Department of Neuroscience, High Throughput Biology Center, and Johns Hopkins Ion Channel Center, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
J Biol Chem. 2011 Sep 23;286(38):33436-46. doi: 10.1074/jbc.M111.274167. Epub 2011 Jul 27.
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.
瞬时受体电位经典型（TRPC）通道是钙通透性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、选择性的 TRPC 通道药理学抑制剂限制了这些通道在生理系统中的作用的研究。本文报道了 ML204 的鉴定和特性，它是一种新型、有效和选择性的 TRPC4 通道抑制剂。通过对 305000 种化合物的高通量荧光筛选，鉴定出了能阻断μ阿片受体刺激后小鼠 TRPC4β 诱导的细胞内 Ca2+升高的化合物。ML204 对 TRPC4β 介导的细胞内 Ca2+升高的抑制作用具有 IC50 值为 0.96μm，对毒蕈碱受体偶联的 TRPC6 通道激活具有 19 倍的选择性。在全细胞膜片钳记录中，ML204 阻断了通过 μ 阿片受体刺激或细胞内 GTPγS 透性化激活的 TRPC4β 电流，表明 ML204 与 TRPC4 通道的直接相互作用，而不是对信号转导途径的任何干扰。选择性研究表明，10-20μm 的 ML204 对 TRPV1、TRPV3、TRPA1 和 TRPM8 以及 KCNQ2 和天然电压门控钠、钾和钙通道没有明显的阻断作用，在小鼠背根神经节神经元中。在分离的豚鼠回肠肌细胞中，ML204 阻断了浴用 carbachol 或细胞内 GTPγS 输注激活的毒蕈碱阳离子电流，证明了它对天然 TRPC4 电流的有效性。因此，ML204 代表了研究 TRPC4 通道功能的一种极好的新型工具，并且可能有助于针对 TRPC4 的治疗药物的开发。
