Developing an Optogenetics Technology Based on Natural Potassium-selective Channelrhodopsins

总结 映射个人对channelrhodopsins类型的抑制性运输作为突触前的目标暂时提出门控的神经回路在大脑中的功能至关重要地依赖于激活和沉默电路组件的能力，以随后评估其对电路的其他部分的影响及其影响行为。在过去的20年里，光门控Na +传导的天然变体和突变体已经被优化，以作为可靶向特定细胞或定位的有效神经元光激活剂，定义了称为光遗传学的技术。然而，与兴奋性工具相比，工具仍然不发达。光驱动的离子泵具有低电导，因为它们的限制是每个光子仅吸收一个离子。阴离子传导通道视紫红质（ACRs）在许多应用中被用作有效的神经元抑制剂。然而，轴突和终末中Cl -浓度的升高使ACRs在突触前轴突投射中成为激活剂而不是抑制剂。该项目的目标是通过开发高传导性、精确的光门控通道来解决当前抑制工具的局限性，这些通道可作为胞体和轴突的光遗传沉默器。我们确实通过一类新的光遗传学抑制工具实现了这一目标，该工具基于我们的团队最近发现和表征的天然K +选择性光通道（“钾通道视紫红质”或KCR），并且机制模拟了神经元中的内源性复极化。c c我们的目标是：（目的1）通过高通量宏基因组筛选天然变体来鉴定和电生理学表征具有改进特征的新型KCR同源物;（目的2）通过四种互补方法对最好的KCR进行蛋白质工程改造以增强其作为光遗传学工具的效用：（i）结构/功能引导的诱变，（ii）自动膜片钳电生理学，（iii）基于荧光的高通量筛选，和（iv）基于机器学习的方法;和（目的3）小鼠初级视觉皮层和丘脑皮层投射中基于KCR的光遗传学抑制的表征和优化，以表征和优化活动物中基于KCR的光遗传学抑制。圣皮埃尔的手机为了实现我们的目标，我们组建了一个由三位主要研究人员领导的专家团队，他们具有互补的专业知识：光生物学家和生物化学家John Spudich，系统神经科学家Mingshan Xue和蛋白质工程师和神经成像专家François St-Pierre。 我们希望为神经科学界提供光遗传学沉默器，通过解决当前的工具，将像ChR 2等神经元光激活剂一样广泛部署。除了它们对于理解健康和患病状态下的大脑的益处之外，KCR还可以导致用于神经元过度兴奋性疾病（例如癫痫和导致神经元过度兴奋性的神经变性疾病，例如帕金森病和阿尔茨海默病）的光遗传学治疗的发展。

SUMMARY Mapping individual on channelrhodopsins types inhibitory transporting as presynaptic objective temporally propose gated whose the function of neural circuits in the brain crucially relies on the ability to both activate and silence circuit components to subsequently assess their impact on other parts of the circuit and their influence behavior. Over the past 20 years, natural variants, and mutants of light-gated Na + -conducting have been optimized to serve as efficient neuron photo-activators targetable to specific cells or localizations, defining the technology called optogenetics. However, compared to excitatory tools, tools remain underdeveloped. Light-driven ion pumps have low conductance given their limitation of only one ion per photon absorbed. Anion-conducting channelrhodopsins (ACRs) have been used as effective neuron suppressors in many applications. However, elevated Cl - concentrations in axons and terminals make ACRs activators rather than inhibitors in presynaptic axonal projections. The goal of this project is to address the limitations of current inhibitory tools by developing highly conductive, precise light-gated channels that function as optogenetic silencers for both somas and axons. We do achieve this aim via a new class of optogenetic inhibitory tools based on natural K + -selective light- channels (“kalium channelrhodopsins”, or KCRs), recently discovered and characterized by our team, and mechanism mimics endogenous repolarization in neurons. c c Our aims are: (Aim 1) the identification and electrophysiological characterization of novel KCR homologs with improved characteristics by high-throughput metagenomic screening for natural variants; (Aim 2) Protein engineering of the best of the KCRs to enhance their utility as optogenetic tools via four complementary approaches: (i) structure/function-guided mutagenesis, (ii) automated patch-clamp electrophysiology, (iii) high-throughput fluorescence-based screening, and (iv) machine-learning-based approaches; and (Aim 3) Characterization and optimization of KCR-based optogenetic inhibition in the mouse primary visual cortex and thalamocortical projection to characterize and optimize KCR- based optogenetic inhibition in living animals. by cellular St-Pierre. limitations to of diseases to accomplish our aims, we have assembled an expert team led by three Principal Investigators with complementary expertise: photobiologist and biochemist John Spudich, system neuroscientist Mingshan Xue, and protein engineer and neuroimaging specialist François St-Pierre. We expect to provide the neuroscience community with optogenetic silencers that, by addressing the current tools, would be deployed as broadly as neuron photo-activators such as ChR2. In addition to their benefits for understanding the brain in healthy and diseased states, KCRs may lead to the development of optogenetic treatments for neuronal hyperexcitability disorders such as epilepsy and neurodegenerative that result in neuronal hyperexcitability such as Parkinson's and Alzheimer's disease.