CRAC Channel Research Articles

Complex molecular and functional outcomes of single versus sequential cytokine stimulation of rat microglia.

BackgroundMicroglia are the “professional” phagocytes of the CNS. Phagocytosis is crucial for normal CNS development and maintenance, but it can be either beneficial or detrimental after injury or disease. For instance, white matter damage releases myelin debris that must be cleared by microglia in order for re-myelination to occur. However, phagocytosis can also produce damaging reactive oxygen species (ROS). Furthermore, microglia can acquire pro-inflammatory (M1) or anti-inflammatory (M2) activation states that affect cell functions. Although microglia are exposed to a changing cytokine environment after injury or disease, little is known about the molecular and functional consequences. Therefore, we applied several microglial activation paradigms, with or without myelin debris. We assessed (i) gene expression changes reflecting microglial activation and inflammatory states, and receptors and enzymes related to phagocytosis and ROS production, (ii) myelin phagocytosis and production of ROS, and (iii) expression and contributions of several ion channels that are considered potential targets for regulating microglial behavior.MethodsPrimary rat microglia were exposed to cytokines, individually or sequentially. First, responses to individual M1 or M2 stimuli were compared: IFN-γ plus TNF-α (“I + T”; M1 activation), interleukin-4 (M2a/alternative activation), and interleukin-10 (M2c/acquired deactivation). Second, sequential cytokine addition was used to assess microglia repolarization and cell functions. The paradigms were M2a→M1, M2c→M1, M1→M2a, and M1→M2c.ResultsM1 stimulation increased pro-inflammatory genes, phagocytosis, and ROS, as well as expression of Kv1.3, KCa3.1, and Kir2.1 channels. M2a stimulation increased anti-inflammatory genes, ROS production, and Kv1.3 and KCa3.1 expression. Myelin phagocytosis enhanced the M1 profile and dampened the M2a profile, and both phagocytosis and ROS production were dependent on NOX enzymes and Kir2.1 and CRAC channels. Importantly, microglia showed some capacity for re-polarization between M1 and M2a states, based on gene expression changes, myelin phagocytosis, and ROS production.ConclusionsIn response to polarizing and re-polarizing cytokine treatments, microglia display complex changes in gene transcription profiles, phagocytic capacity, NOX-mediated ROS production, and in ion channels involved in microglial activation. Because these changes might affect microglia-mediated CNS inflammation, they should be considered in future experimental, pre-clinical studies.

The anti-proliferative effect of cation channel blockers in T lymphocytes depends on the strength of mitogenic stimulation

Ion channels are crucially important for the activation and proliferation of T lymphocytes, and thus, for the function of the immune system. Previous studies on the effects of channel blockers on T cell proliferation reported variable effectiveness due to differing experimental systems. Therefore our aim was to investigate how the strength of the mitogenic stimulation influences the efficiency of cation channel blockers in inhibiting activation, cytokine secretion and proliferation of T cells under standardized conditions.Human peripheral blood lymphocytes were activated via monoclonal antibodies targeting the TCR-CD3 complex and the co-stimulator CD28. We applied the blockers of Kv1.3 (Anuroctoxin), KCa3.1 (TRAM-34) and CRAC (2-Apb) channels of T cells either alone or in combination with rapamycin, the inhibitor of the mammalian target of rapamycin (mTOR). Five days after the stimulation ELISA and flow cytometric measurements were performed to determine IL-10 and IFN-γ secretion, cellular viability and proliferation.Our results showed that ion channel blockers and rapamycin inhibit IL-10 and IFN-γ secretion and cell division in a dose-dependent manner. Simultaneous application of the blockers for each channel along with rapamycin was the most effective, indicating synergy among the various activation pathways. Upon increasing the extent of mitogenic stimulation the anti-proliferative effect of the ion channel blockers diminished. This phenomenon may be important in understanding the fine-tuning of T cell activation.

