplasmic reticulum by cyclopiazonic acid (CPA) resulted in a large sustained calcium entry, which was blocked by SOCC inhibitors. Using the siRNA knockdown approach, we identified STIM1 and Orai1 as primary components of SOCCs in spinal astrocytes. We also observed thapsigargin (TG)- or CPA-induced puncta formation of STIM1 and Orai1. In addition, activation of SOCCs remarkably promoted TNF-α and IL-6 production in spinal astrocytes, which were greatly attenuated by knockdown of STIM1 or Orai1. Importantly, knockdown of STIM2 and Orai1 dramatically decreased lipopolysaccharide-induced TNF-α and IL-6 production without changing cell viability.ConclusionsThis study presents the first evidence that STIM1, STIM2, and Orai1 mediate SOCE and are involved in cytokine production in spinal astrocytes. Our findings provide the basis for future assessment of SOCCs in pain and other central nervous system disorders associated with abnormal astrocyte activities.

Suppression of STIM1 in the early stage after global ischemia attenuates the injury of delayed neuronal death by inhibiting store-operated calcium entry-induced apoptosis in rats

Ca²⁺ overload is considered to be the most important ion imbalance in the neuronal injury. Store-operated Ca²⁺ entry has been suggested to be a significant mechanism of excessive Ca²⁺ influx in many cells. The role of store-operated Ca²⁺ entry in neuronal ischemic injury has yet to be elucidated. The aim of this study was to assess the role of store-operated calcium channel (SOCC) proteins involved with calcium overload in the induction of delayed neuronal death after global ischemia in rats. A transient RNA interference model of global ischemia in rats was established to determine the role of SOCC-induced Ca²⁺ overload in delayed neuronal death. We found that STIM1 and ORAI1 expression in the hippocampus increased continuously after global ischemia and peaked on day 4. These data were consistent with an increase in the intracellular calcium concentration. Using Stim1 siRNA to suppress SOCC activity in the early stage of ischemia significantly inhibited STIM1 and ORAI1 expression and decreased the intracellular calcium concentration in neurons. In addition, the neurological function of rats improved after the Stim1 siRNA injection. High expression of STIM1 and ORAI may be the source of excessive calcium influx after ischemic damage. Blocking of this SOCC-induced calcium influx could lead to an improved neuronal survival. These data suggest that calcium influx through SOCC is another nonexcitotoxicity mechanism of ischemic neuronal death.

Role of calcium channels responsible for phenylephrine-induced contraction in rat aorta 3 days after acute myocardial infarction

BackgroundPhenylephrine (PE) produces tonic contraction through involvement of various calcium channels such as store-operated calcium channels (SOCCs) and voltage-operated calcium channels (VOCCs). However, the relative contribution of each calcium channel to PE-induced contraction has not been investigated in isolated rat aorta of early acute myocardial infarction (AMI).MethodsEndothelium-denuded rat aortic rings from rats 3 days after AMI or sham-operated (SHAM) rats were prepared in an organ chamber with Krebs-Ringer bicarbonate solution for isometric tension recording. We assessed the PE dose-response relationships in 2.5 mM calcium medium for both groups. The same procedure was repeated using rings pretreated with the SOCC inhibitor 2-aminoethoxydiphenyl borate, sarco/endoplasmic-reticulum calcium ATPase inhibitor thapsigargin (TG), diacyl glycerol lipase inhibitor RHC80267, and sodium-calcium exchanger inhibitor 3,4-dichlorobenzamil hydrochloride for 30 minutes before addition of calcium. When ongoing tonic contraction was sustained, dose-response curves to the VOCC inhibitor nifedipine were obtained to assess the relative contribution of each calcium channel under various conditions.ResultsThe effect of SOCC induction with TG pretreatment on PE-induced contraction was significantly lower in the AMI group compared to the SHAM group. In addition, there were significant decreases in the sensitivity and efficacy of the VOCC inhibitor nifedipine on PE-induced contraction in the AMI group.ConclusionsResults suggest that the change of vascular reactivity of PE in rat aorta 3 days after AMI is characterized by a decreased contribution of L-type VOCCs. The enhanced VOCC-independent calcium entry mechanisms after AMI can be mediated by enhanced capacitative calcium entry through the activation of SOCCs.

