Release-activated Ca2 Research Articles

Cholesterol Regulates Orai1 Function

STIM1 and Orai proteins represent the essential molecular components of Ca2+ release-activated Ca2+ (CRAC) channels. Following ER-store depletion, CRAC current activation occurs due to the physical interaction between the STIM1 C-terminus and Orai1 N- and C-termini. Here we focused on an additional role of the extended transmembrane Orai1 N-terminal (ETON) region in cholesterol binding as it contains a cholesterol binding motif. Both chemically induced cholesterol depletion as well as point mutations disrupting the Orai1 cholesterol binding site enhanced store-operated Orai1 currents about 2-fold. Currents were not increased due to enhanced plasma membrane expression as revealed by biotinylation experiments. In addition, Orai1 point mutants that disrupted the cholesterol binding motif were not anymore sensitive to chemically induced cholesterol depletion. In accordance, employing intrinsic fluorescence measurements we detected direct binding of cholesterol to an N-terminal fragment containing the cholesterol binding motif with an equilibrium dissociation constant (KD) of about 2µM, while mutations disrupting it increased the KD 3-5 fold compared to wild-type. In aggregate, we propose a modulatory role of cholesterol on CRAC channel function. (supported by the Austrian Science Fund: FWF project P25210 to I.D., FWF project M01506000 to I.J. and FWF project P25172 to C.R.)

Interplay of Orai1-Loop3 with Extracellular Ca2+ Binding Sites in Loop1 Controls Crac Channel Activity

Ca2+ release-activated Ca2+ (CRAC) channels are a major pathway for Ca2+ entry in a vast majority of cell types. The identification of the ER Ca2+ sensor STIM1 and the plasma membrane channel subunit Orai1 lead to a substantial progress in our understanding of molecular structures underlying the unique permeation properties of CRAC channels. Nevertheless, Ca2+ coordination at the pore entrance and the role of the extracellular loop1 and loop3 of hOrai1 proteins still remain rather unclear. Based on a recent crystal structure of the drosophila Orai channel we generated an all-atom model of human Orai1 including the missing extracellular loops. Using a combined approach of patch-clamp, cysteine-crosslinking and molecular dynamics simulations we investigated the pore entrance of hOrai1 channels. Our experiments showed that the extracellular loop3 of hOrai1 exhibited a high degree of crosslinking along the whole loop compatible with a remarkable flexibility. Crosslinking studies between loop1 and loop3 demonstrated close proximity, suggesting a movement of loop3 affecting the pore entrance. Molecular dynamics simulations indicated that Ca2+ ions bind to the extracellular aspartates located in the first loop. Further, an interaction of the third loop with these negatively charged amino acids seems to compete with the Ca2+ ions, thereby reducing CRAC channel activity. Sequence alignment of Orai1 isoforms identified that the essential residues for hOrai1 loop1-loop3 coupling are conserved in higher mammals. With this work we suggest a pivotal/regulatory role of the third loop in modulating hOrai1 function. Via movement and subsequent coupling of loop3 to aspartates in the first loop it may regulate the Ca2+ sink at the pore entrance and thus permeation through CRAC channels. This work was supported by the Austrian Science Foundation (FWF): P22747, P26067 (R.S.), P25172 (C.R.), V286 (I.F.).

STIM1 Enhances SR Ca2+ Refilling through Activating SERCA2a in Rat Ventricular Myocytes

In cardiac ventricular myocytes, the function of “stromal interaction molecule 1” (STIM1), an ER/SR Ca2+ sensor, is still a mystery regarding its localization, mobilization, and Ca2+ signaling regulation. Here, adult rat ventricular myocytes freshly isolated or in primary culture were examined. Endogenous STIM1 was found by immunofluorescence and shown to be distributed mainly within the Z-disk. Using mcherry tagged expression of STIM1, we found that re-distribution of STIM1 does not occur after SR Ca2+ depletion (using thapsigargin, 2 μM) nor does SR Ca2+ depletion affect STIM1 movement within the SR when evaluated with fluorescence recovery after photobleaching (FRAP). Consistent with this result, native protein electrophoresis showed that STIM1 exists mainly as an oligomer, which is not altered upon SR Ca2+ depletion. Additionally, no store-operated Ca2+ entry (SOCE) or Ca2+ release-activated Ca2+ current (ICRAC) was observed in control or STIM1 overexpressing ventricular myocytes. The overexpression of STIM1 did, however, have dramatic consequences in ventricular myocytes: The SR Ca2+ content did increase as did SR Ca2+ leak. Parallel investigations indicated that STIM1 physically binds to phospholamban (PLN), suggesting that overexpressed STIM1 may activate SERCA2a by regulating PLN and this may underlie the increase in SR Ca2+ content. The molecular signaling and regulatory pathways that may be involved in these pathways are discussed.

