Small-molecule probes for study of CLC-2 chloride-channel function in the central nervous system

项目摘要 CLC氯离子通道家族是一类膜蛋白，其控制CLC的通量。 氯离子穿过细胞膜。九个独特的CLC同源物在哺乳动物中差异表达。 哺乳动物的组织和功能在不同的生理作用，从电刺激， 肌肉和神经元的电解质平衡调节。一种亚型，CLC-2，是一种电压- 在大脑中广泛表达的依赖性通道。尽管大脑中CLC-2的存在 几十年来，这种CLC同源物在神经元信号传导和适当的 大脑功能仍然知之甚少，部分原因是缺乏有效的和选择性的 小分子工具，使研究的分子生理学的这一渠道。最近的一 我们实验室的突破现在打开了开发小分子工具的大门， CLC-2。通过化合物库筛选，我们确定了抑制CLC-2的“命中”化合物 活动我们将其中之一开发成一种有效的选择性CLC-2抑制剂FA 44， 对CLC-2的IC 50为18 nM，对最接近的CLC同系物或对CLC-2的 一组65个CNS通道、受体和转运蛋白。FA 44用于以下的功效和选择性： CLC-2进一步得到了我们对野生型脑切片的电生理记录的支持。 与CLC-2敲除小鼠相比。在这个项目中，我们将继续努力， 开发、表征和使用化学工具化合物来研究CLC-2。在目标1中，我们 确定抑制CLC-2的作用机制和分子决定因素。在目标2中， 我们将开发新的探针，包括小分子激活剂和荧光成像， 探针定位通道表达。在目标3中，我们将利用我们的工具化合物， 质疑CLC-2在兴奋性突触传递和网络兴奋性中的作用， 丘脑，并评估CLC-2功能障碍和癫痫之间的潜在因果关系。 我们团队在合成化学（Du Bois）、离子通道结构-功能 （Maduke），计算（Dror）和细胞神经科学/癫痫（Huguenard）的理想位置 我们要推进这项研究计划。

PROJECT SUMMARY The CLC chloride channel family is a class of membrane proteins that controls the flux of chloride ions across cell membranes. Nine unique CLC homologs are differentially expressed in mammalian tissue and function in diverse physiological roles, ranging from electrical excitation of muscles and neurons to regulation of electrolyte balance. One subtype, CLC-2, is a voltage- dependent channel expressed broadly in the brain. Although the presence of CLC-2 in the brain has been known for decades, the role of this CLC homolog in neuronal signaling and proper brain function remains poorly understood, in part due to the absence of potent and selective small-molecule tools that enable studies of the molecular physiology of this channel. A recent breakthrough in our laboratories now opens the door to developing small molecule tools specific to CLC-2. Through a compound-library screen, we identified ‘hit’ compounds that inhibit CLC-2 activity. We developed one of these into a potent and selective CLC-2 inhibitor, FA44, which has an IC50 of 18 nM for CLC-2 and no off-target effects on the closest CLC homolog or on a panel of 65 CNS channels, receptors, and transporters. The efficacy and selectivity of FA44 for CLC-2 is further supported by our electrophysiological recordings of brain slices from wild-type versus CLC-2 knock-out mice. In this project, we will continue our collaborative efforts to develop, characterize, and use chemical tool compounds for studying CLC-2. In Aim 1, we will identify the mechanism of action and molecular determinants for inhibition of CLC-2. In Aim 2, we will develop novel probes, including small-molecule activators and fluorescent imaging probes for localizing channel expression. In Aim 3, we will leverage our tool compounds to query the role of CLC-2 in excitatory synaptic transmission and network excitability in the thalamus and to evaluate the potential causative link between CLC-2 malfunction and epilepsy. Our team’s combined expertise in synthetic chemistry (Du Bois), ion-channel structure-function (Maduke), computation (Dror), and cellular neuroscience/epilepsy (Huguenard) ideally positions us to advance this research program.