Downregulation of AβPP Enhances Both Calcium Content of Endoplasmic Reticulum and Acidic Stores and the Dynamics of Store Operated Calcium Channel Activity

The amyloid-β protein precursor (AβPP) is a type-1 transmembrane protein involved in Alzheimer's disease (AD). It has become increasingly evident that AβPP, its protein-protein interactions, and its proteolytical fragments may affect calcium homeostasis and vice versa. In addition, there is evidence that calcium dysregulation contributes to AD. To study the role of AβPP in calcium homeostasis, we downregulated its expression in SH-SY5Y cells using shRNA (SH-SY5Y/AβPP-) or increased expression of AβPP695 by transfection (SH-SY5Y/AβPP+). The levels of cytosolic Ca2+ after treatment with thapsigargin, monensin, activation of capacitative calcium entry (CCE), and treatment with SKF, a store operated channel (SOCs) inhibitor, were measured by fura-2AM fluorimetry. SH-SY5Y/AβPP+ cells show reduced response to thapsigargin and reduced CCE, although this reduction is not statistically significant. On the other hand, we found that, relative to SH-SY5Y, SH-SY5Y/AβPP- cells show a significant increase in the response to thapsigargin but not in CCE and their SOCs were more susceptible to SKF inhibition. Additionally, downregulation of AβPP resulted in increased response to monensin that induces calcium release from acidic stores. The increase of calcium release from the endoplasmic reticulum and the acidic stores, when AβPP is downregulated, could be attributed to elevated Ca2+ content or to a dysregulation of Ca2+ transfer through their membranes. These data, along with already existing evidence regarding the role of AβPP in calcium homeostasis and the early occurring structural and functional abnormalities of endosomes, further substantiate the role of AβPP in calcium homeostasis and in AD.

Familial Alzheimer's disease-linked presenilin-1 mutation M146V affects store-operated calcium entry: Does gain look like loss?

Alzheimer's disease (AD) is a neurodegenerative disorder that leads to neuron death and synapse loss in the hippocampus and cortex, with consequent cognitive disability and dementia. Mutations in the presenilin-1 (PS1) gene lead to familial Alzheimer's disease (FAD). Here, we report that the expression of FAD-linked PS1 M146V mutant affects store-operated calcium channel activity (Isoc) in human neuroblastoma SK-N-SH cells. Electrophysiological measurements and calcium imaging experiments have revealed the emergent role of calcium sensor STIM2 in the inhibition of calcium release-activated calcium channel activity (Icrac) and enhancement of intracellular Ca(2+) stores content due to PS1 M146V mutant expression. In general, the results of this study suggest that the pathological inhibition of one type of store-operated calcium channels caused by FAD PS1 mutant expression may be accounted for by preceding gain of spontaneous activity of store-operated calcium channels driven by STIM2.

Location of disorder in coiled coilproteins is influenced by its biological role and subcellular localization: a GO-based study on human proteome

Intrinsic disorder in proteins has been explored to study lack of structure-function aspects of many proteins. The current study focuses on coiled coils which are often linked to intrinsic disorder. We present a sequence level analysis of human coiled coils to find out if this is universally true for all coiled coils. When annotated coiled-coil regions were collected from UniProt and investigated with disorder prediction tools namely-IUPred and DISpro, three patterns were commonly observed-disordered coiled coils (DisCCs), ordered coiled coils (OCCs) and the last one having a disordered region outside the coiled-coil region (DOCCs). Differential enrichment in the gene ontology was seen in these three categories. We found that OCCs are enriched in structural components of the extracellular space including the fibrinogen complex and laminin complex. On the contrary, DisCCs were found to be exclusively over-represented in proteins involved in actin filament, lamellipodium, cell junction, macromolecule complexes, ciliary rootlet and nucleolus. DOCCs are found to be associated with many regulatory and adaptor functions including positive regulation of calcium ion transport via store-operated calcium channel activity, cytoskeletal adaptor activity etc. Other than the GO-based analysis, sequence level analysis showed that disordered coiled-coil regions bear a high proportion of low-complexity regions as compared to ordered coiled coils. The former also has a higher probability of forming a dimer as compared to the ordered counterpart. Our study shows that the in silico approach of mapping of disorder in or around coiled coils in other biological systems or organisms can be applied to understand and rationalize the mode of action of these dynamic motifs.

