Identification of ML204, a Novel Potent Antagonist That Selectively Modulates Native TRPC4/C5 Ion Channels
Identification of ML204, a Novel Potent Antagonist That Selectively Modulates Native TRPC4/C5 Ion Channels
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DOI：
10.1074/jbc.m111.274167
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发表时间：
2011-09-23
影响因子：
4.8
通讯作者：
Zhu, Michael X.
中科院分区：
文献类型：
--
作者：
Miller, Melissa;Shi, Jie;Zhu, Michael X.
瞬时受体电位通道是一类钙离子通透性的非选择性阳离子通道，参与多种生理功能，包括平滑肌收缩和突触传递。然而，缺乏有效的选择性药物抑制TRPC通道，限制了对这些通道在生理系统中的作用的描述。在这里，我们报告了ML204作为一种新的、有效的和选择性的TRPC4通道抑制剂的鉴定和表征。对分子库小分子储存库中的305,000种化合物进行了高通量荧光筛选，以寻找阻止细胞内钙离子升高的抑制剂，这些抑制剂可以阻止小鼠TRPC4β受mU-阿片受体刺激而产生的钙升高。ML204可抑制TRPC4β介导的细胞内钙升高，其IC50值为0.96mU M，并对M受体偶联的TRPC6通道的激活具有19倍的选择性。在全细胞膜片钳记录中，ML204阻断了通过Mu阿片受体刺激或细胞内透析5‘-3-O-(硫代)三磷酸鸟苷(GTP Gamma S)激活的TRPC4β电流，提示ML204与TRPC4通道直接相互作用，而不是干扰信号转导通路。选择性研究表明，10-20MML204的TRPV1、TRPV3、TRPA1和TRPM8以及KCNQ2和自然电压门控性钠、钾、钙通道对小鼠背根神经节神经元无明显阻断作用。在豚鼠回肠肌细胞上，ML204阻断由卡巴胆碱浴或细胞内注入GTP-γS所激活的M胆碱型阳离子电流，证明其对天然TRPC_4电流的作用。因此，ML204是研究TRPC4通道功能的一种很好的新工具，并可能促进针对TRPC4的治疗药物的发展。
Transient receptor potential canonical (TRPC) channels are Ca2+-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 Ca2+ rise in response to stimulation of mouse TRPC4 beta by mu-opioid receptors. ML204 inhibited TRPC4 beta-mediated intracellular Ca2+ rise with an IC50 value of 0.96 mu M and exhibited 19-fold selectivity against muscarinic receptor-coupled TRPC6 channel activation. In whole-cell patch clamp recordings, ML204 blocked TRPC4 beta currents activated through either mu-opioid receptor stimulation or intracellular dialysis of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S), 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 mu M 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 GTP gamma S, 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.