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

用于光遗传学抑制的天然光门控氯离子通道的分子工程

• 批准号: 10237959
• 负责人: JOHN LEE SPUDICH
• 金额: $ 122.86万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-15 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10237959
• 关键词: Action Potentials; Anions; Axon; Behavior; Biophysics; Brain; Chloride Channels; Chlorides; Clinical; Collaborations; Communities; Crystallization; Detection; Effectiveness; Engineering; Epilepsy; Evolution; Exhibits; Goals; Hand; Homologous Gene; Hybrids; Impairment; Investigation; Ion Channel; Ion Pumps; Kinetics; Lead; Light; Mammalian Cell; Membrane; Molecular; Mus; Mutagenesis; Mutation; Nature; Neurons; Neurosciences; Neurosciences Research; Neurotransmitters; Optics; Outcome; Parkinson Disease; Pathologic; Physiology; Potassium Channel; Preparation; Presynaptic Terminals; Protein Engineering; Proteins; Pump; Research; Role; Side; Structure; System; Time; Variant; absorption; attenuation; autism spectrum disorder; base; biophysical properties; brain tissue; chromophore; experimental study; flexibility; high throughput screening; improved; inhibitor/antagonist; insight; light gated; millisecond; nervous system disorder; neuronal cell body; neuroregulation; neurotransmitter release; novel; optogenetics; protein transport; redshift; relating to nervous system; sensor; side effect; spatiotemporal; tool; trafficking; transcriptome; voltage

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.

项目摘要/摘要 神经元活性的靶向调节是基础和临床神经科学研究中的必要方法。 光遗传蛋白，例如光激活的离子通道或泵，可以光控制神经元活性 具有独家时空精度。那就是他们提供了强大的手段来询问神经活动 有助于大脑功能并改变病理活性以治疗神经系统疾病。各种各样 已经开发出兴奋性的光遗传学工具来满足激活范例的不同需求。相比之下， 抑制工具仍然不发达。发达的轻驱动离子泵仍然不够 由于其本质上较低的光效和泵送活性，可有效地沉默神经元。新 发达的轻轨钾通道也遭受了小型照片和慢速电流动力学的影响。 我们发现自然光门控通道，theta theta阴离子渠道1和2 （GTACR1和GTACR2），导致一类新的抑制性光遗传学工具对光高度敏感，具有 杰出的阴离子选择性，暴露的时间常数，毫秒的时间常数，可以产生10-100倍 哺乳动物细胞中的光电流比以前的工具。但是，我们和其他人发现光激活 小鼠神经元中轻门控氯化物通道的去极化，使轴突和突触前末端触发 神经递质释放，即使它抑制了SOMA的动作电位。这种兴奋的动作是由于 轴突和突触前末端中的内源性高浓度氯化物，这会产生一个 通道打开时去极化的氯化物外排。那，轴突兴奋阻碍了神经元的目标 沉默和复杂化使用轻门氯化物通道对实验的解释。其他 重要的限制是，光门口氯化物通道的作用光谱均在蓝色至绿色的内部 光范围限制了它们在深脑组织中的有效性和多重光遗传学的灵活性 申请。因此，该项目的目的是克服这两个主要门控的主要局限性 氯化物通道。我们将利用蛋白质运输机制，基于结构的分子工程，高级 吞吐量筛查和自然界的蛋白质进化，以消除兴奋效果并扩大动作 天然ACR的光谱范围。我们建议利用内源性蛋白质运输机制限制 神经元体体内的ACR（AIM 1），进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Photoscurrents（AIM 2）的ACR变体，并确定 频谱通过天然ACR同源屏幕和高通量诱变转移了ACR变体 屏幕（AIM 3）。拟议的研究资源是建立强大的生物物理学协同合作， 蛋白质工程，高通量筛查，神经元生理和系统神经科学。 该项目的成功完成将呈现给神经科学社区 具有有效效果，最小副作用和潜水光谱灵敏度的抑制光遗传学工具。