TRPC4/TRPC5

ML204 盐酸盐对毒蕈碱受体偶联的 TRPC6 通道激活具有 19 倍的选择性，并抑制 TRPC4β 介导的细胞内 Ca2+ 增加，IC50 值为 0.96 μM（HEK293 细胞）[1]。 ML204 盐酸盐抑制 TRPC4β 活性，该活性由内源性 M3 样毒蕈碱受体触发，通过 μ-阿片类药物、5HT1A 血清素和 M2 毒蕈碱受体刺激 Gq/11 或 Gi/o 激活[1]。 LPS 诱导的 TRPC5 通道活性被 ML204 盐酸盐抑制[3]。
ML204 与毒蕈碱受体偶联 TRPC6 通道激活有 19 倍耦合，并抑制 TRPC4β 介导的细胞内 Ca2+ 升高，IC50 值为 0.96 μM（HEK293 细胞）[1]。 M2 毒蕈碱受体或内源性 M3 样毒蕈碱受体、u-阿片类药物、5HT1A 血红素、Gi/o 和 TRPC4β 对 Gq/11 的激活被 ML204 阻断[1]。 ML204 抑制由 LPS 触发的 TRPC5 通道活动 [3]。

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在MLSMR文库的高通量荧光筛选和活性化合物的SAR分析后，ML204被鉴定为一种新型TRPC4通道抑制剂。ML204抑制了μ阿片受体刺激激活的TRPC4通道的钙内流，IC50值为0.96μM，在类似的荧光检测中对TRPC6通道表现出19倍的选择性。ML204在电生理学检测中阻断了TRPC4通道，IC值为2.6μM，在荧光和电生理检测中也表现活跃，其中TRPC4信道由不同机制激活，表明直接阻断了TRPCV4信道。在荧光和电生理实验中，针对密切相关的TRPC通道和较远相关的TRPV、TRPA和TRPM通道，以及非TRP离子通道，检查了TRPC4通道阻断的选择性。ML204对TRPC6通道具有良好的选择性（19倍），对TRPC3和TRPC5通道具有更温和的选择性（9倍）。很少或没有观察到TRPV、TRPA、TRPM或电压门控离子通道的阻断。ML204表现出可用于各种体外研究的特性[2]。

在注射 LPS 的小鼠中，ML204 盐酸盐（1 mg/kg；皮下注射；每天两次；持续 5 天）会减少腹膜白细胞计数和细胞因子，并导致与体温过低恶化相关的死亡[4]。 ML204对TRPC4/TRPC5的双重阻断增加了硫氧还蛋白处理的LPS小鼠的死亡率和低温，但保留了巨噬细胞的吞噬能力。TRPC5缺失不会改变体温，但会促进硫氧还蛋白给药LPS小鼠腹膜白细胞的额外积聚和炎症介质的释放。硫氧还蛋白减少了野生型巨噬细胞介导的吞噬作用，但没有减少TRPC5敲除动物的吞噬作用。TRPC5消融不影响LPS诱导的反应。然而，在注射LPS的小鼠中，ML204导致了与低温加剧和腹膜白细胞数量和细胞因子减少相关的死亡率。这些结果表明，LPS刺激下的细菌硫氧还蛋白效应是由TRPC4和TRPC5介导的，从而揭示了细菌毒力的其他机制以及这些受体的病理生理作用。 [4]

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值得注意的是，ML204灌注的小鼠免受PS诱导的FPE的影响（图7A）。通过计算TEM图像中GBM测量长度上的FP数量（每组n=90-105张图像），以盲法量化这些结果。通过这一分析，ML204灌注小鼠对PS的影响显示出显著的保护作用（图7B）。这些结果与我们在Trpc5 KO小鼠中的观察结果一致。[3]
接下来，我们测试了ML204在LPS模型中的作用。在注射LPS后，每隔12小时注射两次ML204（20mg/kg/d i.p.）。对照组注射PBS的小鼠没有可观察到的结构变化，也没有蛋白尿（图7，C-E）。LPS注射导致FPE（图7D），尽管这比PS诱导的FPE更温和。重要的是，经ML204处理的小鼠免受LPS诱导的FPE和蛋白尿的影响[3]。

