Structural investigation of the gating and regulatory mechanism of voltage-gated Ca2+ channels

钙离子（Ca 2+）在收缩、分泌等多种生理过程中发挥着关键作用， 神经传递、基因转录和细胞死亡。电压门控钙（Cav）通道打开时， 膜去极化，将膜电信号转化为细胞内Ca 2+介导的事件。 Cav通道的功能障碍或失调与广泛的神经系统疾病有关， 心血管和肌肉疾病。尽管Cav在生理和病理生理上具有重要意义， 由于缺乏结构性信息，进一步的进展受到严重限制。的确， 任何真核Cav通道的可用结构是Cav 1.1通道复合物，我的小组 使用单粒子电子冷冻显微镜（cryo-EM）测定。Cav频道被多个 FDA批准的用于治疗神经和心血管疾病的药物，其活性是 由各种肽毒素调节。这些配体可用于稳定各种细胞中的Cav通道。 功能状态，便于门控机制的解剖。反过来，Cav的结构解析 与药物和毒素复合的通道将阐明其作用模式的分子基础。 这些结构将指导诱变的功能和机制表征，作为一种新的生物学方法。 同源建模、配体对接和分子动力学模拟分析的重要框架，以及 最终促进潜在的药物发现。该提案的总体目标是实现更好的 通过高分辨率的Cav1.1结构测定， 使用单颗粒cryo-EM与各种调节配体复合。在目标1中，我们将进一步改善 通过优化冷冻样品制备和硬件，Cav1.1通道的分辨率超过3 μ m 配置.分辨率的提高将为分子动力学提供更精确的结构模板 仿真分析在目标2中，我们将生物化学地概括纯化的Cav1.1之间的相互作用。 通道和各种药物和毒素，并阐明Cav1.1的结构与明确定义的 配体。这些结构将指导突变的设计，用于功能表征和机制分析。 基于细胞的电生理学测定的研究。在目标3中，我们将研究 通过衔接蛋白Stac 3调节Cav1.1。这项研究将包括交联，质谱 分析，和新的算法cryo-EM解开Stac 3和Cav1.1之间的识别。完成 这项研究将进一步加深我们对Cav的功能和致病机制的认识， 渠道，以及促进未来的药物发现。

Calcium ions (Ca2+) play a critical role in diverse physiological processes such as contraction, secretion, neurotransmission, gene transcription, and cell death. The voltage-gated calcium (Cav) channels open upon membrane depolarization, converting the membrane electrical signals to intracellular Ca2+-mediated events. Malfunction or dysregulation of Cav channels is associated with a broad spectrum of neurological, cardiovascular, and muscular disorders. Despite the physiological and pathophysiological significance of Cav channels, further progress has been acutely limited by the dearth of structural information. Indeed, the only available structure of any eukaryotic Cav channel is that of the Cav1.1 channel complex, which my group determined using single-particle electron cryo-microscopy (cryo-EM). Cav channels are targeted by multiple FDA-approved drugs for the treatment of neurological and cardiovascular disorders, and their activity is modulated by various peptide toxins. These ligands could be used to stabilize the Cav channels in various functional states, facilitating the dissection of the gating mechanism. In turn, structural elucidation of Cav channels in complex with the drugs and toxins will elucidate the molecular basis for their modes of action. These structures will guide mutagenesis for functional and mechanistic characterizations, serve as an important framework for homology modeling, ligand docking, and molecular dynamics simulation analyses, and eventually facilitate potential drug discovery. The overarching goal of this proposal is to achieve an improved mechanistic understanding of Cav channels through high-resolution structural determination of Cav1.1 in complex with various modulatory ligands using single-particle cryo-EM. In Aim 1, we will further improve the resolution of the Cav1.1 channel to beyond 3 Å by optimizing cryo-sample preparation and hardware configuration. Improved resolution will afford a more accurate structural template for molecular dynamics simulation analysis. In Aim 2, we will biochemically recapitulate the interactions between the purified Cav1.1 channel and various drugs and toxins, and elucidate the structures of Cav1.1 in complex with well-defined ligands. These structures will guide the design of mutations for functional characterizations and mechanistic investigations in cell-based electrophysiological assays. In Aim 3, we will investigate the structural basis for the modulation of Cav1.1 by the adaptor protein Stac3. This study will encompass crosslinking, mass spectrometric analysis, and new algorithms for cryo-EM to unravel the recognition between Stac3 and Cav1.1. Completion of the proposed research will advance our understanding of the function and disease-causing mechanisms of Cav channels as well as facilitate future drug discovery.