Orai1 Concatemers Reveal a Hexameric Orai1 Channel Assembly

The multimeric assembly of the CRAC channel has remained a contentious issue despite strong crystallographic evidence of a hexameric structure for both Drosophila and human Orai1. Recent studies suggested concatemeric tetramers of Orai1 mediate authentic CRAC current whereas equivalent hexameric concatemers form only non-selective cation channels. We expressed concatenated human Orai1 constructs with intervening 36-aa linkers. We observed that expression of dimers, trimers, tetramers, pentamers and hexamers of human Orai1 in HEK cells stably expressing STIM1 (HEK-STIM1 cells), all gave rise to similar high levels of Ca2+ entry. All constructs were C-terminally tagged with tdTomato and all were observed to be exclusively PM-expressed. Each construct also gave similar Ca2+ entry when expressed in MEFS derived from Orai1 knockout mice, the MEFS stably expressing STIM1. Expressed in HEK-STIM1 cells, the Orai1 dimers, trimers, tetramers and hexamers all gave rise to authentic CRAC channel activity with similar inward rectification and reversal potential. Substituting the pore-inactive E106A mutant in tetramers, we observed that the initial two N-terminal residues of the tetramer are crucial for channel activity. The remaining two C-terminal residues in tetramers are inconsequential for function. In hexamers, the position of inactive mutants in the concatemer are not specific. Substitution with a single E106A mutant monomer in the hexamer has the same effect in reducing channel function at each position. Substitution with two E106A residues results in little remaining channel function. We interpret these results to reveal that hexamers are likely the true functional Orai1 channel unit. Tetramers likely feed dimers into a hexameric structure, with the two C-terminal residues outside the hexameric ring, explaining why these last two residues in the tetramer can be E106A without loss of activity. Certainly, the hexameric concatemer gives a fully functional CRAC channel.

Impact of STIM1 R304W Mutant on Intra- and Intermolecular Cytosolic Coiled-Coil Interactions

STIM1 and Orai1 are key components of the Ca2+-release activated Ca2+ (CRAC) current which plays an important role in a broad range of cellular/physiological processes including T cell activation as well as cell proliferation, growth and apoptosis. Activation of Orai1 - the CRAC channel forming subunit - occurs via a physical interaction with the ER resident Ca2+ sensor protein STIM1. Upon ER Ca2+ store depletion, STIM1 undergoes extensive structural rearrangements resulting in an activated extended conformation. Interhelical rearrangements between the three cytosolic STIM1 coiled-coil (CC) domains drive this process therefore exposing SOAR/CAD allowing puncta formation and coupling to Orai1 in the cell periphery.Two years ago, the genetically inherited Stormorken Syndrome disease has been linked to the constitutively active human STIM1 R304W mutant (first published by Nesin et al.). In the present study, we focused on intra- and inter-molecular interactions specifically between the three cytosolic STIM1 CC domains comparing the STIM1 R304W mutant to the wildtype system as well as the constitutively active STIM1 L251S mutant employing electrophysiology, the YFP-OASF-CFP-FRET sensor and the recently described FRET based FIRE system. Our results revealed new insights into the mechanism how the disease related mutant R304W yields constitutive activity pointing to a novel effect distinct to the previously described constitutively active STIM1 CC1/CC3 mutants. (Supported by Austrian Science Fund (FWF) and science department of Upper Austria: P281203 to M.F and FWF-P27263 to C.R.)

Molecular mechanisms of STIM/Orai communication.

Ca2+ entry into the cell via store-operated Ca2+ release-activated Ca2+ (CRAC) channels triggers diverse signaling cascades that affect cellular processes like cell growth, gene regulation, secretion, and cell death. These store-operated Ca2+ channels open after depletion of intracellular Ca2+ stores, and their main features are fully reconstituted by the two molecular key players: the stromal interaction molecule (STIM) and Orai. STIM represents an endoplasmic reticulum-located Ca2+ sensor, while Orai forms a highly Ca2+-selective ion channel in the plasma membrane. Functional as well as mutagenesis studies together with structural insights about STIM and Orai proteins provide a molecular picture of the interplay of these two key players in the CRAC signaling cascade. This review focuses on the main experimental advances in the understanding of the STIM1-Orai choreography, thereby establishing a portrait of key mechanistic steps in the CRAC channel signaling cascade. The focus is on the activation of the STIM proteins, the subsequent coupling of STIM1 to Orai1, and the consequent structural rearrangements that gate the Orai channels into the open state to allow Ca2+ permeation into the cell.