Rac1激活试验。[3]
Rac1活化试验如前所述进行，但有一些修改。足细胞用300μg/ml PS处理1小时，然后进行收获和Rac1下拉实验。在ML204实验中，在施加PS之前，用30μMML204预处理细胞20分钟。根据制造商的说明，使用与人PAK-1的p21结合结构域（PBD；残基67-150）相对应的GST标记的融合蛋白，用商业Rac1活化测定试剂盒 分析活化的Rac1。下拉后，使用小鼠单克隆Rac1抗体通过免疫印迹检测洗脱的活性Rac1。在用于下拉研究的细胞裂解物中测量总Rac1和GADPH，并作为负载对照。

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电生理学。[3]
膜片钳电生理学是在全细胞结构或外向贴片上进行的。用P-97拉拔器从硼硅酸盐玻璃中拉出电阻为3-4MΩ的贴片移液管，并填充含有135 mM CH3SO3Cs、10 mM CsCl、3 mM MgATP、0.2 mM NaGTP、0.2 mM EGTA、0.13 mM CaCl2和10 mM HEPES（pH 7.3）的CsOH溶液。浴液含有135 mM CH3SO3Na、5 mM CsCl、2 mM CaCl2、1 mM MgCl2、10 mM HEPES和10 mM葡萄糖（pH 7.4）以及NaOH。将血管紧张素II（500 nM）、LPS（100μg/ml）和ML204（10μM）应用于浴液。在400毫秒内记录了从-100 mV到+100 mV的全细胞电流，电压斜坡为-60 mV。对于外向配置的单通道记录，我们使用了以20 mV的间隔和0 mV的保持电位进行的-100 mV到+100 mV的电压阶跃协议。填充有移液管溶液的平均移液管电阻为3-5 MΩ。数据以10kHz采样，以5kHz滤波。在分析之前，单通道数据在500Hz下进一步离线滤波。在单通道迹线中，使用手动定义的振幅标准对电流进行理想化，以分配离子通道的打开和关闭过渡。集合平均值表示为Po（平均电流除以单位电流振幅和每个斑块的通道数），并绘制为直方图。所有数据均在室温下采集，并使用pClamp 10进行分析。

PS、LPS和Cch处理培养的足细胞。[3] 以>90%融合生长的分化培养足细胞与1-30μM ML204一起孵育20分钟，然后根据需要暴露于300μg/ml PS、100μg/ml LPS或100μM Cch。对于PS实验，一旦通过光学显微镜观察到细胞形态的变化（70-90分钟），就用PBS中的4%多聚甲醛固定细胞15分钟，然后用0.1%Triton X-100透化10分钟。对于LPS和Cch实验，在处理后24小时如上所述固定细胞。为了进行免疫染色，足细胞与突触蛋白“NT”抗体一起孵育，并用Alexa Fluor 488偶联的二抗进行检测。如前所述，肌动蛋白结构用Alexa Fluor 594偶联的鬼笔环肽标记。分析了3项独立试验，每项试验中每种条件3个培养皿，每皿10张图像，细胞密度相当。PS/ML204实验共分析1600-2000个细胞，PS/KD实验共分析1400-1600个细胞。共分析了1000个细胞用于LPS实验，1400个细胞用于Cch实验。通过DAPI染色和ImageJ中的自动脚本分析细胞数量，随后手动校正。如前所述，受影响的细胞被定义为具有非常明亮、凝聚的肌动蛋白染色的塌陷细胞（用于PS实验），或没有明显可见应力纤维的细胞（用于LPS和Cch实验）。图像是用蔡司LSM510立式共聚焦显微镜采集的。使用蔡司Pascal软件采集3-4μm光学切片的图像。通过方差分析和Dunnett多重比较检验评估统计显著性。

Animal/Disease Models: Nonfasted male C57BL/6 (2 -3 months)[4]
Doses: 1 mg/kg
Route of Administration: subcutaneous (sc) injection, twice a day, for 5 days (prior to LPS injection)
Experimental Results: Induces mortality associated with increased hypothermia in mice with LPS-induced systemic inflammatory response.

