Structural and mechanistic basis of channelrhodopsin function

项目摘要 本项目的目标是了解通道视紫红质的门控和功能的机制基础， 类似于脊椎动物视觉蛋白的视网膜结合蛋白，形成光门控离子通道， 游动藻类的趋光性。自首次克隆以来的近20年里，通道视紫红质 成为理解膜蛋白结构、功能和生物物理学的重要模型， 利用光遗传学中的分子工具，在遗传靶向细胞中异源表达 能够对光的膜电位和电兴奋性进行控制。在这里，我们将应用低温电子 显微镜以确定不同功能状态和电生理状态下的通道视紫红质的结构 记录基于结构的变体，以了解通道门控的基础和关键的决定因素 频道属性我们的目标是捕捉不同的开放和封闭构象的结构快照， 识别促进不同状态的刺激条件和通道变体的组合。我们将 利用这些结构见解来探究不同动力学、电导和电导的分子基础 通道视紫红质之间光谱敏感性，并导出门控和功能特性的物理模型。 我们将集中我们的努力，两个通道视紫红质是最有效的成员，两个去极化 广泛用于光遗传学的阳离子通道视紫红质（CCR）和细菌视紫红质样通道家族 阳离子通道视紫红质（BCCRs）。CCR和BCCR共享共同的架构，但是在结构上， 在进化上和机械上都是不同的。这两个通道视紫红质家族的比较分析将 因此，提供了额外的洞察光能是如何转换成门控构象变化， 通道活动的分子基础。由于最初的表征和克隆的通道视紫红质， 光遗传学工具箱已经通过工程化具有多种和 改进的性能。然而，这些努力迄今为止仍然受到对环境的不完全理解的限制。 渠道功能结构和机制基础。除了提供基本的 机械洞察通道视紫红质门控和活动，这项工作将作为一个合理的基础， 设计新的具有改性性质的通道视紫红质变体， 光遗传学操作。这样的工具可以在更大的规模，更深的组织，更大的范围内进行新的实验。 生物，精度更高。它们还可能导致治疗疾病的新临床方法 包括神经系统和心血管系统。

PROJECT SUMMARY The goal of this project is to understand the mechanistic basis for gating and function in channelrhodopsins, retinal-binding proteins that are similar to vertebrate visual proteins and form light-gated ion channels to control phototaxis in motile algae. In the nearly two decades since they were first cloned, channelrhodopsins have become important models for understanding membrane protein structure, function, and biophysics and widely utilized molecular tools in optogenetics, in which their heterologous expression in genetically targeted cells enables control of membrane potential and electrical excitability with light. Here, we will apply cryo-electron microscopy to determine structures of channelrhodopsins in different functional states and electrophysiological recordings of structure-based variants to understand the basis for channel gating and determinants for key channel properties. We aim to capture structural snapshots of different open and closed conformations by identifying combinations of stimulation conditions and channel variants that promote different states. We will leverage these structural insights to interrogate the molecular basis for diverse kinetics, conductance, and spectral sensitivity among channelrhodopsins and derive physical models for gating and functional properties. We will focus our efforts on two channelrhodopsins that are the most potent members of the two depolarizing channel families widely used in optogenetics, the cation channelrhodopsins (CCRs) and bacteriorhodopsin-like cation channelrhodopsins (BCCRs). CCRs and BCCRs share a common architecture, but are structurally, evolutionarily, and mechanistically distinct. Comparative analyses of these two channelrhodopsin families will therefore provide additional insight into how light energy is converted into gating conformational changes and the molecular basis for channel activity. Since the initial characterization and cloning of channelrhodopsins, the optogenetic toolbox has been greatly expanded by the engineering of novel channelrhodopsins with varied and improved properties. Still, these efforts have been limited to date by an incomplete understanding of the structural and mechanistic basis for channel function. Therefore, in addition to providing fundamental mechanistic insight into channelrhodopsin gating and activity, this work will serve as a basis for the rational design of new channelrhodopsin variants with modified properties that further expand the potential of optogenetic manipulations. Such tools could enable new experiments at larger scale, in deeper tissue, in larger organisms, and with higher precision. They could also lead to new clinical approaches for treating disease including those of the nervous and cardiovascular systems.