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 central to Ca²⁺ signaling in astrocytes. We aimed to elucidate the molecular mechanisms of storage-operated calcium influx in mouse Müller cells, involving ER calcium replenishment. Calcium depletion induced by blocking calcium chelate transporters in calcium-free saline synergistically activated classical transient receptor potential 1 (TRPC1) and Orai channels. We identified storage-operated TRPC1 channels using electrophysiological properties, pharmacological inhibitors, and Trpc1 gene knockout. We characterized the calcium release activation current (ICRAC) by ion permeability, voltage dependence, and sensitivity to the selective Orai antagonists Synta66 and GSK7975A. Depletion-induced calcium influx originates at the terminal foot and apical processes of Müller cells, triggering centrifugal propagation of Ca²⁺ waves into the cell body. Electron microscopy analysis of the terminal foot region showed that high-density endoplasmic reticulum cisterns obscured the cell bodies and axons of retinal ganglion cells (RGCs), as well as the endoplasmic reticulum-mitochondrial junctions on the surface of protoplasmic astrocytes, vascular endothelial cells, and terminal foot vitreous bodies. Mouse retinas expressed transcripts encoding Stim and all known Orai genes; Müller glial cells mainly expressed matrix interaction molecule 1 (STIM1), while STIM2 was mainly confined to the outer plexiform layer and RGC layer. The loss of TRPC1 promoted Müller glial proliferation induced by intraocular pressure elevation, suggesting that TRPC channels may play a neuroprotective role during mechanical stress. By characterizing the properties of storage manipulative signaling pathways in Müller cells, these studies expanded the current understanding of the functional roles of these cells in retinal physiology and pathology, and also provided further evidence for the complexity of calcium signaling mechanisms in astrocytes of the central nervous system. [1]

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Objective: Platelet Orai1 channels mediate storage manipulative Ca(2+) influx (SOCE), which is essential for procoagulant activity and arterial thrombosis. Pharmacological blocking of these pathways may provide a novel antithrombotic treatment approach. Therefore, this study aimed to determine the thromboprotective effect of SOCE blockers targeting Orai1 in platelets. Methods and Results: Candidate inhibitors were screened for their effects on SOCE in washed human platelets. Antagonists tested included known compounds SKF96365, 2-aminoethyl diphenylboronic acid ester, and MRS1845, as well as novel compounds Synta66 and GSK-7975A. The order of SOCE inhibitory potency was: Synta66, 2-aminoethyl diphenylboronic acid ester, GSK-7975A, SKF96365, MRS1845. The specificity of the first three compounds was validated using platelets from Orai1-deficient mice. The inhibitory effects of these compounds on procoagulant activity and high-shear thrombus formation were evaluated in plasma and whole blood. In the presence of plasma, all three compounds inhibited platelet response and thrombus formation under flow conditions. Using a mouse stroke model, arterial thrombosis was induced in vivo by transient middle cerebral artery occlusion. Postoperative administration of 2-aminoethyl diphenylboronic acid significantly reduced the infarct area. Conclusion: Plasma-soluble SOCE blockers, such as 2-aminoethyl diphenylboronic acid, can inhibit platelet-dependent coagulation and thrombosis. Platelet Orai1 channels are a novel target for preventing thrombotic events leading to cerebral infarction. [2]

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Abnormal activation of mast cells via FcεRI receptors leads to the release of inflammatory mediators and symptoms of allergic diseases. Calcium influx is a key regulator of mast cell signaling and is essential for the exocytosis of pre-formed mediators and the synthesis of arachidic acid, cytokines and chemokines. Studies of rodent and human mast cells have confirmed that Orai calcium channels are key factors in the release of mediators initiated by FcεRI. However, the role of TRPC calcium channels in FcεRI-mediated human mast cell signaling has not been reported to date. This study confirmed the expression of Orai 1, 2, and 3, as well as TRPC1 and 6, in primary human lung mast cells and the LAD2 human mast cell line. However, we found that only Orai channels, not TRPC channels, functionally contribute to FcεRI-mediated calcium influx. Calcium imaging experiments using the Orai selective antagonist (Synta66) showed that Orai is involved in FcεRI-mediated signaling in human mast cells. However, no evidence was found of TRPC6 involvement in FcεRI-mediated calcium signaling in human mast cells using TRPC3/6 selective antagonists and agonists (GSK-3503A and GSK-2934A, respectively). Similarly, in human mast cells, STIM1-regulated TRPC1 inactivation (tested by transfecting cells with the STIM1-KK684-685EE-TRPC1 gated mutant) failed to alter FcεRI-mediated calcium signaling in LAD2 human mast cells. Mediator release experiments confirmed that FcεRI-mediated calcium ion influx via Orai is essential for histamine and TNFα release, but plays different roles in the production of cytokines and arachidic acid. [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.)