Complex Function of Stim1 in the Activation of Store-Independent Orai Channels

Orai proteins contribute to Ca2+ entry pathways through store-dependent, Ca2+ release-activated Ca2+ (CRAC) channels (Orai1), and store-independent, arachidonic acid (AA)-regulated Ca2+ (ARC) or LeukotrieneC4-regulated Ca2+ (LRC) channels (Orai1 and Orai3). Remarkably, although activated by fundamentally different mechanisms, both CRAC and ARC/LRC channels share a requirement for STIM1 expression. To date the role of endoplasmic reticulum-resident STIM1 (ER-STIM1) in the activation of CRAC channels is well appreciated. There is a minor pool of STIM1 at the plasma membrane (PM-STIM1) that was shown to be necessary for ARC current activation in HEK293 cells. Using pharmacological tools targeting AA synthesis and metabolism, Ca2+ imaging, whole-cell and perforated patch clamp electrophysiological recordings we demonstrate that both Orai1 and Orai3 are required for ARC and LRC current activation in both primary vascular smooth muscle cells (VSMCs) and HEK293 cells. Surprisingly, while PM-STIM1 is required for ARC and LRC current activation under whole cell patch clamp recordings in both cell types, ER-STIM1 is sufficient for both ARC and LRC channel activation when intact cells are considered. These results are first to demonstrate ARC channel function in primary VSMCs, highlight the complexity of STIM1 regulation of store-independent Orai channels and demonstrate that ARC and LRC currents are mediated by the same channels.

Orai3 TM3 Point Mutation G158C Alters Kinetics of 2-Apb-Induced Gating by Disulfide Bridge Formation with TM2 C101

After endoplasmic reticulum (ER) Ca2+ store depletion, Orai channels in the plasma membrane (PM) are activated directly by ER-resident STIM proteins to form the Ca2+-selective Ca2+ release-activated Ca2+ (CRAC) channel. However, in the absence of Ca2+ store depletion and STIM interaction, the mammalian homolog Orai3 can be activated by 2-aminoethyl diphenylborinate (2-APB), resulting in a nonselective cation conductance characterized by biphasic inward and outward rectification. Here, we use site-directed mutagenesis and patch-clamp analysis to better understand the mechanism by which 2-APB activates Orai3. We find that point mutation of glycine 158 in the third transmembrane (TM) segment to cysteine, but not alanine, slows the kinetics of 2-APB activation and prevents complete channel closure upon 2-APB washout. Orai3-G158C channels are trapped in an open state, as indicated by a lack of recovery upon 2-APB washout. The “slow” phenotype exhibited by Orai3 mutant G158C reveals distinct open states, characterized by reversal potentials that shift gradually from 47 to 7 mV during activation. The slow phenotype can be reversed by application of the reducing reagent bis(2-mercaptoethylsulfone) (BMS) in a state-dependent manner, only during 2-APB activation. Moreover, the double mutant C101G/G158C, in which an endogenous TM2 cysteine is changed to glycine, does not exhibit altered kinetics of 2-APB activation. We suggest that a disulfide bridge, formed between the introduced cysteine at TM3 position 158 and the endogenous cysteine at TM2 position 101, hinders transitions between Orai3 2-APB-induced open and closed states. Our data provide functional confirmation of the proximity of these two residues and suggest a location within the Orai3 protein that mediates activation and gating by 2-APB.

