CLC-2 voltage-gated chloride channel structure and ligand recognition

在中枢神经系统中，电压门控离子通道在动作电位的形成中起着重要作用 击发阳离子选择性电压门控通道-钠（Na+）、钾（K+）和钙（Ca 2+）通道 - 在过去的几十年里受到了严格的审查。相反，氯离子（Cl-）选择性电压门控 尽管CLC-2在整个CNS的神经元和神经胶质中广泛表达，但对CLC-2通道的了解较少。 对CLC-2对CNS功能的贡献的完整理解将包括对CLC-2的 分子结构在结构上，CLC渠道拥有独特的双管架构， 通过不同的门控（打开/关闭）机制，这些机制与研究充分的Na+，K+， Ca 2+通道除了为研究CLC-2通道门控提供关键框架外， 渗透机制，确定CLC-2结构的另一个令人信服的理由是它的价值， 理解配体相互作用和指导小分子探针的设计。这样的探测器将是伟大的 在CLC-2神经生理学研究中的价值。因此，本R21项目的目标是开发表达 和CLC-2的纯化方案，并使用冷冻电子显微镜来确定CLC-2在细胞中的结构。 不存在和存在选择性CLC-2抑制剂AK-42。

In the central nervous system (CNS), voltage-gated ion channels play central roles in shaping action-potential firing. The cation-selective voltage-gated channels – sodium (Na+), potassium (K+), and calcium (Ca2+)) channels – have received intense scrutiny over the past decades. In contrast, the chloride (Cl–)-selective voltage-gated channel, CLC-2, is less well understood, despite its broad expression in neurons and glia throughout the CNS. A complete understanding of CLC-2’s contribution to CNS function will include an understanding of CLC-2’s molecular structure. Structurally, CLC channels possess a unique double-barreled architecture and operate through distinct gating (opening/closing) mechanisms that differ markedly from those of the well-studied Na+, K+, and Ca2+ channels. In addition to providing a critical framework for studying CLC-2 channel gating and permeation mechanisms, another compelling rationale for determining the CLC-2 structure is its value for understanding ligand interactions and guiding design of small-molecule probes. Such probes would be of great value in investigations of CLC-2 neurophysiology. Accordingly, the goal of this R21 project is develop expression and purification protocols for CLC-2 and to use cryo-electron microscopy to determine CLC-2 structures in the absence and presence of the selective CLC-2 inhibitor AK-42.