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

基本信息

批准号：
10413162
负责人：
JOHN LEE SPUDICH
金额：
$ 116.72万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
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/10413162
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 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 dysfunction brain tissue chromophore experimental study flexibility high throughput screening improved inhibitor 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

项目摘要

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。光遗传学蛋白，例如光激活离子通道或泵，可以对神经元活动进行光学控制因此，它们提供了询问神经活动如何的强大手段。有助于大脑功能并改变病理活动以治疗各种神经系统疾病。兴奋性光遗传学工具已被开发以满足激活范式的不同需求。最完善的光驱动离子泵仍然不够发达。由于其本质上较低的光效率和泵浦活性，可以有效地抑制神经元。发达的光门控钾通道还存在光电流小和电流动力学慢的问题。我们发现天然光门控氯离子通道，Guillardia theta 阴离子通道视紫红质 1 和 2 （GtACR1 和 GtACR2），导致了一类对光高度敏感的新型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 倍大的阴离子哺乳动物细胞中的光电流比以前的工具更好。然而，我们和其他人发现光激活。小鼠神经元中的光门控氯离子通道使轴突和突触前末梢去极化以触发神经递质释放，即使它抑制体细胞的动作电位，这种兴奋作用是由于。轴突和突触前末梢内源性高浓度氯化物，产生通道打开时使氯离子流出去极化因此，轴突兴奋阻碍了神经元的目标。使用光门控氯离子通道使实验沉默并使解释变得复杂。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，限制了它们在深层脑组织中的有效性和多重光遗传学的灵活性因此，该项目的目标是克服光门控的这两个主要限制。我们将利用蛋白质运输机制、基于结构的分子工程、高科技。通量筛选，以及自然界中的蛋白质进化，以消除兴奋效应并扩大作用我们建议利用内源性蛋白质运输机制来限制天然 ACR 的光谱范围。神经元体树突域内的 ACR（目标 1）执行结构引导的高通量诱变屏幕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转移的 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、神经生理学和系统神经科学。该项目的成功完成将为神经科学界呈现一套大大改进的抑制性光遗传学工具具有强大的功效、最小的副作用和多样化的光谱敏感性。