Developing an Optogenetics Technology Based on Natural Potassium-selective Channelrhodopsins

摘要 将个体映射到通道视紫红质类型抑制性运输作为突触前目标，暂时提出门控，其大脑中神经回路的功能关键依赖于激活和沉默回路组件的能力，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 Na 传导的自然变体和突变体已经过优化，可作为针对特定细胞或定位的高效神经元光激活剂，从而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。光驱动离子泵的电导较低，因为其限制为每个吸收的光子只能有一个离子。阴离子传导视紫红质通道 (ACR) 已在许多应用中用作有效的神经元抑制剂。然而，轴突和末端的 Cl - 浓度升高使得 ACR 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导电性、精确的光门控通道来解决当前抑制工具的局限性，这些通道可作为体细胞和轴突的光遗传学消音器。我们确实通过我们团队最近发现和表征的基于天然 K 选择性光通道（“钾通道视紫红质”或 KCR）的新型光遗传学抑制工具实现了这一目标，其机制模拟了神经元的内源性复极化。 c c 我们的目标是：（目标 1）通过高通量宏基因组筛选天然变异体来鉴定和电生理学表征具有改进特性的新型 KCR 同系物； （目标 2）对最好的 KCR 进行蛋白质工程，通过四种互补方法增强其作为光遗传学工具的实用性：（i）结构/功能引导诱变，（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； （目标 3）表征和优化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，以表征和优化活体动物中基于 KCR 的光遗传学抑制。由蜂窝圣皮埃尔。为了实现我们的目标，我们组建了一个由三位首席研究员领导的专家团队，他们具有互补的专业知识：光生物学家和生物化学家 John Spudich、系统神经科学家薛明山以及蛋白质工程师和神经影像专家 François St-Pierre。 我们期望为神经科学界提供光遗传学消音器，通过解决当前的工具，这些消音器将像 ChR2 等神经元光激活器一样广泛部署。除了有助于了解健康和患病状态下的大脑之外，KCR 还可能促进光遗传学疗法的发展，用于治疗神经元过度兴奋性疾病，例如癫痫和神经退行性疾病，这些疾病会导致帕金森氏症和阿尔茨海默氏病等神经元过度兴奋。

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.