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LPS-induced albuminuria.[3]
Induction of albuminuria in male WT and Trpc5-KO mice (20–25 g BW) by LPS injection was done as previously described, with some modifications. At 48 hours prior to injection, baseline urine was collected for 24 hours in metabolic cages. LPS (15 μg/g i.p., 1 mg/ml) was injected twice, at the 0- and 24-hour time points. PBS was injected i.p. twice, at 12 and 36 hours, to avoid dehydration. ML204 (20 mg/kg/d i.p.) was injected at 12 and 24 hours. Urine was collected for a 24-hour period beginning 24 hours after initial LPS injection using metabolic cages. To quantify the levels of albuminuria, 10 μl urine was analyzed by SDS-PAGE. Bovine serum albumin standards (0.25, 0.5, 1.0, 2.5, and 5.0 μg) were run on the same gel and used to identify and quantify urinary albumin bands. Coomassie signals were quantified using ImageJ. The resulting values — the product of area size and mean gray value of each albumin standard band — were used for construction of a standard curve and its associated mathematical function. Subsequently, the values of the sample bands were translated into albumin concentrations, which were extrapolated to the 24-hour total urine volume. Results were assessed by ANOVA and Bonferroni’s multiple-comparison test.
For preparation of glomerular lysates for Western blotting, mice were treated as above and killed 36 hours after initial LPS injection. Mouse kidneys were perfused through the renal artery with Dynabeads for magnetic isolation of highly purified glomeruli. Protein extraction from isolated glomeruli, SDS-PAGE, and Western blotting was done as described previously, and proteins were detected with appropriate primary and secondary antibodies.

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PS model.[3]
Adult WT (n = 15) and Trpc5-KO (n = 7) littermate mice were anesthetized with pentobarbital and placed on a heat pad set at 37°C, and their kidneys were perfused in situ through the renal artery at a pressure of approximately 240 mm Hg and an infusion rate of 9 ml/min as previously described (58), with some modifications. First, kidneys were flushed with HBSS or with HBSS plus 10 μM ML204 at 37°C for 2 minutes, followed by perfusion with 2 mg/ml PS in HBSS or with PS plus ML204 at 37°C for 15 minutes. All vascular perfusion solutions were kept at 37°C throughout the duration of the experiment.

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Pharmacological Treatments[4]
C57BL/6, TRPC5+/+, and TRPC5−/− mice received a subcutaneous (s.c.) injection of phosphate-buffered saline (PBS) containing bacterial Trx (20 μg/150 μl/animal, twice a day; from E. coli) for 3 days prior to the induction of SIRS. In order to assess the role of TRPC4 and TRPC5 complexes in LPS-induced responses, C57BL/6 mice received ML204 [16, 21] (1 mg/kg, 150 μl/animal, twice a day) for 5 days and then LPS. In a separate set of experiments, C57BL/6 animals received ML204 (1 mg/kg, twice a day; in 6% dimethyl sulfoxide (DMSO) in PBS) for 2 days alone, and then, this drug was coinjected with bacterial Trx (20 μg/animal, twice a day) for another 3 days prior to LPS challenge. Vehicle-treated mice were used as controls.

[1]. Identification of ML204, a novel potent antagonist that selectively modulates native TRPC4/C5 ion channels. J Biol Chem. 2011 Sep 23;286(38):33436-46.

[2]. Novel Chemical Inhibitor of TRPC4 Channels. Probe Reports from the NIH Molecular Libraries Program.

[3]. Inhibition of the TRPC5 ion channel protects the kidney filter. J Clin Invest. 2013 Dec 2; 123(12): 5298–5309.

[4]. Transient Receptor Potential Canonical Channels 4 and 5 Mediate Escherichia coli-Derived Thioredoxin Effects in Lipopolysaccharide-Injected Mice. Oxid Med Cell Longev. 2018 Jun 10;2018:4904696.