Calcium signaling in mouse oocyte maturation: the roles of STIM1, ORAI1 and SOCE

Calcium handling is critical for the oocyte function, since the first steps of fertilization are dependent on the appropriate Ca(2+) mobilization to originate transient spikes of the cytosolic Ca(2+) concentration. It is well known that the Ca(2+) influx from the extracellular milieu is required to maintain this signaling in mammalian oocytes. However, the regulation of the Ca(2+) channels involved in this process is still unknown in oocytes. STIM1, a key regulator of store-operated Ca(2+) entry (SOCE), relocates in the mouse oocyte shortly after sperm stimulation, suggesting that SOCE is involved in the maintenance of cytosolic Ca(2+)-spiking in the fertilized oocyte. Here, we show that there is an up-regulation of the expression of STIM1 at the germinal vesicle breakdown stage, and this expression remains steady during following maturation stages. We found that oocytes express ORAI1, a store-operated Ca(2+) channel, and that ORAI1 expression level was stable during oocyte maturation. Immature oocytes showed no Ca(2+) entry and no increase in STIM1-ORAI1 colocalization in response to the store depletion induced by thapsigargin. On the contrary, in mature oocytes, STIM1-ORAI1 colocalization is enhanced 3-fold by depletion of Ca(2+) stores, enabling the activation of store-operated calcium channels and therefore Ca(2+) entry. Finally, the correlation between SOCE activation during the maturation of oocytes and STIM1-ORAI1 colocalization strongly suggests that ORAI1 is involved in the Ca(2+) entry pathway in the mature oocyte. SOCE up-regulation in the final stage of maturation is further evidence of a major role for SOCE in fully mature oocytes, and therefore in Ca(2+) signaling at fertilization.

P4.14 Investigating the role of calcium-independent phospholipase [formula omitted] in store-operated calcium entry in mdx muscle

Duchenne muscular dystrophy (DMD) is caused by lack of dystrophin. Characteristic signs of dystrophic muscles are membrane fragility and abnormally elevated levels of intracellular calcium, detrimental to the cells. Increased activity of store-operated calcium channels (SOC) has been proposed as a possible mechanism for the enhanced calcium concentration. Studies of SOC have indicated the enzyme calcium-independent phospholipase A2β (iPLA2β) as an important mediator of calcium influx. We have previously shown that SOC entry in FDB fibres isolated from mdx mice is reduced in the presence of bromoenol lactone (BEL), a suicide inhibitor of iPLA2.

Tunicamycin desensitizes store-operated Ca 2+ entry to ATP and mitochondrial potential

Tunicamycin effect on thapsigargin-induced store-operated calcium entry was investigated. Ca 2+ influx was stimulated by 50% upon exposure of Jurkat cells to tunicamycin. Moreover, tunicamycin efficiently prevented the inhibition of store-operated calcium entry caused by dissipation of mitochondrial membrane potential. Protective action of tunicamycin on store-operated Ca 2+ entry was also partially preserved in Jurkat cells depleted of ATP, while Ca 2+ entry into ATP-deprived cells grown in tunicamycin-free medium was almost completely inhibited. Tunicamycin-evoked changes in cellular Ca 2+ fluxes coincided with decreased glycosylation of STIM1 protein. Although the latter observation is correlative and needs additional confirmation it may suggest that deglycosylation of STIM1 protein deprives store-operated calcium entry system of an important regulatory mechanism. This study suggests a novel mechanism of modulation of the activity of store-operated calcium channels in lymphoidal cells.

Linoleic Acid Induces Calcium Signaling, Src Kinase Phosphorylation, and Neurotransmitter Release in Mouse CD36-positive Gustatory Cells

We have recently demonstrated that the cells expressing CD36, localized apically on the taste buds of mouse lingual circumvallate papillae, act as gustatory cells. In the present study we isolated these CD36-positive cells from mouse circumvallate papillae and investigated intracellular signaling events, triggered by a long-chain polyunsaturated fatty acid, i.e. linoleic acid (LA). LA induced increases in free intracellular calcium concentrations, [Ca(2+)](i), by recruiting calcium from endoplasmic reticulum pool via inositol 1,4,5-triphosphate production followed by calcium influx via opening of store-operated calcium (SOC) channels. LA also induced phosphorylation of Src-protein-tyrosine kinases (Src-PTKs), particularly of Fyn(59) and Yes(62). LA-evoked phosphorylation of Fyn(59) and Yes(62) was implicated in the activation of SOC channels. Reverse transcription-quantitative PCR revealed that the CD36-positive gustatory cells possessed mRNA of enzymes like tryptophan hydroxylase-1, l-aromatic amino acid decarboxylase, tyrosine hydroxylase, and dopamine beta-hydroxylase, involved in the synthesis of monoamine neurotransmitters. Interestingly, the addition of LA to these cells induced the release of 5-hydroxytryptamine and noradrenalin to the extracellular environment. The LA-induced release of these neurotransmitters was curtailed by SOC channel blockers and Src-PTK inhibitors. These results altogether demonstrate that LA binds to mouse CD36-positive gustatory cells, induces Src-PTKs phosphorylation, triggers calcium signaling, and evokes the release of 5-hydroxytryptamine and noradrenalin, which in turn may be implicated in the downstream signaling to the afferent nerve fibers, thus transmitting the output signal from taste buds to the central nervous system.

