Store-operated calcium entry channel Orai

The CRAC channel pore is formed by Orai, which is inhibited by Synta66. In Müller glia, Synta66 (10 μM) reduces peak SOCE. In Trpc1−/− Müller cells, synta66 (10 μM) blocks orai channels from mediating residual SOC currents [1]. The Ca2+ entry signal caused by the injection of CaCl2 is nearly entirely blocked by Synta66 (10 μM), whereas the mobilization of stored Ca2+ in platelets is only slightly decreased by 10% to 30%. Human platelet activation in plasma and whole blood thrombosis is inhibited by Synta66 (10 μM). In mice, Synta66 (10 μM) also prevents thrombosis and the platelet response [2]. Synta66 (10 μM) suppresses human mast cell lines' expression of LAD2. Synta66 (10 μM) has varied effects on FcεRI-stimulated prostaglandin D2 and cytokine release in human lung mast cells (HLMC) and strongly suppresses FcεRI-stimulated histamine and TNFα production [3].

SOCE Blockers Suppress Human Platelet Activation in Plasma and Whole-Blood Thrombus Formation[2]
In plasma or whole blood systems, lipophilic inhibitors often need to be added at 10× to 50× higher concentrations than in nonplasma-based buffer systems to affect platelet function.28 This also appeared to be the case for the SOCE inhibitors. When added to platelet-rich plasma, concentrations of 100 μmol/L Synta66, 2APB, or GSK-7975A were required for inhibition of convulxin-induced Ca2+ rises and PS exposure with 41% to 49% (data not shown). To verify that these inhibitors influenced platelet procoagulant activity, the effects of Synta66, 2APB, or GSK-7975A (100 μmol/L) in platelet-rich plasma were measured on thrombin generation. Upon triggering with 1 pmol/L tissue factor, peak heights of thrombin generation were reduced with Synta66, 2APB, and GSK-7975A to 29±2%, 58±2%, and 28±2% of control, respectivel

CRAC channels are activated in a STIM-dependent fashion following the reduction of free [Ca2+]ER (Prakriya and Lewis, 2015). To assess the contribution of these Ca2+-selective channels to Müller glial SOCE, we depleted ER stores in the presence of putative selective inhibitors Synta66 and GSK7975A. Synta66 (10 μm) attenuated peak SOCE from 511.0 ± 78.5 to 349.9 ± 40.1 nm (N = 2; p < 0.01), whereas the antagonist had no significant effect on basal [Ca2+]i (221.5 ± 29.2 nm in untreated control and 251.7 ± 31.3 nm in Synta66-treated cells, respectively; Fig. 5). Likewise, the SOCE response in wild-type cells was partially antagonized by GSK-7975A (10 μm; Fig. 5C–E). The residual SOCE in Orai-inhibited cells was abolished by 2-APB/SKF 96365/Gd3+ (Fig. 5A,B).[1]

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Core body temperatures activate STIM1, decouple STIM1 from Orai1 (Xiao et al., 2011), and may stimulate the TRPV4 thermochannel expressed in Müller cells (Ryskamp et al., 2014). Thus, ICRAC is suboptimally activated at RT (Somasundaram et al., 1996). To determine whether glial SOCE is regulated by temperature, we compared the amplitudes of overshoot responses in control and Synta66-treated cells. The increase in temperature from RT to 32°C resulted in a modest increase in SOCE that was not statistically significant (Fig. 5C). The SOCE amplitude at 32°C was 0.723 ± 0.165 and was reduced to 0.371 ± 0.058 in the presence of Synta66 (n = 6/9 cells). Despite a ∼49% reduction, the result did not reach significance due to the large variability in the response. These data suggest that the relative fraction of Orai versus TRPC [Ca2+]SOCE response is likely to persist at the core body temperature.[1]

Wild-type or chimeric Orai1−/− mice were injected with vehicle solution or 2APB (3 mg/kg) as indicated. In blood samples isolated 60 minutes after injection, collagen-induced thrombus formation was measured, as described above. To induce focal cerebral ischemia in mice, the middle cerebral artery (MCA) was transiently occluded for 60 minutes using an intraluminal filament as described elsewhere (transient MCA occlusion model).20 Immediately after reperfusion of the MCA territory, vehicle solution or 2APB (3 mg/kg) was injected postoperatively. Animals were euthanized on day 1 after transient MCA occlusion, and brain sections were stained with 2% 2,3,5-triphenyltetrazolium chloride to quantify the ischemic brain volume (corrected for edema).[2]

[1]. Store-Operated Calcium Entry in Müller Glia Is Controlled by Synergistic Activation of TRPC and Orai Channels. J Neurosci. 2016 Mar 16;36(11):3184-98.

[2]. Antithrombotic potential of blockers of store-operated calcium channels in platelets. Arterioscler Thromb Vasc Biol. 2012 Jul;32(7):1717-23.

[3]. Orai and TRPC channel characterization in FcεRI-mediated calcium signaling and mediator secretion in human mast cells. Physiol Rep. 2017 Mar;5(5).