Transient receptor potential canonical (TRPC) channels are Ca(2+)-permeable nonselective cation channels implicated in diverse physiological functions, including smooth muscle contractility and synaptic transmission. However, lack of potent selective pharmacological inhibitors for TRPC channels has limited delineation of the roles of these channels in physiological systems. Here we report the identification and characterization of ML204 as a novel, potent, and selective TRPC4 channel inhibitor. A high throughput fluorescent screen of 305,000 compounds of the Molecular Libraries Small Molecule Repository was performed for inhibitors that blocked intracellular Ca(2+) rise in response to stimulation of mouse TRPC4β by μ-opioid receptors. ML204 inhibited TRPC4β-mediated intracellular Ca(2+) rise with an IC(50) value of 0.96 μm and exhibited 19-fold selectivity against muscarinic receptor-coupled TRPC6 channel activation. In whole-cell patch clamp recordings, ML204 blocked TRPC4β currents activated through either μ-opioid receptor stimulation or intracellular dialysis of guanosine 5'-3-O-(thio)triphosphate (GTPγS), suggesting a direct interaction of ML204 with TRPC4 channels rather than any interference with the signal transduction pathways. Selectivity studies showed no appreciable block by 10-20 μm ML204 of TRPV1, TRPV3, TRPA1, and TRPM8, as well as KCNQ2 and native voltage-gated sodium, potassium, and calcium channels in mouse dorsal root ganglion neurons. In isolated guinea pig ileal myocytes, ML204 blocked muscarinic cation currents activated by bath application of carbachol or intracellular infusion of GTPγS, demonstrating its effectiveness on native TRPC4 currents. Therefore, ML204 represents an excellent novel tool for investigation of TRPC4 channel function and may facilitate the development of therapeutics targeted to TRPC4.[1]

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ML204 was identified as a novel TRPC4 channel inhibitor following a high throughput fluorescent screen of the MLSMR library and SAR analysis of active compounds. ML204 inhibited calcium influx through TRPC4 channels activated by μ-opioid receptor stimulation with an IC50 value of 0.96 μM and exhibited 19-fold selectivity against TRPC6 channels in similar fluorescent assays. ML204 blocked TRPC4 channels in an electrophysiological assay with an IC value of 2.6 μM and was also active in fluorescent and electrophysiological assays in which TRPC4 channels were activated by different mechanisms, indicating direct block of TRPC4 channels. Selectivity for block of TRPC4 channels was examined in fluorescent and electrophysiological experiments against closely related TRPC channels and more distantly related TRPV, TRPA and TRPM channels, and against non-TRP ion channels. ML204 afforded good selectivity (19-fold) against TRPC6 channels and more modest selectivity against TRPC3 and TRPC5 (9-fold) channels. Little or no block of TRPV, TRPA, TRPM or voltage-gated ion channels was observed. ML204 exhibited properties useful for a variety of in vitro investigations.[2]

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An intact kidney filter is vital to retention of essential proteins in the blood and removal of waste from the body. Damage to the filtration barrier results in albumin loss in the urine, a hallmark of cardiovascular disease and kidney failure. Here we found that the ion channel TRPC5 mediates filtration barrier injury. Using Trpc5-KO mice, a small-molecule inhibitor of TRPC5, Ca2+ imaging in isolated kidney glomeruli, and live imagining of podocyte actin dynamics, we determined that loss of TRPC5 or its inhibition abrogates podocyte cytoskeletal remodeling. Inhibition or loss of TRPC5 prevented activation of the small GTP-binding protein Rac1 and stabilized synaptopodin. Importantly, genetic deletion or pharmacologic inhibition of TRPC5 protected mice from albuminuria. These data reveal that the Ca2+-permeable channel TRPC5 is an important determinant of albuminuria and identify TRPC5 inhibition as a therapeutic strategy for the prevention or treatment of proteinuric kidney disease.[3]

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Thioredoxin plays an essential role in bacterial antioxidant machinery and virulence; however, its regulatory actions in the host are less well understood. Reduced human Trx activates transient receptor potential canonical 5 (TRPC5) in inflammation, but there is no evidence of whether these receptors mediate bacterial thioredoxin effects in the host. Importantly, TRPC5 can form functional complexes with other subunits such as TRPC4. Herein, E. coli-derived thioredoxin induced mortality in lipopolysaccharide- (LPS-) injected mice, accompanied by reduction of leukocyte accumulation, regulation of cytokine release into the peritoneum, and impairment of peritoneal macrophage-mediated phagocytosis.[4]

C15H19CLN2

262.777762651443

262.123

2070015-10-8

5465-86-1;2070015-10-8 (HCl);

49786978

White to off-white solid powder

16.1

1

2

1

18

247

0

Cl.N1(C2C=C(C)C3C=CC=CC=3N=2)CCCCC1

QCABWRXQHZUBPW-UHFFFAOYSA-N

InChI=1S/C15H18N2.ClH/c1-12-11-15(17-9-5-2-6-10-17)16-14-8-4-3-7-13(12)14;/h3-4,7-8,11H,2,5-6,9-10H2,1H3;1H

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)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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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：
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