Ph Dependence of Orai1 and Orai3 Store-Operated Current

Calcium influx through the Ca2+ release-activated Ca2+ (CRAC) channel plays a critical role in human T-cell activation. As the prototypical store-operated Ca2+ channel, CRAC channel opening requires direct molecular interaction between the endoplasmic reticulum-resident Ca2+ sensor STIM1 and plasma membrane pore-forming Orai subunits. Aside from Orai1, mammals express two homologs: Orai2 and Orai3. In this study we found that unlike Orai1, which is blocked by acidic pH and activated by alkaline pH, the Orai3 Stim1-activated current is potentiated by protons. Following current development, replacement of Ringer solution to pH 6.0 alters the I-V shape, inducing a “bulge” of inward current at −30 mV. Ion substitution experiments revealed that this current is mostly conducted by Na+ and is blocked by external Ca2+. Orai1 and Orai3 are identical through TM1 and the critical glutamate that confers Ca2+ selectivity; yet only Orai3 exhibits the acid-induced increase of sodium permeability. Using Orai1-Orai3 chimeras we identified several parts of the channel responsible for pH sensitivity. Among the three Orai homologs, Orai1 is unique in being glycosylated. Point mutation of the N223 glycosylation site (N223A) results in acid-induced Na+ current, suggesting that glycosylation can fine-tune the ion selectivity of Orai1. Orai3 with the internal second loop taken from Orai1 gains alkaline sensitivity but the reciprocal chimera is indistinguishable from Orai1, suggesting that there should be at least two sites responsible for alkaline activation. Orai3 with TM2-loop2-TM3 taken from Orai1 loses acid sensitivity but the reciprocal chimera does not gain it, indicating the presence of at least two sites sensitive to protons. This novel property of Orai3 - the ability of STIM1-operated Na+ current to be activated by acidic pH - may provide clues for its possible physiological functions.

Atomistic Molecular Dynamics Simulations of Drosophila Orai in a Hydrated Lipid Bilayer

Orai channels are prominent (calcium) Ca2+ signal mediators in many cell types. In T lymphocytes, Orai1 and STIM1 form the Ca2+ release-activated Ca2+ (CRAC) channel. Following endoplasmic reticulum (ER) Ca2+ store depletion, ER membrane-resident Ca2+ sensor STIM1 physically interacts with plasma membrane-bound, pore-forming Orai1 subunits. The STIM1-Orai1 interaction results in Ca2+ influx and ultimately changes in gene expression that underlie the immune response. In 2012, the Drosophila Orai (dOrai) crystal structure was solved to 3.34 A resolution. To develop an atomistic view of Orai channel function, given the high sequence identity (73%) between dOrai and human Orai1, we constructed a complete dOrai structure by building in flexible loops missing in the crystal structure and adjusting the protonation state of several pore residues. The model was placed into a lipid bilayer with excess hydration and simulated for over 200 ns. We find shifted pKa values for several titratable residues in the pore interior, and that changing protonation states of these key residues is essential to maintain pore stability. To explore the mechanism of channel block, we also simulated the model with either Ca2+ or (gadolinium) Gd3+ in the selectivity filter. Taken together, our results help to address the stability of the closed structure in a lipid bilayer while also yielding novel atomistic insights into channel block and pH sensitivity.This work was supported by NIH grants F30CA171717 to M.L.W., P01GM86685 to D.J.T., and NS-14609 to M.D.C.1. Hou, X, et al. Science. (2012); 338; 1308-13.

Key Components of Store-Operated Ca2+ Entry in Non-Excitable Cells

Store-operated Ca(2+) entry (SOCE) is a ubiquitous Ca(2+) entry pathway in non-excitable cells. It is activated by the depletion of Ca(2+) from intracellular Ca(2+) stores, notably the endoplasmic reticulum (ER). In the past 9 years, it has been established that two key proteins, stromal interacting molecule 1 (STIM1) and Orai1, play critical roles in SOCE. STIM1 is a single-pass transmembrane protein located predominantly in the ER that serves as a Ca(2+) sensor within the ER, while Orai1 is a tetraspanning plasma membrane (PM) protein that functions as the pore-forming subunit of store-operated Ca(2+) channels. A decrease in the ER Ca(2+) concentration induces translocation of STIM1 into puncta close to the PM. STIM1 oligomers directly interact with Orai1 channels and activates them. This review summarizes the molecular basis of the interaction between STIM1 and Orai1 in SOCE. Further, we describe current findings on additional regulatory proteins, such as Ca(2+) release-activated Ca(2+) regulator 2A and septin, novel roles of STIM1, and modulation of SOCE by protein phosphorylation.