Orai1 pore residues control CRAC channel inactivation independently of calmodulin.

Ca(2+) entry through CRAC channels causes fast Ca(2+)-dependent inactivation (CDI). Previous mutagenesis studies have implicated Orai1 residues W76 and Y80 in CDI through their role in binding calmodulin (CaM), in agreement with the crystal structure of Ca(2+)-CaM bound to an Orai1 N-terminal peptide. However, a subsequent Drosophila melanogaster Orai crystal structure raises concerns about this model, as the side chains of W76 and Y80 are predicted to face the pore lumen and create a steric clash between bound CaM and other Orai1 pore helices. We further tested the functional role of CaM using several dominant-negative CaM mutants, none of which affected CDI. Given this evidence against a role for pretethered CaM, we altered side-chain volume and charge at the Y80 and W76 positions to better understand their roles in CDI. Small side chain volume had different effects at the two positions: it accelerated CDI at position Y80 but reduced the extent of CDI at position W76. Positive charges at Y80 and W76 permitted partial CDI with accelerated kinetics, whereas introducing negative charge at any of five consecutive pore-lining residues (W76, Y80, R83, K87, or R91) completely eliminated CDI. Noise analysis of Orai1 Y80E and Y80K currents indicated that reductions in CDI for these mutations could not be accounted for by changes in unitary current or open probability. The sensitivity of CDI to negative charge introduced into the pore suggested a possible role for anion binding in the pore. However, although Cl(-) modulated the kinetics and extent of CDI, we found no evidence that CDI requires any single diffusible cytosolic anion. Together, our results argue against a CDI mechanism involving CaM binding to W76 and Y80, and instead support a model in which Orai1 residues Y80 and W76 enable conformational changes within the pore, leading to CRAC channel inactivation.

The inactivation domain of STIM1 is functionally coupled with the Orai1 pore to enable Ca2+-dependent inactivation.

The inactivation domain of STIM1 (ID(STIM): amino acids 470-491) has been described as necessary for Ca(2+)-dependent inactivation (CDI) of Ca(2+) release-activated Ca(2+) (CRAC) channels, but its mechanism of action is unknown. Here we identify acidic residues within IDSTIM that control the extent of CDI and examine functional interactions of ID(STIM) with Orai1 pore residues W76 and Y80. Alanine scanning revealed three IDSTIM residues (D476/D478/D479) that are critical for generating full CDI. Disabling ID(STIM) by a triple alanine substitution for these three residues ("STIM1 3A") or by truncation of the entire domain (STIM1(1-469)) reduced CDI to the same residual level observed for the Orai1 pore mutant W76A (approximately one third of the extent seen with full-length STIM1). Results of noise analysis showed that STIM11-469 and Orai1 W76A mutants do not reduce channel open probability or unitary Ca(2+) conductance, factors that determine local Ca(2+) accumulation, suggesting that they diminish CDI instead by inhibiting the CDI gating mechanism. We tested for functional coupling between ID(STIM) and the Orai1 pore by double-mutant cycle analysis. The effects on CDI of mutations disabling ID(STIM) or W76 were not additive, demonstrating that ID(STIM) and W76 are strongly coupled and act in concert to generate full-strength CDI. Interestingly, disabling ID(STIM) and W76 separately gave opposite results in Orai1 Y80A channels: channels with W76 but lacking ID(STIM) generated approximately two thirds of the WT extent of CDI but those with ID(STIM) but lacking W76 completely failed to inactivate. Together, our results suggest that Y80 alone is sufficient to generate residual CDI, but acts as a barrier to full CDI. Although ID(STIM) is not required as a Ca(2+) sensor for CDI, it acts in concert with W76 to progress beyond the residual inactivated state and enable CRAC channels to reach the full extent of inactivation.

Selective ORAI1 Inhibition Ameliorates Autoimmune Central Nervous System Inflammation by Suppressing Effector but Not Regulatory T Cell Function.

