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

项目摘要/摘要 靶向调节神经活动是基础和临床神经科学研究的基本方法。 光遗传蛋白，如光激活离子通道或泵，能够实现对神经元活动的光学控制 有着精致的时空精确度。因此，它们提供了强有力的手段来询问神经活动是如何 有助于大脑功能和改变病理活动，以治疗神经疾病。各种各样的 兴奋性光遗传工具已经被开发出来，以满足激活范例的不同需求。相比之下， 抑制工具仍然不发达。最发达的光驱动离子泵仍然不够 由于其内在的低光效和泵送活性，有效地使神经元沉默。新开 发达的光门控钾通道也存在光电流小和电流动力学慢的问题。 我们发现的天然光门氯离子通道，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工具，具有 出色的阴离子选择性，表现出毫秒的时间常数，并可产生10-100倍的 在哺乳动物细胞中的光电流比以前的工具要好。然而，我们和其他人发现光激活 小鼠神经元上的光门氯离子通道使轴突和突触前终末去极化以触发 神经递质的释放，即使它抑制了胞体的动作电位。这种兴奋行为是由于 轴突和突触前终末的内源性高浓度氯，产生了 通道打开时，去极化氯化物外流。因此，轴突兴奋阻碍了神经元的目标。 沉默并使使用光门氯离子通道的实验的解释复杂化。另一个 重要的限制是光门氯离子通道的作用光谱都在蓝到绿的范围内。 光线范围，限制了其在深部脑组织中的有效性和多重光遗传的灵活性 申请。因此，本项目的目标就是克服光门的这两大局限性 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，高度... 通过筛选，并在自然界中对蛋白质的进化消除兴奋作用而扩大作用 天然ACR的光谱范围。我们建议利用内源性蛋白运输机制来限制 神经元躯体树突域内的ACRs(Aim 1)，进行结构导向的高通量突变 筛选产生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同源筛选和高通量突变获得光谱移位的ACR变体 屏幕(目标3)。这项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。这个 该项目的成功完成将向神经科学界展示一套大大改进的 具有强大功效、最小副作用和多种光谱敏感度的抑制性光遗传工具。

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.