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

项目概要 CLC 氯离子通道家族是一类膜蛋白，控制着氯离子的通量。 氯离子穿过细胞膜。九个独特的 CLC 同源物在 哺乳动物的组织和功能具有多种生理作用，包括电激发 肌肉和神经元调节电解质平衡。一种亚型 CLC-2 是一种电压- 依赖性通道在大脑中广泛表达。尽管大脑中存在 CLC-2 几十年来，人们已经知道这种 CLC 同源物在神经元信号传导和适当的作用中的作用 大脑功能仍然知之甚少，部分原因是缺乏有效和选择性的 小分子工具可以研究该通道的分子生理学。最近的一个 我们实验室的突破现在为开发特定的小分子工具打开了大门 至 CLC-2。通过化合物库筛选，我们鉴定了抑制 CLC-2 的“热门”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 FA44， CLC-2 的 IC50 为 18 nM，对最接近的 CLC 同系物或 由 65 个 CNS 通道、受体和转运蛋白组成的面板。 FA44 的功效和选择性 我们对野生型脑切片的电生理记录进一步支持了 CLC-2 与 CLC-2 敲除小鼠相比。在这个项目中，我们将继续共同努力 开发、表征和使用化学工具化合物来研究 CLC-2。在目标 1 中，我们将 确定抑制 CLC-2 的作用机制和分子决定因素。在目标 2 中， 我们将开发新型探针，包括小分子激活剂和荧光成像 用于定位通道表达的探针。在目标 3 中，我们将利用我们的工具化合物来 查询 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 功能障碍与癫痫之间的潜在因果关系。 我们团队综合了合成化学 (Du Bois)、离子通道结构功能方面的专业知识 (Maduke)、计算 (Dror) 和细胞神经科学/癫痫 (Huguenard) 的理想位置 我们来推进这项研究计划。

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.