The function of CD4(+) T cells is dependent on Ca(2+) influx through Ca(2+) release-activated Ca(2+) (CRAC) channels formed by ORAI proteins. To investigate the role of ORAI1 in proinflammatory Th1 and Th17 cells and autoimmune diseases, we genetically and pharmacologically modulated ORAI1 function. Immunization of mice lacking Orai1 in T cells with MOG peptide resulted in attenuated severity of experimental autoimmune encephalomyelitis (EAE). The numbers of T cells and innate immune cells in the CNS of ORAI1-deficient animals were strongly reduced along with almost completely abolished production of IL-17A, IFN-γ, and GM-CSF despite only partially reduced Ca(2+) influx. In Th1 and Th17 cells differentiated in vitro, ORAI1 was required for cytokine production but not the expression of Th1- and Th17-specific transcription factors T-bet and RORγt. The differentiation and function of induced regulatory T cells, by contrast, was independent of ORAI1. Importantly, induced genetic deletion of Orai1 in adoptively transferred, MOG-specific T cells was able to halt EAE progression after disease onset. Likewise, treatment of wild-type mice with a selective CRAC channel inhibitor after EAE onset ameliorated disease. Genetic deletion of Orai1 and pharmacological ORAI1 inhibition reduced the leukocyte numbers in the CNS and attenuated Th1/Th17 cell-mediated cytokine production. In human CD4(+) T cells, CRAC channel inhibition reduced the expression of IL-17A, IFN-γ, and other cytokines in a dose-dependent manner. Taken together, these findings support the conclusion that Th1 and Th17 cell function is particularly dependent on CRAC channels, which could be exploited as a therapeutic approach to T cell-mediated autoimmune diseases.

The STIM1: Orai Interaction.

Ca(2+) influx via store-operated Ca(2+) release activated Ca(2+) (CRAC) channels represents a main signalling pathway for a variety of cell functions, including T-cell activation as well as mast-cell degranulation. Depletion of [Ca(2+)]ER results in activation of Ca(2+) channels within the plasmamembrane that mediate sustained Ca(2+) influx which is required for refilling Ca(2+) stores and down-stream Ca(2+) signalling. The CRAC channel is the best characterized store-operated channel (SOC) with well-defined electrophysiological properties. In recent years, the molecular components of the CRAC channel have been defined. The ER - located Ca(2+)-sensor, STIM1 and the Ca(2+)-selective ion pore, Orai1 in the membrane are sufficient to fully reconstitute CRAC currents. Stromal interaction molecule (STIM) 1 is localized in the ER, senses [Ca(2+)]ER and activates the CRAC channel upon store depletion by direct binding to Orai1 in the plasmamembrane. The identification of STIM1 and Orai1 and recently the structural resolution of both proteins by X-ray crystallography and nuclear magnetic resonance substantiated many findings from structure-function studies which has substantially improved the understanding of CRAC channel activation. Within this review, we summarize the functional and structural mechanisms of CRAC channel regulation, present a detailed overview of the STIM1/Orai1 signalling pathway where we focus on the critical domains mediating interactions and on the ion permeation pathway. We portray a mechanistic view of the steps in the dynamics of CRAC channel signalling ranging from STIM1 oligomerization over STIM1-Orai1 coupling to CRAC channel activation and permeation.

Role of TRPC Channels in Store-Operated Calcium Entry.

Store-operated calcium entry (SOCE) is a ubiquitous Ca(2+) entry pathway that is activated in response to depletion of Ca(2+) stores within the endoplasmic reticulum (ER) and contributes to the control of various physiological functions in a wide variety of cell types. The transient receptor potential canonical (TRPC) channels (TRPCs 1-7), that are activated by stimuli leading to PIP2 hydrolysis, were first identified as molecular components of SOCE channels. TRPC channels show a miscellany of tissue expression, physiological functions and channel properties. However, none of the TRPC members display currents that resemble I CRAC. Intensive search for the CRAC channel component led to identification of Orai1 and STIM1, now established as being the primary constituents of the CRAC channel. There is now considerable evidence that STIM1 activates both Orai1 and TRPC1 via distinct domains in its C-terminus. Intriguingly, TRPC1 function is not only dependent on STIM1 but also requires Orai1. The critical functional interaction between TRPC1 and Orai1, which determines the activation of TRPC1, has also been identified. In this review, we will discuss current concepts regarding the role of TRPC channels in SOCE, the physiological functions regulated by TRPC-mediated SOCE, and the complex mechanisms underlying the regulation of TRPCs, including the functional interactions with Orai1 and STIM1.

