A2764 diHCl

Activated mTRESK channel (IC50 = 6.8 μM)

将 A2764 (100 µM) 应用于表达 mTRESK 的卵母细胞可导致背景 K+ 电流抑制 42.8±11.5%[1]。在离子霉素诱导的 mTRESK 电流中，A2764 (100 µM) 与激活通道的 IC50 相比表现出增强的抑制活性。之后，使用A2764显着降低电流（77.8±3.5%）[1]。在静息条件下，A2764 (100 µM) 可使 TRESK 电流降低 42.8±11.5%，而在活动期间，它会抑制 TRESK 电流 77.8±3.5% [1]。

双电极电压钳和膜片钳测量。[1]
如前所述，在将cRNA显微注射到爪蟾卵母细胞中1-3天后进行了双电极电压钳实验（Czirják等人，2004）。对于每种通道类型，对n个数有贡献的卵母细胞（确切的n个数在文本或图中标明）来自至少两个，但通常是三个单独的青蛙。保持电位为0 mV。在每4秒施加300毫秒长的电压阶跃至-100 mV后，测量背景钾电流。低钾记录溶液含有以下物质（单位为mM）：NaCl 95.4、KCl 2、CaCl2 1.8和HEPES 5，pH值为7.5，用NaOH调节。高钾溶液含有80mM K+（低钾溶液中的78mM NaCl被KCl替代）。为了测量TREK-1、TREK-2和TRAAK电流，高钾溶液含有40 mM K+。使用重力驱动灌注系统将溶液施加到卵母细胞上。实验在室温（21°C）下进行
电压钳配置中的全细胞膜片钳实验如前所述进行（Lengyel等人，2016）。在无电流注入的电流钳模式下记录静息膜电位（I=0模式）。通过每4秒注入去极化电流（以100 pA为增量，高达1500 pA）1秒来测定流变碱。分离后1至2天，将分离的DRG神经元用于实验。对于DRG神经元的电流钳研究，如先前的研究所述，只接受膜电位在-45至-70 mV之间的细胞（Petruska等人，2000）。由于本研究的重点是检查我们新的氯氧喹类似物对分离的DRG神经元电生理参数的影响，因此没有使用其他选择标准。将八极点贝塞尔滤波器的截止频率调整为200Hz，并在1kHz下采集数据。移液管溶液含有（以mM计）：140 KCl、3 MgCl2、0.05 EGTA、1 Na2 ATP、0.1 Na2 GTP和10 HEPES。低钾溶液含有（以mM计）：140 NaCl、3.6 KCl、0.5 MgCl2、2 CaCl2、11葡萄糖和10 HEPES。高钾溶液含有30mM KCl（低钾溶液的26.4mM NaCl被KCl代替）。用NaOH将浴溶液的pH值调节至7.4。实验在室温（21°C）下进行。

Animal Husbandry, Preparation, and Microinjection of Xenopus Oocytes. Generation of a TRESK Knockout Mouse Line: Isolation of DRG Neurons.[1]
Xenopus laevis oocytes were prepared as previously described (Czirják and Enyedi, 2002). For the expression of the different channels, the oocytes were injected with 57 pg to 4 ng of cRNA (depending on the channel type) 1 day after defolliculation. Injection was performed with a Nanoliter Injector. X. laevis frogs were housed in 50-l tanks with continuous filtering and water circulation. Room temperature was 19°C. Frogs were anesthetized with 0.1% tricaine solution and killed by decerebration and pithing.
FVB/Ant (FVB.129P2-Pde6b+Tyrc-ch/Ant) mice were obtained commercially. TRESK knockout (KO) animals were generated by the transcription activator–like effector nuclease (TALEN) technique using plasmids ordered from Addgene. Mouse TRESK (mTRESK)-specific TALEN recognition sites were designed for the genomic sequence of the first exon (corresponding to the N-terminal intracellular domain of the channel) using the 5′-TN19 N14−20 N19A-3′ formula, with the following sequences: left TALEN recognition site, 5′tgaggagccacctgaggcca; right TALEN recognition site, 5′ccctggggaaggccagggga; and an 18 base pair (5′ggagatgctgtcctgagg) FokI nuclease dimerization and cutting sequence in between. mTRESK recognizing TALEN plasmids were assembled according to the protocol of Sanjana et al. (2012). TALEN mRNAs were produced using the Ambion mMESSAGE mMACHINE T7 in vitro transcription kit (Ambion). TALEN mRNAs were microinjected at a concentration of 20–20 ng/μl into the pronuclei of fertilized eggs of FVB/Ant mice. Pups were analyzed with Surveyor assay plus sequencing. In 12 mice, among the 61 born pups the TRESK (KCNK18) gene was changed, and a founder bearing a 33 base pair deletion and also a mutation introducing a stop codon was chosen to establish a colony.
Adult female wild-type and TRESK KO mice (2–3 months of age) were used for the patch-clamp experiments in this study. The animals were maintained on a 12-hour light/dark cycle with free access to food and water in a specific pathogen-free animal facility. Mice were killed humanely by CO2 exposure (CO2 was applied until death of the animals). DRGs were dissected from the thoracic and lumbar levels of the spinal cord and collected in sterile PBS (137 mM NaCl, 2.7 mM KCl, and 10 mM NaH2PO4, pH adjusted to 7.4 with NaOH) at 4°C. Ganglia were incubated in PBS containing 2 mg/ml collagenase enzyme (type I) for 30 minutes with gentle shaking at 37°C. For further details regarding the isolation and culturing of the cells, see Braun et al. (2015). All experimental procedures using animals were conducted in accordance with the Guide for the Care and Use of Laboratory Animals as adopted by the National Institutes of Health, local state laws, and institutional regulations. All animal experiments were approved by the Animal Care and Ethics Committee of Semmelweis University (approval ID: XIV-I-001/2154-4/2012).