Akt2- and ETS1-Dependent IP3 Receptor 2 Expression in Dendritic Cell Migration

Background/Aims: The protein kinase Akt2/PKBβ is a known regulator of macrophage and dendritic cell (DC) migration. The mechanisms linking Akt2 activity to migration remained, however, elusive. DC migration is governed by Ca²⁺ signaling. We thus explored whether Akt2 regulates DC Ca²⁺ signaling. Methods: DCs were derived from bone marrow of Akt2-deficient mice (akt2^-/-) and their wild type littermates (akt2^+/+). DC maturation was induced by lipopolysaccharides (LPS) and evaluated by flow cytometry. Cytosolic Ca²⁺ concentration was determined by Fura-2 fluorescence, channel activity by whole cell recording, transcript levels by RT-PCR, migration utilizing transwells. Results: Upon maturation, chemokine CCL21 stimulated migration of akt2^+/+ but not akt2^-/- DCs. CCL21-induced increase in cytosolic Ca²⁺ concentration, thapsigargin-induced release of Ca²⁺ from intracellular stores with subsequent store-operated Ca²⁺ entry (SOCE), ATP-induced inositol 1,4,5-trisphosphate (IP₃)-dependent Ca²⁺ release as well as Ca²⁺ release-activated Ca²⁺ (CRAC) channel activity were all significantly lower in mature akt2^-/- than in mature akt2^+/+ DCs. Transcript levels of IP₃ receptor IP₃R2 and of IP₃R2 regulating transcription factor ETS1 were significantly higher in akt2^+/+ than in akt2^-/- DCs prior to maturation and were upregulated by LPS stimulation (1h) in akt2^+/+ and to a lower extent in akt2^-/- DCs. Following maturation, protein abundance of IP₃R2 and ETS1 were similarly higher in akt2^+/+ than in akt2^-/- DCs. The IP₃R inhibitor Xestospongin C significantly decreased CCL21-induced migration of akt2^+/+DCs and abrogated the differences between genotypes. Finally, knock-down of ETS1 with siRNA decreased IP₃R2 mRNA abundance, thapsigargin- and ATP-induced Ca²⁺ release, SOCE and CRAC channel activation, as well as DC migration. Conclusion: Akt2 upregulates DC migration at least in part by ETS1-dependent stimulation of IP₃R2 transcription.

TRIC-A Prevents Store-Overload Induced Calcium Release Through Interaction with the Cardiac Ryanodine Receptor

TRIC-A and TRIC-B are trimeric intracellular cation channels located at the sarcoplasmic reticulum (SR) or endoplasmic reticulum (ER) of multiple cell types. These channels regulate the permeability of K ions across the SR/ER and consequently the movement of Ca ions during excitation-contraction coupling. Previously we showed that genetic ablation of TRIC led to compromised K-permeability and Ca release across the SR membrane, supporting the hypothesis that TRIC could function as counter-ion channels that allows the flow of K ions into the SR during the acute phase of Ca release. In the absence of TRIC, overload of Ca inside the ER/SR causes instability of Ca storage and release, leading to stress-induced dysfunction of multiple tissues. Spontaneous Ca waves, also called store overload-induced Ca release (SOICR) mediated by the type 2 ryanodine receptor (RyR2), evoke ventricular tachyarrhythmia in individuals with heart failure. Our biochemical studies revealed that the carboxyl-tail domain of TRIC-A could interact with the RyR channel, suggesting the possibility that TRIC-A may directly regulate the Ca release activity. We found that TRIC-A, but not TRIC-B, prevented the appearance of SOICR in HEK293 cells expressing RyR2. Cytosolic Ca measurement by Fura-2 and ER luminal Ca measurement by D1ER revealed that expression of TRIC-A in HEK293 cells could prevent overload of Ca inside the ER by targeting the RyR2 channel function. Such effect was translated into suppression of SOICR. These effects appeared to be specific for TRIC-A, as co-expression of RyR2 with TRIC-B did not affect SOICR. Together, our data suggest that functional interaction between TRIC-A and RyR can modulate the Ca release process from internal stores and regulate Ca homeostasis across the ER/SR.

