Developing an Optogenetics Technology Based on Natural Potassium-selective Channelrhodopsins

开发基于天然钾选择性通道视紫红质的光遗传学技术

• 批准号: 10731153
• 负责人: JOHN LEE SPUDICH
• 金额: $ 314.51万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10731153
• 关键词: Address; Alzheimer' s Disease; Animals; Anions; Axon; Behavior; Biological Assay; Biomedical Research; Brain; Cardiac; Cells; Characteristics; Chimera organism; Communities; Cryoelectron Microscopy; Development; Disease; Dorsal; Electrophysiology (science); Embryo; Engineering; Epilepsy; Family; Family member; Fluorescence; Goals; Homologous Gene; Homology Modeling; Human; Individual; Ion Channel; Ion Pumps; Ions; Kidney; Kinetics; Knowledge; Lateral Geniculate Body; Light; Lighting; Machine Learning; Maps; Metagenomics; Molecular; Mus; Mutagenesis; Nerve Degeneration; Nervous System; Neurons; Neurophysiology - biologic function; Neurosciences; Neurosciences Research; Parkinson Disease; Performance; Permeability; Photons; Potassium; Presynaptic Terminals; Principal Investigator; Property; Protein Engineering; Proteins; Protocols documentation; Pump; Research Project Grants; Retinal Pigments; Rhinosporidium; Rhodopsin; Roentgen Rays; Role; Scientist; Slice; Specialist; Specificity; Structural Models; Structure; Structure-Activity Relationship; System; Technology; Testing; Thalamic structure; Tinnitus; Variant; Visual Cortex; absorption; area striata; behavior influence; desensitization; experimental study; extracellular; improved; in vivo; inhibitor; light gated; machine learning algorithm; microbial; mutant; nervous system disorder; neural; neural circuit; neuroimaging; neuronal cell body; neuroregulation; novel; optogenetics; painful neuropathy; patch clamp; photoactivation; presynaptic; protein transport; redshift; screening; spatiotemporal; structural determinants; tool; Address; Alzheimer' s Disease; Animals; Anions; Axon; Behavior; Biological Assay; Biomedical Research; Brain; Cardiac; Cells; Characteristics; Chimera organism; Communities; Cryoelectron Microscopy; Development; Disease; Dorsal; Electrophysiology (science); Embryo; Engineering; Epilepsy; Family; Family member; Fluorescence; Goals; Homologous Gene; Homology Modeling; Human; Individual; Ion Channel; Ion Pumps; Ions; Kidney; Kinetics; Knowledge; Lateral Geniculate Body; Light; Lighting; Machine Learning; Maps; Metagenomics; Molecular; Mus; Mutagenesis; Nerve Degeneration; Nervous System; Neurons; Neurophysiology - biologic function; Neurosciences; Neurosciences Research; Parkinson Disease; Performance; Permeability; Photons; Potassium; Presynaptic Terminals; Principal Investigator; Property; Protein Engineering; Proteins; Protocols documentation; Pump; Research Project Grants; Retinal Pigments; Rhinosporidium; Rhodopsin; Roentgen Rays; Role; Scientist; Slice; Specialist; Specificity; Structural Models; Structure; Structure-Activity Relationship; System; Technology; Testing; Thalamic structure; Tinnitus; Variant; Visual Cortex; absorption; area striata; behavior influence; desensitization; experimental study; extracellular; improved; in vivo; inhibitor; light gated; machine learning algorithm; microbial; mutant; nervous system disorder; neural; neural circuit; neuroimaging; neuronal cell body; neuroregulation; novel; optogenetics; painful neuropathy; patch clamp; photoactivation; presynaptic; protein transport; redshift; screening; spatiotemporal; structural determinants; tool

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.

概括 将个体映射在通道旋转蛋白上类型的抑制性转运作为突触前物镜暂时提出的门控，其神经元在大脑中的功能完全取决于激活和沉默的电路成分的能力，以评估其对电路其他部位的影响及其影响行为。在过去的20年中，已对轻轨Na +传导的自然变体和突变体进行了优化，可作为可靶向特定细胞或局部化的有效神经元光激活剂，定义了称为光遗传学的技术。但是，与兴奋性工具相比，工具仍然不发达。鉴于每个光子吸收的一个离子仅限制，光驱动的离子泵的电导率较低。在许多应用中，阴离子传导通道旋转（ACR）已被用作有效的神经元补充剂。然而，轴突和终端中的Cl浓度升高会使突触前轴突项目中的ACRS激活剂而不是抑制剂。该项目的目的是通过开发高导电性，精确的轻门通道来解决当前抑制工具的局限性，这些通道可作为SOMAS和SOMAS和轴突的光遗传消音器。我们确实通过基于天然K +选择光通道（“ Kalium ChannelRhopopsins”或KCRS）的新的光遗传学抑制工具实现了这一目标，该工具最近被我们的团队发现和表征，并模仿了神经元中的内源性重塑。 c我们的目标是：（目标1）通过对自然变异的高通量元基因组筛选，具有改善特征的新型KCR同源物的鉴定和电生理表征； （AIM 2）通过四种完整的方法，最佳KCR的蛋白质工程以增强其作为光遗传学工具的实用性：（i）结构/功能引导的诱变，（ii）自动化斑块钳电生理学，（iii）高电流荧光筛选和基于机器学习方法的高直发荧光筛选； （AIM 3）在小鼠初级视觉皮层和丘脑皮层投影中表征和优化基于KCR的光遗传学抑制，以表征和优化活动物中基于KCR的光遗传学抑制。通过细胞st-pierre。疾病的局限性以实现我们的目标，我们组建了一个由三名具有完整专业知识的主要研究人员领导的专家团队：光生物学家和生物化学家John Spudich，System Neuroscientist Mingshan Xue以及Protein Engineer和Protein Engineer和Neurotimaging专家FrançoisFrançoisFrançoisFrançoisStpierre。我们希望为神经科学界提供光遗传的消音器，通过解决当前工具，将像CHR2这样的神经元光激活剂一样广泛地部署。除了了解健康和厌恶状态下的大脑的好处外，KCR还可能导致神经元过度兴奋性疾病的光遗传学治疗，例如癫痫和神经退行性疾病，从而导致神经元过度刺激性，例如帕金森氏症和阿尔茨海默氏病。