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

在中枢神经系统(CNS)中，电压门控离子通道在动作电位的形成中起着核心作用 开火。阳离子选择性电压门控通道--钠(Na+)、钾(K+)和钙(Ca+)通道 -在过去的几十年里受到了严格的审查。相比之下，氯离子(Cl-)选择性电压门控 尽管CLC-2在整个中枢神经系统的神经元和神经胶质细胞中广泛表达，但人们对它的了解较少。 全面了解《中图法》2号S对中枢神经系统功能的贡献，将包括理解《中图法》2号的S 分子结构。在结构上，CLC通道拥有独特的双管式体系结构并运行 通过不同的门控(打开/关闭)机制，与那些研究得很好的Na+，K+， 和钙离子通道。除了为研究CLC-2通道选通和 渗透机制，确定ClC-2结构的另一个令人信服的理由是它对 了解配体相互作用并指导小分子探针的设计。这样的调查将是非常有意义的 ClC-2在神经生理学研究中的价值因此，这个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确定CLC-2的结构 选择性ClC-2抑制剂AK-42的存在和缺失。

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.