An EF hand mutation in Stim1 causes premature platelet activation and bleeding in mice

Changes in cytoplasmic Ca2+ levels regulate a variety of fundamental cellular functions in virtually all cells. In nonexcitable cells, a major pathway of Ca2+ entry involves receptor-mediated depletion of intracellular Ca2+ stores followed by the activation of store-operated calcium channels in the plasma membrane. We have established a mouse line expressing an activating EF hand motif mutant of stromal interaction molecule 1 (Stim1), an ER receptor recently identified as the Ca2+ sensor responsible for activation of Ca2+ release-activated (CRAC) channels in T cells, whose function in mammalian physiology is not well understood. Mice expressing mutant Stim1 had macrothrombocytopenia and an associated bleeding disorder. Basal intracellular Ca2+ levels were increased in platelets, which resulted in a preactivation state, a selective unresponsiveness to immunoreceptor tyrosine activation motif-coupled agonists, and increased platelet consumption. In contrast, basal Ca2+ levels, but not receptor-mediated responses, were affected in mutant T cells. These findings identify Stim1 as a central regulator of platelet function and suggest a cell type-specific activation or composition of the CRAC complex.

Regulation of Ca2+ Signaling in Mast Cells by Tyrosine-Phosphorylated and Unphosphorylated Non-T Cell Activation Linker

Engagement of the FcepsilonRI in mast cells and basophils leads to a rapid tyrosine phosphorylation of the transmembrane adaptors LAT (linker for activation of T cells) and NTAL (non-T cell activation linker, also called LAB or LAT2). NTAL regulates activation of mast cells by a mechanism, which is incompletely understood. Here we report properties of rat basophilic leukemia cells with enhanced or reduced NTAL expression. Overexpression of NTAL led to changes in cell morphology, enhanced formation of actin filaments and inhibition of the FcepsilonRI-induced tyrosine phosphorylation of the FcepsilonRI subunits, Syk kinase and LAT and all downstream activation events, including calcium and secretory responses. In contrast, reduced expression of NTAL had little effect on early FcepsilonRI-induced signaling events but inhibited calcium mobilization and secretory response. Calcium response was also repressed in Ag-activated cells defective in Grb2, a major target of phosphorylated NTAL. Unexpectedly, in cells stimulated with thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+) ATPase, the amount of cellular NTAL directly correlated with the uptake of extracellular calcium even though no enhanced tyrosine phosphorylation of NTAL was observed. The combined data indicate that NTAL regulates FcepsilonRI-mediated signaling at multiple steps and by different mechanisms. At early stages NTAL interferes with tyrosine phosphorylation of several substrates and formation of signaling assemblies, whereas at later stages it regulates the activity of store-operated calcium channels through a distinct mechanism independent of enhanced NTAL tyrosine phosphorylation.

Calcium-dependent and calcium-sensitizing pathways in the mature and immature ductus arteriosus

Studies performed in sheep and baboons have shown that after birth, the normoxic muscle media of ductus arteriosus (DA) becomes profoundly hypoxic as it constricts and undergoes anatomic remodeling. We used isolated fetal lamb DA (pretreated with inhibitors of prostaglandin and nitric oxide production) to determine why the immature DA fails to remain tightly constricted during the hypoxic phase of remodeling. Under normoxic conditions, mature DA constricts to 70% of its maximal active tension (MAT). Half of its normoxic tension is due to Ca(2+) entry through calcium L-channels and store-operated calcium (SOC) channels. The other half is independent of extracellular Ca(2+) and is unaffected by inhibitors of sarcoplasmic reticulum (SR) Ca(2+) release (ryanodine) or reuptake [cyclopiazonic acid (CPA)]. The mature DA relaxes slightly during hypoxia (to 60% MAT) due to decreases in calcium L-channel-mediated Ca(2+) entry. Inhibitors of Rho kinase and tyrosine kinase inhibit both Ca(2+)-dependent and Ca(2+)-independent DA tension. Although Rho kinase activity may increase during gestation, immature DA develop lower tensions than mature DA, primarily because of differences in the way they process Ca(2+). Calcium L-channel expression increases with advancing gestation. Under normoxic conditions, differences in calcium L-channel-mediated Ca(2+) entry account for differences in tension between immature (60% MAT) and mature (70% MAT) DA. Under hypoxic conditions, differences in both calcium L-channel-dependent and calcium L-channel-independent Ca(2+) entry, account for differences in tension between immature (33% MAT) and mature (60% MAT) DA. Stimulation of Ca(2+) entry through reverse-mode Na(+)/Ca(2+) exchange or CPA-induced SOC channel activity constrict the DA and eliminate differences between immature and mature DA during both hypoxia and normoxia.