Developing an Optogenetics Technology Based on Natural Potassium-selective Channelrhodopsins

摘要 视紫红质抑制转运蛋白作为突触前的突触前目标暂时提出了GATED，它的神经回路的功能关键依赖于激活和抑制回路成分的能力，以随后评估它们对回路其他部分的影响及其影响行为。在过去的20年里，光门控钠传导的自然变体和突变体已经被优化，成为有效的神经元光激活剂，针对特定的细胞或局部，定义了被称为光遗传学的技术。然而，与激励性工具相比，工具仍然不发达。光驱动离子泵具有低电导，因为它们的限制是每个吸收的光子只能有一个离子。阴离子传导通道视紫红质(ACRs)作为一种有效的神经元抑制因子在许多领域有着广泛的应用。然而，在突触前轴突投射中，轴突和终末的氯离子浓度升高使ACRS成为激活剂而不是抑制物。该项目的目标是通过开发高导电性、精确的光门通道来解决当前抑制工具的局限性，这些通道可以作为SoMAS和轴突的光遗传消音器。我们确实通过一类新的光遗传抑制工具来实现这一目标，该工具基于我们团队最近发现并表征的天然K-选择性光通道(“钾通道视紫红质”，或KCRs)，其机制模仿神经元的内源性复极。我们的目标是：(1)通过高通量自然变异体的元基因组筛选来鉴定和鉴定具有改进特性的新的KCR同源物；(2)通过四种互补的方法对KCR进行蛋白质工程，以增强其作为光遗传工具的实用性：(I)结构/功能导向的突变，(Ii)自动膜片钳电生理学，(Iii)基于高通量荧光的筛选，以及(Iv)基于机器学习的方法；以及(目的3)小鼠初级视皮层和丘脑皮质投射中基于KCR的光遗传抑制的特征和优化，以表征和优化活体动物中基于KCR的光遗传抑制。由蜂窝圣皮埃尔。为了实现我们的目标，我们组建了一个专家团队，由三名具有互补专业知识的首席研究人员领导：光生物学家和生物化学家John Spudich，系统神经学家薛明山，以及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消音器，通过解决当前的工具，将像ChR2等神经元光激活器一样广泛地部署。除了在健康和疾病状态下了解大脑的益处外，KCR还可能导致开发光遗传疗法来治疗神经元过度兴奋障碍，如癫痫和导致神经元过度兴奋的神经退行性疾病，如帕金森氏症和阿尔茨海默病。

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.