Distribution and Ca2+ signalling of fibroblast‐like (PDGFRα+) cells in the murine gastric fundus

Platelet-derived growth factor receptor α positive (PDGFRα(+)) cells are suggested to mediate purinergic inputs in GI muscles, but the responsiveness of these cells to purines in situ has not been evaluated. We developed techniques to label and visualize PDGFRα(+) cells in murine gastric fundus, load cells with Ca(2+) indicators, and follow their activity via digital imaging. Immunolabelling demonstrated a high density of PDGFRα(+) cells in the fundus. Cells were isolated and purified by fluorescence-activated cell sorting (FACS) using endogenous expression of enhanced green fluorescent protein (eGFP) driven off the Pdgfra promoter. Quantitative PCR showed high levels of expression of purinergic P2Y1 receptors and SK3 K(+) channels in PDGFRα(+) cells. Ca(2+) imaging was used to characterize spontaneous Ca(2+) transients and responses to purines in PDGFRα(+) cells in situ. ATP, ADP, UTP and β-NAD elicited robust Ca(2+) transients in PDGFRα(+) cells. Ca(2+) transients were also elicited by the P2Y1-specific agonist (N)-methanocarba-2MeSADP (MRS-2365), and inhibited by MRS-2500, a P2Y1-specific antagonist. Responses to ADP, MRS-2365 and β-NAD were absent in PDGFRα(+) cells from P2ry1((-/-)) mice, but responses to ATP were retained. Purine-evoked Ca(2+) transients were mediated through Ca(2+) release mechanisms. Inhibitors of phospholipase C (U-73122), IP3 (2-APB), ryanodine receptors (Ryanodine) and SERCA pump (cyclopiazonic acid and thapsigargin) abolished Ca(2+) transients elicited by purines. This study provides a link between purine binding to P2Y1 receptors and activation of SK3 channels in PDGFRα(+) cells. Activation of Ca(2+) release is likely to be the signalling mechanism in PDGFRα(+) cells responsible for the transduction of purinergic enteric inhibitory input in gastric fundus muscles.

ICRAC controls the rapid androgen response in human primary prostate epithelial cells and is altered in prostate cancer

Labelled 5α-dihydrotestosterone (DHT) binding experiments have shown that expression levels of (yet unidentified) membrane androgen receptors (mAR) are elevated in prostate cancer and correlate with a negative prognosis. However, activation of these receptors which mediate a rapid androgen response can counteract several cancer hallmark functions such as unlimited proliferation, enhanced migration, adhesion and invasion and the inability to induce apoptosis. Here, we investigate the downstream signaling pathways of mAR and identify rapid DHT induced activation of store-operated Ca2+ entry (SOCE) in primary cultures of human prostate epithelial cells (hPEC) from non-tumorous tissue. Consequently, down-regulation of Orai1, the main molecular component of Ca2+ release-activated Ca2+ (CRAC) channels results in an almost complete loss of DHT induced SOCE. We demonstrate that this DHT induced Ca2+ influx via Orai1 is important for rapid androgen triggered prostate specific antigen (PSA) release. We furthermore identified alterations of the molecular components of CRAC channels in prostate cancer. Three lines of evidence indicate that prostate cancer cells down-regulate expression of the Orai1 homolog Orai3: First, Orai3 mRNA expression levels are significantly reduced in tumorous tissue when compared to non-tumorous tissue from prostate cancer patients. Second, mRNA expression levels of Orai3 are decreased in prostate cancer cell lines LNCaP and DU145 when compared to hPEC from healthy tissue. Third, the pharmacological profile of CRAC channels in prostate cancer cell lines and hPEC differ and siRNA based knock-down experiments indicate changed Orai3 levels are underlying the altered pharmacological profile. The cancer-specific composition and pharmacology of CRAC channels identifies CRAC channels as putative targets in prostate cancer therapy.