Genetically targeted single-channel optical recording reveals multiple Orai1 gating states and oscillations in calcium influx

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

Pharmacodynamic evaluation of RP3128, a novel and potent CRAC channel inhibitor in guinea pig models of allergic asthma

The increase in intracellular Ca2+ levels through the activation of Ca2+ release-activated Ca2+ (CRAC) channels is essential for mediating a wide scale of immune cell responses. Emerging evidence indicates an involvement of abnormal CRAC channel activity in human diseases such as certain types of immunodeficiency, autoimmunity and allergic disorders. This objective of this study was to evaluate the therapeutic potency of a novel CRAC channel inhibitor, RP3128, in experimental models of allergic asthma using guinea pigs.Ovalbumin-induced allergic airway inflammation was determined upon acute and long-term (14 days) oral administration of RP3128. In vivo changes in specific airways resistance (sRaw) and amplitude of isometric contraction (mN) of ASM (in vitro) were estimated to evaluate bronchodilatory effect upon acute and long-term administration of RP3128 or salbutamol. Exhaled nitric oxide (eNO), immunohistochemical and histological analysis of cellular infiltration in airways tissue, and levels of cytokines in plasma as well as bronchoalveolar lavage fluid (BALF), were determined using Bio-Plex® 200 System (BIO-RAD, USA). Ciliary beat frequency (CBF, in Hz) was estimated using a high-speed video camera and LabVIEW™ Software. Additionally, the impact of RP3128 and budesonide on mucociliary clearance was determined.Acute and long-term administration of RP3128 resulted in significant bronchodilation. Long-term administration of RP3128 exceeded the bronchodilatory effect of salbutamol and significantly decreased eNO and cytokine levels in plasma and BALF, which together with histological and immunohistochemical analysis validated its anti-inflammatory effect compared to budesonide. Data demonstrate the therapeutic potential of RP3128 in respiratory diseases causally associated with allergic inflammation.

Crac Channel Deletion in Leukemic Cells Delays Progression of Leukemia and Prolongs Survival of Mice with Notch-1-Induced T-Cell Acute Lymphoblastic Leukemia

Introduction: Ca2+ release-activated Ca2+ (CRAC) channels and their activators stromal interaction molecule (STIM) 1 and 2 are the main regulators of calcium entry in T Lymphocytes through a process known as store-operated Ca2+ entry (SOCE). SOCE results in the activation of calcineurin and other downstream signals with important effects on lymphocyte function. Notch-1 is a protein that is essential for T lymphocyte development. Activating mutations of Notch-1 occurs in about 60% of T-cell acute lymphoblastic leukemia (T-ALL). Introduction of constitutively active forms of Notch-1 in hematopoietic stem cells (HSC) induces T-ALL in mice, providing a useful animal model for the study of leukemia.Methods: To study the role of CRAC channels in T-ALL, we used a mouse model in which c-kit+ HSC from wild-type (WT) and STIM1/STIM2-deficient mice (DKO) were retrovirally transduced with the intracellular Notch-1 domain (ICN1). Transduced HSC were injected into lethally irradiated C57BL/6 mice. Following leukemia development, mice were analyzed for survival and cellular and molecular activity of leukemic cells using various techniques including histology, flow cytometry, RT-PCR and gene array expression analysis. In addition, we used the human T-ALL cell line CEM, in which we introduced a dominant negative form of the CRAC channel subunit ORAI1 (ORAI1-DN) that abolishes CRAC channel function and SOCE, for coculture with the human bone marrow stromal cell line HS5.Results: Mice injected with wild-type HSC transduced with ICN1 succumbed from T-ALL characterized by the presence of CD4+ CD8+ leukemic T cell blasts in the blood, bone marrow and infiltrating organs within 3 to 4 weeks after transfer of HSC. By contrast, mice that had received ICN1 transduced STIM1/2 deficient HSC lived approximately twice as long. The survival benefit was not due to differences in leukemic cell numbers or in proliferation and apoptosis of leukemic cells. Histologies of the bone marrow and spleen of WT leukemic mice showed necrotic lesions, pronounced neutrophil infiltration, the presence of histiocytes engulfing red blood cells (RBC) indicative of severe inflammation. No signs of necrosis and inflammation were present in DKO leukemic mice. Paralleling the inflammation and destruction of the bone marrow environment, WT leukemic mice showed greatly diminished presence of erythroid precursors (EP) in the bone marrow whereas EP frequencies in DKO leukemic mice were similar to those in non-leukemic mice. In line with findings in mice, we observed that human leukemic CEM T cells reduced the viability of HS5 stromal cells in a contact-dependent manner. This cytotoxic effect of CEM cells depended on CRAC channel function as CEM cells transduced with ORAI1-DN had little effect on HS5 viability.Conclusion: These results suggest that CRAC channels are important for the function of T-ALL cells and their effects on the organs they infiltrate, most notably the bone marrow. Inhibition of CRAC channel function prolongs survival of mice with T-ALL potentially by attenuating the cytotoxic effects of leukemic T cells on their environment and on hematopoiesis. Further studies are underway to understand the mechanisms by which CRAC channels regulate leukemic T cell function. DisclosuresFeske:Calcimedica: Consultancy, Equity Ownership, Honoraria, Patents & Royalties: CRAC Channel Inibitors.