The endoplasmic reticulum (ER) is at the epicenter of astrocyte Ca(2+) signaling. We sought to identify the molecular mechanism underlying store-operated calcium entry that replenishes ER stores in mouse Müller cells. Store depletion, induced through blockade of sequestration transporters in Ca(2+)-free saline, induced synergistic activation of canonical transient receptor potential 1 (TRPC1) and Orai channels. Store-operated TRPC1 channels were identified by their electrophysiological properties, pharmacological blockers, and ablation of the Trpc1 gene. Ca(2+) release-activated currents (ICRAC) were identified by ion permeability, voltage dependence, and sensitivity to selective Orai antagonists Synta66 and GSK7975A. Depletion-evoked calcium influx was initiated at the Müller end-foot and apical process, triggering centrifugal propagation of Ca(2+) waves into the cell body. EM analysis of the end-foot compartment showed high-density ER cisternae that shadow retinal ganglion cell (RGC) somata and axons, protoplasmic astrocytes, vascular endothelial cells, and ER-mitochondrial contacts at the vitreal surface of the end-foot. The mouse retina expresses transcripts encoding both Stim and all known Orai genes; Müller glia predominantly express stromal interacting molecule 1 (STIM1), whereas STIM2 is mainly confined to the outer plexiform and RGC layers. Elimination of TRPC1 facilitated Müller gliosis induced by the elevation of intraocular pressure, suggesting that TRPC channels might play a neuroprotective role during mechanical stress. By characterizing the properties of store-operated signaling pathways in Müller cells, these studies expand the current knowledge about the functional roles these cells play in retinal physiology and pathology while also providing further evidence for the complexity of calcium signaling mechanisms in CNS astroglia.[1]

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Objective: Platelet Orai1 channels mediate store-operated Ca(2+) entry (SOCE), which is required for procoagulant activity and arterial thrombus formation. Pharmacological blockage of these channels may provide a novel way of antithrombotic therapy. Therefore, the thromboprotective effect of SOCE blockers directed against platelet Orai1 is determined. Methods and results: Candidate inhibitors were screened for their effects on SOCE in washed human platelets. Tested antagonists included the known compounds, SKF96365, 2-aminoethyl diphenylborate, and MRS1845 and the novel compounds, Synta66 and GSK-7975A. The potency of SOCE inhibition was in the order of Synta66>2-aminoethyl diphenylborate>GSK-7975A>SKF96365>MRS1845. The specificity of the first 3 compounds was verified with platelets from Orai1-deficient mice. Inhibitory activity on procoagulant activity and high-shear thrombus formation was assessed in plasma and whole blood. In the presence of plasma, all 3 compounds suppressed platelet responses and restrained thrombus formation under flow. Using a murine stroke model, arterial thrombus formation was provoked in vivo by transient middle cerebral artery occlusion. Postoperative administration of 2-aminoethyl diphenylborate markedly diminished brain infarct size. Conclusions: Plasma-soluble SOCE blockers such as 2-aminoethyl diphenylborate suppress platelet-dependent coagulation and thrombus formation. The platelet Orai1 channel is a novel target for preventing thrombotic events causing brain infarction.[2]

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Inappropriate activation of mast cells via the FcεRI receptor leads to the release of inflammatory mediators and symptoms of allergic disease. Calcium influx is a critical regulator of mast cell signaling and is required for exocytosis of preformed mediators and for synthesis of eicosanoids, cytokines and chemokines. Studies in rodent and human mast cells have identified Orai calcium channels as key contributors to FcεRI-initiated mediator release. However, until now the role of TRPC calcium channels in FcεRI-mediated human mast cell signaling has not been published. Here, we show evidence for the expression of Orai 1,2, and 3 and TRPC1 and 6 in primary human lung mast cells and the LAD2 human mast cell line but, we only find evidence of functional contribution of Orai and not TRPC channels to FcεRI-mediated calcium entry. Calcium imaging experiments, utilizing an Orai selective antagonist (Synta66) showed the contribution of Orai to FcεRI-mediated signaling in human mast cells. Although, the use of a TRPC3/6 selective antagonist and agonist (GSK-3503A and GSK-2934A, respectively) did not reveal evidence for TRPC6 contribution to FcεRI-mediated calcium signaling in human mast cells. Similarly, inactivation of STIM1-regulated TRPC1 in human mast cells (as tested by transfecting cells with STIM1-KK684-685EE - TRPC1 gating mutant) failed to alter FcεRI-mediated calcium signaling in LAD2 human mast cells. Mediator release assays confirm that FcεRI-mediated calcium influx through Orai is necessary for histamine and TNFα release but is differentially involved in the generation of cytokines and eicosanoids.[3]

C20H17N2O3F

352.359

352.122

835904-51-3

11337104

Typically exists as White to gray solids at room temperature

1.3±0.1 g/cm3

422.4±45.0 °C at 760 mmHg

209.3±28.7 °C

0.0±1.0 mmHg at 25°C

1.611

2.52

1

5

5

26

456

0

O=C(C1C(F)=CN=CC=1)NC1C=CC(C2C(OC)=CC=C(OC)C=2)=CC=1

GFEIWXNLDKUWIK-UHFFFAOYSA-N

InChI=1S/C20H17FN2O3/c1-25-15-7-8-19(26-2)17(11-15)13-3-5-14(6-4-13)23-20(24)16-9-10-22-12-18(16)21/h3-12H,1-2H3,(H,23,24)

N-[4-(2,5-dimethoxyphenyl)phenyl]-3-fluoropyridine-4-carboxamide

Synta-66; CHEMBL3403742; SCHEMBL1829334; CHEBI:231608; GLXC-03244; GSK1349571A;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~77.5 mg/mL (~219.95 mM)

Solubility in Formulation 1: 2.58 mg/mL (7.32 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with heating and sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.58 mg/mL (7.32 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: 2.58 mg/mL (7.32 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)