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

钙离子(Ca~(2+))在多种生理过程中起着关键作用，如收缩、分泌、 神经传递、基因转录和细胞死亡。电压门控钙(Cav)通道在 膜去极化，将膜电信号转换为细胞内钙介导的事件。 CAV通道的功能障碍或失调与广泛的神经系统疾病有关， 心血管疾病和肌肉疾病。尽管CAV具有生理和病理生理学意义 由于缺乏结构性信息，进一步的进展受到了严重限制。事实上，唯一的 任何真核细胞Cav通道的可用结构都是Cav1.1通道复合体的结构，我的团队 用单粒子电子冷冻显微镜(Cryo-EM)测定。CAV渠道被多个 FDA批准的治疗神经和心血管疾病的药物，它们的活性是 受各种多肽毒素的调节。这些配体可以用来稳定不同类型的Cav通道。 功能状态，便于门控机构的解剖。反过来，Cav的结构阐明 与药物和毒素形成复合体的通道将阐明它们作用模式的分子基础。 这些结构将指导功能和机械特性的突变，作为一种 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。这项建议的首要目标是实现一个更好的 通过Cav1.1的高分辨率结构测定对CAV通道的机制认识 利用单粒子冷冻-EM技术合成了具有多种调节配体的复合体。在目标1中，我们将进一步完善 通过优化低温样品制备和硬件，将Cav1.1通道的分辨率提高到Beyond 3？ 配置。提高分辨率将为分子动力学提供更准确的结构模板 仿真分析。在目标2中，我们将以生物化学的方式概括纯化的Cav1.1之间的相互作用 通道和各种药物和毒素，并阐明了Cav1.1的结构与明确 配基。这些结构将指导功能表征和机械学突变的设计 基于细胞的电生理检测的研究。在目标3中，我们将调查 适配蛋白Stac3对Cav1.1的调控。这项研究将包括交联剂、质谱学 分析，以及低温EM解开Stac3和Cav1.1之间识别的新算法。完成 这项研究将促进我们对CAV的功能和致病机制的理解 并为未来的药物发现提供便利。

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.