Dental enamel cells express functional SOCE channels

Dental enamel formation requires large quantities of Ca2+ yet the mechanisms mediating Ca2+ dynamics in enamel cells are unclear. Store-operated Ca2+ entry (SOCE) channels are important Ca2+ influx mechanisms in many cells. SOCE involves release of Ca2+ from intracellular pools followed by Ca2+ entry. The best-characterized SOCE channels are the Ca2+ release-activated Ca2+ (CRAC) channels. As patients with mutations in the CRAC channel genes STIM1 and ORAI1 show abnormal enamel mineralization, we hypothesized that CRAC channels might be an important Ca2+ uptake mechanism in enamel cells. Investigating primary murine enamel cells, we found that key components of CRAC channels (ORAI1, ORAI2, ORAI3, STIM1, STIM2) were expressed and most abundant during the maturation stage of enamel development. Furthermore, inositol 1,4,5-trisphosphate receptor (IP3R) but not ryanodine receptor (RyR) expression was high in enamel cells suggesting that IP3Rs are the main ER Ca2+ release mechanism. Passive depletion of ER Ca2+ stores with thapsigargin resulted in a significant raise in [Ca2+]i consistent with SOCE. In cells pre-treated with the CRAC channel blocker Synta-66 Ca2+ entry was significantly inhibited. These data demonstrate that enamel cells have SOCE mediated by CRAC channels and implicate them as a mechanism for Ca2+ uptake in enamel formation.

The Intervention of CRAC Channels Alleviates Inflammatory Responses in Nasal Polyps

Background: Chronic rhinosinusitis with nasal polyps (CRSwNP) is associated with Th2-dominant inflammation. However, effective treatments for CRSwNP have not yet been found. This study aimed to investigate the expression of Orai1 in nasal polyps (NP) and the influence on them of the intervention of Ca²⁺ release-activated Ca²⁺ (CRAC) channels. Materials and Methods: Nasal samples were obtained from normal subjects or subjects with CRSwNP. We studied the distribution of Orai1 protein in NP and normal mucosa (normal group) using immunohistochemistry. These tissues in cultures were then maintained in the absence (control group) or presence of 2-aminoethoxydiphenyl borate (2-APB) for 24 h. Orai1 was examined by means of enzyme-linked immunosorbent assay (ELISA) and real-time reverse transcription-polymerase chain reaction (RT-PCR). The Ca²⁺ mean fluorescence intensity (MFI) was evaluated by flow cytometry, and the inflammatory mediators, including interleukin (IL)-4, IL-5, IL-13, Dermatophagoides pteronyssinus-specific IgE, leukotriene C4 and eosinophil cation protein in cultures, were analyzed with ELISA and real-time RT-PCR. Results: The expression of Orai1 was localized to the cytoplasmic membrane of inflammatory cells, and upregulated in NP compared to that in the normal group. However, Orai1 protein was decreased in polyp tissues after the 2-APB treatment. The levels of Ca²⁺ MFI and above inflammatory mediators were also elevated in NP, and reduced after the 2-APB administration compared to those in the control group. Conclusions: Orai1 and CRAC channels may play a crucial role in NP formation and development, and the 2-APB intervention of Orai1 protein may alleviate inflammatory responses in NP.