Chimeras of sperm PLC reveal disparate protein domain functions in the generation of intracellular Ca2+ oscillations in mammalian eggs at fertilization

Phospholipase C-zeta (PLCζ) is a sperm-specific protein believed to cause Ca(2+) oscillations and egg activation during mammalian fertilization. PLCζ is very similar to the somatic PLCδ1 isoform but is far more potent in mobilizing Ca(2+) in eggs. To investigate how discrete protein domains contribute to Ca(2+) release, we assessed the function of a series of PLCζ/PLCδ1 chimeras. We examined their ability to cause Ca(2+) oscillations in mouse eggs, enzymatic properties using in vitro phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and their binding to PIP2 and PI(3)P with a liposome interaction assay. Most chimeras hydrolyzed PIP2 with no major differences in Ca(2+) sensitivity and enzyme kinetics. Insertion of a PH domain or replacement of the PLCζ EF hands domain had no deleterious effect on Ca(2+) oscillations. In contrast, replacement of either XY-linker or C2 domain of PLCζ completely abolished Ca(2+) releasing activity. Notably, chimeras containing the PLCζ XY-linker bound to PIP2-containing liposomes, while chimeras containing the PLCζ C2 domain exhibited PI(3)P binding. Our data suggest that the EF hands are not solely responsible for the nanomolar Ca(2+) sensitivity of PLCζ and that membrane PIP2 binding involves the C2 domain and XY-linker of PLCζ. To investigate the relationship between PLC enzymatic properties and Ca(2+) oscillations in eggs, we have developed a mathematical model that incorporates Ca(2+)-dependent InsP3 generation by the PLC chimeras and their levels of intracellular expression. These numerical simulations can for the first time predict the empirical variability in onset and frequency of Ca(2+) oscillatory activity associated with specific PLC variants.

Amelioration of Muc5b mucin hypersecretion is enhanced by IL-33 after 2-APB administration in a murine model of allergic rhinitis

We attempted to clarify whether hypersecretion of Muc5b mucin from mouse nasal submucosal glands that is enhanced by interleukin (IL)-33 under allergic conditions can be ameliorated by administration of 2-APB. Immunohistochemistry was used to examine both the distribution of T cells in the nasal mucosa of an allergic rhinitis mouse model and expressions of IL-33 receptor ST2 and Muc5b protein in mouse submucosal gland cells. The amounts of protein and mRNA of Orai1, Muc5b, IL-4, IL-5, IL-13 and IL-33 in mouse nasal lavage fluid (NLF) and nasal mucosa were determined using enzyme-linked immunosorbent assay and real-time reverse transcription-polymerase chain reaction. Expressions of Orai1, Muc5b, IL-4, IL-5, IL-13 and IL-33 were up-regulated in the allergic state and IL-33 increased the levels of Muc5b, IL-4, IL-5 and IL-13, but did not influence proliferation of T cells; however, ST2 was diminished in nasal submucosal gland cells. 2-APB reduced proliferation of T cells and the Orai1 level in the nasal mucosa. It also reduced the concentrations of IL-4, IL-5 and IL-13 in NLF and nasal mucosa, and hypersecretion of Muc5b from glandular cells that was enhanced by IL-33, but did not affect IL-33 production. 2-APB decreased Muc5b mucin hypersecretion from submucosal gland that was enhanced by IL-33 in allergic mice by limiting Ca2+ release-activated Ca2+ channel activity in which Orai1 plays a crucial role in the gland cells and/or by controlling channel activation in T cells and proliferation of these cells.

Effects of the Inflammatory Cytokines TNF-α and IL-13 on Stromal Interaction Molecule–1 Aggregation in Human Airway Smooth Muscle Intracellular Ca2+ Regulation

