Molecular Engineering of Natural Light-Gated Chloride Channels for Optogenetic Inhibition

项目概要/摘要 神经活动的靶向调节是基础和临床神经科学研究的重要方法。 光遗传蛋白，如光激活离子通道或泵，使神经元活动的光学控制 精确的时空信息因此，它们提供了强有力的手段来询问神经活动如何 有助于大脑功能和改变病理活动，以治疗神经系统疾病。各种 已经开发了兴奋性光遗传学工具以满足激活范例的不同需要。与此相反， 抑制性工具仍然不发达。最发达的光驱动离子泵仍然不足以 由于其固有的低光效率和泵送活性，有效地沉默神经元。新 已开发的光门控钾通道也受到其小光电流和慢电流动力学的影响。 我们发现了天然光门控氯离子通道，Guillardia theta阴离子通道视紫红质1和2 （GtACR 1和GtACR 2），导致一类新的对光高度敏感的抑制性光遗传学工具， 出色的阴离子选择性，表现出毫秒级的时间常数，并且可以产生10-100倍大的 哺乳动物细胞中的光电流比以前的工具。然而，我们和其他人发现， 光门控氯离子通道在小鼠神经元的去极化轴突和突触前末梢触发 尽管它抑制了索马的动作电位，但它仍然抑制了神经递质的释放。这种兴奋作用是由于 轴突和突触前末梢中的内源性高浓度氯化物， 通道开放时去极化氯离子流出。因此，轴突兴奋阻碍了神经元的目标， 沉默，并使使用光门控氯离子通道的实验的解释复杂化。另一 重要的限制是光门控氯离子通道的作用光谱都在蓝色到绿色范围内。 光范围，限制了它们在深部脑组织中的有效性和多重光遗传学中的灵活性。 应用.因此，本项目的目标是克服光选通的这两大局限性 氯离子通道我们将利用蛋白质运输机制，基于结构的分子工程，高- 通过筛选和蛋白质进化消除自然界中的兴奋效应并扩大作用 天然ACR的光谱范围。我们建议利用内源性蛋白质运输机制来限制 神经元体树突结构域内的ACR（Aim 1），执行结构引导的高通量诱变 屏幕，以创建具有强大的外向整流和光电流的ACR变体（目标2），并识别 通过天然ACR同源物筛选和高通量诱变实现光谱移位的ACR变体 屏幕（目标3）。拟议的研究利用了生物物理学的强大协同合作， 蛋白质工程、高通量筛选、神经元生理学和系统神经科学。的 这个项目的成功完成将为神经科学界提供一套改进的 抑制性光遗传学工具具有有效的功效、最小的副作用和多样的光谱灵敏度。

PROJECT SUMMARY/ABSTRACT Targeted modulation of neural activity is an essential approach in basic and clinical neuroscience research. Optogenetic proteins, such as light-activated ion channels or pumps, enable optical control of neuronal activity with exquisite spatiotemporal precision. Thus, they provide powerful means to interrogate how neural activity contributes to brain functions and alter pathological activity to treat neurological disorders. A variety of excitatory optogenetic tools have been developed to meet different needs of activation paradigms. In contrast, inhibitory tools remain underdeveloped. The most well-developed light-driven ion pumps are still not sufficiently effective in silencing neurons due to their intrinsically low photoefficiency and pumping activity. Newly developed light-gated potassium channels also suffer from their small photocurrents and slow current kinetics. Our discovery of natural light-gated chloride channels, Guillardia theta anion channelrhodopsins 1 and 2 (GtACR1 and GtACR2), led to a new class of inhibitory optogenetic tools that are highly sensitive to light, have outstanding anion selectivity, exhibit time constants of milliseconds, and can generate 10–100-fold larger photocurrents in mammalian cells than previous tools. However, we and others discovered that light activation of light-gated chloride channels in mouse neurons depolarizes the axon and presynaptic terminals to trigger neurotransmitter release even though it inhibits action potentials at the soma. This excitatory action is due to the endogenous high concentrations of chloride in the axon and presynaptic terminals, which create a depolarizing chloride efflux upon channel opening. Thus, axonal excitation impedes the goal of neuronal silencing and complicates the interpretation of experiments using light-gated chloride channels. Another important limitation is that the action spectra of light-gated chloride channels are all within the blue to green- light ranges, limiting their effectiveness in deep brain tissues and flexibility in multiplex optogenetic applications. Therefore, the objective of this project is to overcome these two major limitations of light-gated chloride channels. We will harness protein trafficking machinery, structure-based molecular engineering, high- throughput screening, and protein evolution in nature to eliminate the excitatory effect and expand the action spectra range of natural ACRs. We propose to exploit endogenous protein trafficking mechanisms to restrict ACRs within neuronal somatodendritic domain (Aim 1), perform structure-guided high-throughput mutagenesis screens to create ACR variants with robust outward rectification and photocurrents (Aim 2), and identify spectrally shifted ACR variants through natural ACR homolog screens and high-throughput mutagenesis screens (Aim 3). The proposed research capitalizes on a powerful synergistic collaboration of biophysics, protein engineering, high-throughput screening, neuronal physiology, and system neuroscience. The successful completion of this project will present to the neuroscience community a set of much improved inhibitory optogenetic tools with potent efficacy, minimal side effects, and diverse spectral sensitivities.