Diseases caused by mutations in ORAI1 and STIM1.

Ca(2+) release-activated Ca(2+) (CRAC) channels mediate a specific form of Ca(2+) influx called store-operated Ca(2+) entry (SOCE) that contributes to the function of many cell types. CRAC channels are composed of ORAI1 proteins located in the plasma membrane, which form its ion-conducting pore. ORAI1 channels are activated by stromal interaction molecule (STIM) 1 and STIM2 located in the endoplasmic reticulum. Loss- and gain-of-function gene mutations in ORAI1 and STIM1 in human patients cause distinct disease syndromes. CRAC channelopathy is caused by loss-of-function mutations in ORAI1 and STIM1 that abolish CRAC channel function and SOCE; it is characterized by severe combined immunodeficiency (SCID)-like disease, autoimmunity, muscular hypotonia, and ectodermal dysplasia, with defects in sweat gland function and dental enamel formation. The latter defect emphasizes an important role of CRAC channels in tooth development. By contrast, autosomal dominant gain-of-function mutations in ORAI1 and STIM1 result in constitutive CRAC channel activation, SOCE, and increased intracellular Ca(2+) levels that are associated with an overlapping spectrum of diseases, including nonsyndromic tubular aggregate myopathy (TAM) and York platelet and Stormorken syndromes. The latter two syndromes are defined, besides myopathy, by thrombocytopenia, thrombopathy, and bleeding diathesis. The fact that myopathy results from both loss- and gain-of-function mutations in ORAI1 and STIM1 highlights the importance of CRAC channels for Ca(2+) homeostasis in skeletal muscle function. The cellular dysfunction and clinical disease spectrum observed in mutant patients provide important information about the molecular regulation of ORAI1 and STIM1 proteins and the role of CRAC channels in human physiology.

Optogenetic control of endogenous Ca(2+) channels in vivo.

Calcium (Ca(2+)) signals that are precisely modulated in space and time mediate a myriad of cellular processes, including contraction, excitation, growth, differentiation and apoptosis. However, study of Ca(2+) responses has been hampered by technological limitations of existing Ca(2+)-modulating tools. Here we present OptoSTIM1, an optogenetic tool for manipulating intracellular Ca(2+) levels through activation of Ca(2+)-selective endogenous Ca(2+) release-activated Ca(2+) (CRAC) channels. Using OptoSTIM1, which combines a plant photoreceptor and the CRAC channel regulator STIM1 (ref. 4), we quantitatively and qualitatively controlled intracellular Ca(2+) levels in various biological systems, including zebrafish embryos and human embryonic stem cells. We demonstrate that activating OptoSTIM1 in the CA1 hippocampal region of mice selectively reinforced contextual memory formation. The broad utility of OptoSTIM1 will expand our mechanistic understanding of numerous Ca(2+)-associated processes and facilitate screening for drug candidates that antagonize Ca(2+) signals.

The role of CRAC channel in asthma

Asthma is increasing globally and current treatments only manage a proportion of patients. There is an urgent need to develop new therapies. Lymphocytes are thought to play a central role in the pathophysiology of asthma through the production of inflammatory mediators. This is thought to be via the transcription factor NFAT which in turn can be activated through Ca2+ release-activated Ca2+ (CRAC) channels. The aim of this work was to investigate the role of CRAC in clinical and pre-clinical models of allergic asthma.Initial data demonstrated that the NFAT pathway is increased in stimulated lymphocytes from asthmatics. To confirm a role for the channel we showed that a selective inhibitor, Synta 66, blocked mediator production from lymphocytes. Synta 66 inhibited CD2/3/28 induced IL-2, IL-7, IL-13 & IFNΥ in a concentration-dependent manner in healthy and severe asthma donors, with over 60% inhibition observed for all cytokines. NFAT pathway was also increased in a pre-clinical asthma model. In this model we have demonstrated that CRAC played a central role in the airway inflammation and late asthmatic response (LAR). In conclusion, our data provides evidence that suggests targeting CRAC channels could be of therapeutic benefit for asthma sufferers.