Inflammation elevates intracellular Ca(2+) ([Ca(2+)]i) concentrations in airway smooth muscle (ASM). Store-operated Ca(2+) entry (SOCE) is an important source of [Ca(2+)]i mediated by stromal interaction molecule-1 (STIM1), a sarcoplasmic reticulum (SR) protein. In transducing SR Ca(2+) depletion, STIM1 aggregates to form puncta, thereby activating SOCE via interactions with a Ca(2+) release-activated Ca(2+) channel protein (Orai1) in the plasma membrane. We hypothesized that STIM1 aggregation is enhanced by inflammatory cytokines, thereby augmenting SOCE in human ASM cells. We used real-time fluorescence microscopic imaging to assess the dynamics of STIM1 aggregation and SOCE after exposure to TNF-α or IL-13 in ASM cells overexpressing yellow fluorescent protein-tagged wild-type STIM1 (WT-STIM1) and STIM1 mutants lacking the Ca(2+)-sensing EF-hand (STIM1-D76A), or lacking the cytoplasmic membrane binding site (STIM1ΔK). STIM1 aggregation was analyzed by monitoring puncta size during the SR Ca(2+) depletion induced by cyclopiazonic acid (CPA). We found that puncta size was increased in cells expressing WT-STIM1 after CPA. However, STIM1-D76A constitutively formed puncta, whereas STIM1ΔK failed to form puncta. Furthermore, cytokines increased basal WT-STIM1 puncta size, and the SOCE triggered by SR Ca(2+) depletion was increased in cells expressing WT-STIM1 or STIM1-D76A. Meanwhile, SOCE in cells expressing STIM1ΔK and STIM1 short, interfering RNA (siRNA) was decreased. Similarly, in cells overexpressing STIM1, the siRNA knockdown of Orai1 blunted SOCE. However, exposure to cytokines increased SOCE in all cells, increased basal [Ca(2+)]i, and decreased SR Ca(2+) content. These data suggest that cytokines induce a constitutive increase in STIM1 aggregation that contributes to enhanced SOCE in human ASM after inflammation. Such effects of inflammation on STIM1 aggregations may contribute to airway hyperresponsiveness.

The Extended Transmembrane Orai1 N-terminal (ETON) Region Combines Binding Interface and Gate for Orai1 Activation by STIM1

STIM1 and Orai1 represent the two molecular key components of the Ca(2+) release-activated Ca(2+) channels. Their activation involves STIM1 C terminus coupling to both the N terminus and the C terminus of Orai. Here we focused on the extended transmembrane Orai1 N-terminal (ETON, aa73-90) region, conserved among the Orai family forming an elongated helix of TM1 as recently shown by x-ray crystallography. To identify "hot spot" residues in the ETON binding interface for STIM1 interaction, numerous Orai1 constructs with N-terminal truncations or point mutations within the ETON region were generated. N-terminal truncations of the first four residues of the ETON region or beyond completely abolished STIM1-dependent Orai1 function. Loss of Orai1 function resulted from neither an impairment of plasma membrane targeting nor pore damage, but from a disruption of STIM1 interaction. In a complementary approach, we monitored STIM1-Orai interaction via Orai1 V102A by determining restored Ca(2+) selectivity as a consequence of STIM1 coupling. Orai1 N-terminal truncations that led to a loss of function consistently failed to restore Ca(2+) selectivity of Orai1 V102A in the presence of STIM1, demonstrating impairment of STIM1 binding. Hence, the major portion of the ETON region (aa76-90) is essential for STIM1 binding and Orai1 activation. Mutagenesis within the ETON region revealed several hydrophobic and basic hot spot residues that appear to control STIM1 coupling to Orai1 in a concerted manner. Moreover, we identified two basic residues, which protrude into the elongated pore to redound to Orai1 gating. We suggest that several hot spot residues in the ETON region contribute in aggregate to the binding of STIM1, which in turn is coupled to a conformational reorientation of the gate.

Interference with Ca2+ release activated Ca2+ (CRAC) channel function delays T‐cell arrest in vivo

Entry of lymphocytes into secondary lymphoid organs (SLOs) involves intravascular arrest and intracellular calcium ion ([Ca(2+)]i) elevation. TCR activation triggers increased [Ca(2+)]i and can arrest T-cell motility in vitro. However, the requirement for [Ca(2+)]i elevation in arresting T cells in vivo has not been tested. Here, we have manipulated the Ca(2+) release-activated Ca(2+) (CRAC) channel pathway required for [Ca(2+)]i elevation in T cells through genetic deletion of stromal interaction molecule (STIM) 1 or by expression of a dominant-negative ORAI1 channel subunit (ORAI1-DN). Interestingly, the absence of CRAC did not interfere with homing of naïve CD4(+) T cells to SLOs and only moderately reduced crawling speeds in vivo. T cells expressing ORAI1-DN lacked TCR activation induced [Ca(2+)]i elevation, yet arrested motility similar to control T cells in vitro. In contrast, antigen-specific ORAI1-DN T cells had a twofold delayed onset of arrest following injection of OVA peptide in vivo. CRAC channel function is not required for homing to SLOs, but enhances spatiotemporal coordination of TCR signaling and motility arrest.

