Structure/Function of Channelrhodopsins and Related Retinylidene Proteins

视紫红质通道蛋白和相关视黄基蛋白的结构/功能

• 批准号: 10166003
• 负责人: JOHN LEE SPUDICH
• 金额: $ 62.74万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2026-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10166003
• 关键词: Action Potentials; Algae; Anions; Basic Science; Biomedical Research; Cardiac Myocytes; Cations; Cell membrane; Cells; Chlamydomonas reinhardtii; Color; Coupled; Cryoelectron Microscopy; Crystallization; Disease; Distant; Electrophysiology (science); Engineering; Epilepsy; Family; Genetic Techniques; Genome; Heart Diseases; Image; In Vitro; Investigational Therapies; Ion Channel Gating; Ions; Kinetics; Knowledge; Laboratories; Light; Maps; Microbial Rhodopsins; Mining; Molecular; Molecular Conformation; Mutagenesis; Neurons; Neurosciences Research; Optics; Parkinson Disease; Pathway interactions; Physiological; Proteins; Research; Resources; Retinal Pigments; Rhodopsin; Role; Seminal; Spectrum Analysis; Structure; Tachycardia; Techniques; Technology; Therapeutic Clinical Trial; Tissues; Visually Impaired Persons; Work; X-Ray Crystallography; base; brain circuitry; constriction; heart rhythm; in vivo; inhibitor/antagonist; innovation; light gated; microbial; nervous system disorder; neural circuit; optogenetics; painful neuropathy; photoactivation; programs; receptor; response; sight restoration; tool; vibration

My laboratory focuses on the structure, function, and mechanisms of microbial rhodopsins, widespread visual pigment-like proteins with diverse functions. Over the past decade, a subfamily, light-gated ion channels (channelrhodopsins), have had exceptional impact because of their central role in the transformative technology of optogenetics. We originally found them in the chlorophyte alga Chlamydomonas reinhardtii as phototaxis receptors that depolarize the cell membrane by producing cation currents in response to light. Subsequently neuroscientists found that these light-gated cation channelrhodopsins (CCRs) expressed in neurons produce depolarizing currents that enable light to trigger action potentials. Targeted photoactivation of neurons enabled by expression of CCRs in neural circuits has proven to be a powerful technique transforming many aspects of neuroscience research. Nevertheless, their light-gated channel activity is one of the least understood rhodopsin functions in terms of molecular mechanisms. Several advances in our work over the past 5 years, coupled to our knowledge and expertise over decades of research on microbial rhodopsins, guide our current research strategy. In 2015 we discovered exclusively anion-conducting (physiologically Cl-) channelrhodopsins (ACRs) in the distant phylum of cryptophyte algae. A breakthrough for optogenetics, ACRs enable efficient light-induced hyperpolarization and therefore are potent inhibitors of neuron firing. Also seminal to our research plans, our recent crystal structure of the most used ACR in optogenetics (GtACR1 from Guillardia theta) revealed a preexisting tunnel in the closed dark state that we propose is the channel closed by 3 well-defined constrictions. The GtACR1 tunnel is the only candidate ion pathway imaged in a channelrhodopsin, and provides a valuable resource for elucidating the mystery of channel gating by light. Principles learned from our study will likely enhance our understanding also of other microbial rhodopsins. Our current research investigates the diversity and molecular mechanisms of channelrhodopsins by: (i) ongoing genome mining to expand our knowledge and also advance optogenetics, focused on ACRs, but including CCRs (e.g. possible K+ and Ca++ channels). Recently we identified two new ACR families and long-sought red-shifted ACRs (“RubyACRs”) activated by tissue-penetrating long wavelengths, valuable for optogenetics and opening the way to elucidating color tuning mechanisms of channelrhodopsins; (ii) unraveling the relationship of electrical steps in channel function to photochemical transitions by structure-based mutagenesis, photo-electrophysiology in vivo, and kinetic optical and vibrational spectroscopy in vitro; and (iii) determination of atomic structures by X-ray crystallography and cryoEM, including innovative approaches to image the transient open-channel conformation. Elucidating mechanisms of channelrhodopsins will advance basic science and also facilitate engineering to optimize and tailor them for new optogenetic applications.

我的实验室专注于微生物视紫红质的结构、功能和机制，广泛的视觉 在过去的十年中，具有多种功能的色素样蛋白亚家族，光门控离子通道。 （视紫红质通道蛋白）由于其在变革性技术中的核心作用而产生了非凡的影响 我们最初在叶绿藻莱茵衣藻中发现它们具有趋光性。 随后通过响应光产生阳离子电流来使细胞膜去极化。 神经科学家发现，这些在神经元中表达的光门控阳离子通道视紫红质（CCR）产生 去极化电流使光能够触发神经元的定向光激活。 通过在神经回路中表达 CCR 已被证明是一种强大的技术，可以改变许多方面 然而，它们的光门控通道活性是人们最不了解的视紫红质之一。 过去 5 年我们的工作取得了一些进展，再加上我们的研究成果。 几十年来微生物视紫红质研究的知识和专业知识指导着我们当前的研究策略。 2015 年，我们在 作为光遗传学的一项突破，ACR 能够实现高效的光诱导。 超极化因此是神经元放电的有效抑制剂，对我们的研究计划和我们的研究计划也具有重要意义。 光遗传学中最常用的 ACR（来自 Guillardia theta 的 GtACR1）的最新晶体结构揭示了 我们提出的处于封闭黑暗状态的预先存在的隧道是由 3 个明确定义的收缩封闭的通道。 GtACR1 通道是视紫红质通道中成像的唯一候选离子通道，并提供了有价值的 从我们的研究中学到的原理可能会成为阐明光通道门控之谜的资源。 也增强了我们对其他微生物视紫红质的了解。我们目前的研究调查了多样性。 和视紫红质通道蛋白的分子机制：（i）持续的基因组挖掘以扩展我们的知识和 还推进光遗传学，重点关注 ACR，但包括 CCR（例如可能的 K+ 和 Ca++ 通道）。 最近，我们发现了两个新的 ACR 家族和长期寻找的红移 ACR（“RubyACR”）， 组织穿透的长波长，对于光遗传学很有价值，并为阐明颜色调节开辟了道路 (ii) 揭示通道功能中电步骤与 基于结构的诱变、体内光电生理学和动力学光学的光化学转变 和体外振动光谱；以及 (iii) 通过 X 射线晶体学测定原子结构和 冷冻电镜，包括对瞬态开放通道构象进行成像的创新方法。 视紫红质通道蛋白的机制将推动基础科学的发展，并促进工程的优化和应用 为新的光遗传学应用定制它们。