Store-Operated Ca2+ Release-Activated Ca2+ Channels Regulate PAR2-Activated Ca2+ Signaling and Cytokine Production in Airway Epithelial Cells.

The G-protein-coupled protease-activated receptor 2 (PAR2) plays an important role in the pathogenesis of various inflammatory and auto-immune disorders. In airway epithelial cells (AECs), stimulation of PAR2 by allergens and proteases triggers the release of a host of inflammatory mediators to regulate bronchomotor tone and immune cell recruitment. Activation of PAR2 turns on several cell signaling pathways of which the mobilization of cytosolic Ca(2+) is likely a critical but poorly understood event. In this study, we show that Ca(2+) release-activated Ca(2+) (CRAC) channels encoded by stromal interaction molecule 1 and Orai1 are a major route of Ca(2+) entry in primary human AECs and drive the Ca(2+) elevations seen in response to PAR2 activation. Activation of CRAC channels induces the production of several key inflammatory mediators from AECs including thymic stromal lymphopoietin, IL-6, and PGE2, in part through stimulation of gene expression via nuclear factor of activated T cells (NFAT). Furthermore, PAR2 stimulation induces the production of many key inflammatory mediators including PGE2, IL-6, IL-8, and GM-CSF in a CRAC channel-dependent manner. These findings indicate that CRAC channels are the primary mechanism for Ca(2+) influx in AECs and a vital checkpoint for the induction of PAR2-induced proinflammatory cytokines.

Discovery and structural optimization of 1-phenyl-3-(1-phenylethyl)urea derivatives as novel inhibitors of CRAC channel.

Ca(2+)-release-activated Ca(2+) (CRAC) channel, a subfamily of store-operated channels, is formed by calcium release-activated calcium modulator 1 (ORAI1), and gated by stromal interaction molecule 1 (STIM1). CRAC channel may be a novel target for the treatment of immune disorders and allergy. The aim of this study was to identify novel small molecule CRAC channel inhibitors. HEK293 cells stably co-expressing both ORAI1 and STIM1 were used for high-throughput screening. A hit, 1-phenyl-3-(1-phenylethyl)urea, was identified that inhibited CRAC channels by targeting ORAI1. Five series of its derivatives were designed and synthesized, and their primary structure-activity relationships (SARs) were analyzed. All derivatives were assessed for their effects on Ca(2+) influx through CRAC channels on HEK293 cells, cytotoxicity in Jurkat cells, and IL-2 production in Jurkat cells expressing ORAI1-SS-eGFP. A total of 19 hits were discovered in libraries containing 32 000 compounds using the high-throughput screening. 1-Phenyl-3-(1-phenylethyl)urea inhibited Ca(2+) influx with IC50 of 3.25±0.17 μmol/L. SAR study on its derivatives showed that the alkyl substituent on the α-position of the left-side benzylic amine (R1) was essential for Ca(2+) influx inhibition and that the S-configuration was better than the R-configuration. The derivatives in which the right-side R3 was substituted by an electron-donating group showed more potent inhibitory activity than those that were substituted by electron-withdrawing groups. Furthermore, the free N-H of urea was not necessary to maintain the high potency of Ca(2+) influx inhibition. The N,N'-disubstituted or N'-substituted derivatives showed relatively low cytotoxicity but maintained the ability to inhibit IL-2 production. Among them, compound 5b showed an improved inhibition of IL-2 production and low cytotoxicity. 1-Phenyl-3-(1-phenylethyl)urea is a novel CRAC channel inhibitor that specifically targets ORAI1. This study provides a new chemical scaffold for design and development of CRAC channel inhibitors with improved Ca(2+) influx inhibition, immune inhibition and low cytotoxicity.