[1]. Chemically Modified Derivatives of the Activator Compound Cloxyquin Exert Inhibitory Effect on TRESK (K2P18.1) Background Potassium Channel.Mol Pharmacol. 2019 Jun;95(6):652-660.

Cloxyquin has been reported as a specific activator of TRESK [TWIK-related spinal cord K+ channel (also known as K2P18.1)] background potassium channel. In this study, we have synthetized chemically modified analogs of cloxyquin and tested their effects on TRESK and other K2P channels. The currents of murine K2P channels, expressed heterologously in Xenopus oocytes, were measured by two-electrode voltage clamp, whereas the native background K+ conductance of mouse dorsal root ganglion (DRG) neurons was examined by the whole-cell patch-clamp method. Some of the analogs retained the activator character of the parent compound, but, more interestingly, other derivatives inhibited mouse TRESK current. The inhibitor analogs (A2764 and A2793) exerted state-dependent effects. The degree of inhibition by 100 µM A2764 (77.8% ± 3.5%, n = 6) was larger in the activated state of TRESK (i.e., after calcineurin-dependent stimulation) than in the resting state of the channel (42.8% ± 11.5% inhibition, n = 7). The selectivity of the inhibitor compounds was tested on several K2P channels. A2793 inhibited TWIK-related acid-sensitive K+ channel (TASK)-1 (100 µM, 53.4% ± 13, 5%, n = 5), while A2764 was more selective for TRESK, it only moderately influenced TREK-1 and TWIK-related alkaline pH-activated K+ channel. The effect of A2764 was also examined on the background K+ currents of DRG neurons. A subpopulation of DRG neurons, prepared from wild-type animals, expressed background K+ currents sensitive to A2764, whereas the inhibitor did not affect the currents in the DRG neurons of TRESK-deficient mice. Accordingly, A2764 may prove to be useful for the identification of TRESK current in native cells, and for the investigation of the role of the channel in nociception and migraine. SIGNIFICANCE STATEMENT: TRESK background potassium channel is a potential pharmacological target in migraine and neuropathic pain. In this study, we have identified a selective inhibitor of TRESK, A2764. This compound can inhibit TRESK in native cells, leading to cell depolarization and increased excitability. This new inhibitor may be of use to probe the role of TRESK channel in migraine and nociception.[1]

C15H20CL2N2O

315.238101959229

350.071

861038-72-4

146013302

Typically exists as Light yellow to yellow solids at room temperature

25.4Ų

2

3

6

21

261

0

ZVRDPULCSHGKBD-UHFFFAOYSA-N

InChI=1S/C15H19ClN2O.2ClH/c1-3-18(4-2)10-11-19-14-8-7-13(16)12-6-5-9-17-15(12)14;;/h5-9H,3-4,10-11H2,1-2H3;2*1H

2-(5-chloroquinolin-8-yl)oxy-N,N-diethylethanamine;dihydrochloride

A 2764 diHCl; A-2764 diHCl; A2764 dihydrochloride; 2-((5-Chloroquinolin-8-yl)oxy)-N,N-diethylethanamine dihydrochloride; A2764 (dihydrochloride); 2-((5-Chloroquinolin-8-yl)oxy)-N,N-diethylethanaminedihydrochloride; 2-(5-chloroquinolin-8-yl)oxy-N,N-diethylethanamine;dihydrochloride; A2764 dihydrochloride?; TRESK inhibitor A2764;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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

H2O : ≥ 100 mg/mL (~284.33 mM)
DMSO : ~41.67 mg/mL (~118.48 mM)

配方 1 中的溶解度: ≥ 2.08 mg/mL (5.91 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 20.8 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (5.91 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。