The role of Ca2+ in the pathophysiology of pancreatitis

Acute pancreatitis is a human disease in which the pancreatic pro-enzymes, packaged into the zymogen granules of acinar cells, become activated and cause autodigestion. The main causes of pancreatitis are alcohol abuse and biliary disease. A considerable body of evidence indicates that the primary event initiating the disease process is the excessive release of Ca2+ from intracellular stores, followed by excessive entry of Ca2+ from the interstitial fluid. However, Ca2+ release and subsequent entry are also precisely the processes that control the physiological secretion of digestive enzymes in response to stimulation via the vagal nerve or the hormone cholecystokinin. The spatial and temporal Ca2+ signal patterns in physiology and pathology, as well as the contributions from different organelles in the different situations, are therefore critical issues. There has recently been significant progress in our understanding of both physiological stimulus–secretion coupling and the pathophysiology of acute pancreatitis. Very recently, a promising potential therapeutic development has occurred with the demonstration that the blockade of Ca2+ release-activated Ca2+ currents in pancreatic acinar cells offers remarkable protection against Ca2+ overload, intracellular protease activation and necrosis evoked by a combination of alcohol and fatty acids, which is a major trigger of acute pancreatitis.

Mechanisms of STIM1 Activation of Store-Independent Leukotriene C4-Regulated Ca2+ Channels

We recently showed, in primary vascular smooth muscle cells (VSMCs), that the platelet-derived growth factor activates canonical store-operated Ca(2+) entry and Ca(2+) release-activated Ca(2+) currents encoded by Orai1 and STIM1 genes. However, thrombin activates store-independent Ca(2+) selective channels contributed by both Orai3 and Orai1. These store-independent Orai3/Orai1 channels are gated by cytosolic leukotriene C4 (LTC4) and require STIM1 downstream LTC4 action. However, the source of LTC4 and the signaling mechanisms of STIM1 in the activation of this LTC4-regulated Ca(2+) (LRC) channel are unknown. Here, we show that upon thrombin stimulation, LTC4 is produced through the sequential activities of phospholipase C, diacylglycerol lipase, 5-lipo-oxygenease, and leukotriene C4 synthase. We show that the endoplasmic reticulum-resident STIM1 is necessary and sufficient for LRC channel activation by thrombin. STIM1 does not form sustained puncta and does not colocalize with Orai1 either under basal conditions or in response to thrombin. However, STIM1 is precoupled to Orai3 and Orai3/Orai1 channels under basal conditions as shown using Forster resonance energy transfer (FRET) imaging. The second coiled-coil domain of STIM1 is required for coupling to either Orai3 or Orai3/Orai1 channels and for LRC channel activation. We conclude that STIM1 employs distinct mechanisms in the activation of store-dependent and store-independent Ca(2+) entry pathways.

An aromatic amino acid in the coiled-coil 1 domain plays a crucial role in the auto-inhibitory mechanism of STIM1

STIM1 (stromal interaction molecule 1) is one of the key elements that mediate store-operated Ca²⁺ entry via CRAC (Ca²⁺- release-activated Ca²⁺) channels in immune and non-excitable cells. Under physiological conditions, the intramolecular auto-inhibitions in STIM1 C- and STIM1 N-termini play essential roles in keeping STIM1 in an inactive state. However, the auto-inhibitory mechanism of the STIM1 C-terminus is still unclear. In the present study, we first predicted a short inhibitory domain (residues 310-317) in human STIM1 that might determine the different localizations of human STIM1 from Caenorhabditis elegans STIM1 in resting cells. Next, we confirmed the prediction and further identified an aromatic amino acid residue, Tyr³¹⁶, that played a crucial role in maintaining STIM1 in a closed conformation in quiescent cells. Full-length STIM1-Y316A formed constitutive clusters near the plasma membrane and activated the CRAC channel in the resting state when co-expressed with Orai1. The introduction of a Y316A mutation caused the higher-order oligomerization of the in vitro purified STIM1 fragment containing both the auto-inhibitory domain and CAD(CRAC-activating domain).We propose that the Tyr³¹⁶ residue may be involved in the auto-inhibitory mechanism of the STIM1 C-terminus in the quiescent state. This inhibition could be achieved either by interacting with the CAD using hydrogen and/or hydrophobic bonds, or by an intermolecular interaction using repulsive forces, which maintained a dimeric STIM1.