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: 10576389
• 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/10576389
• 关键词: Action Potentials; Algae; Anions; Basic Science; Biomedical Research; Brain Mapping; Cardiac Myocytes; Cations; Cell membrane; Cells; Chlamydomonas reinhardtii; Clinical Trials; Color; Coupled; Cryoelectron Microscopy; Darkness; Disease; Distant; Electrophysiology (science); Engineering; Epilepsy; Family; Genetic Techniques; Genome; Heart Diseases; Image; In Vitro; Investigational Therapies; Ion Channel Gating; Ions; Kinetics; Knowledge; Laboratories; Learning; Light; Microbial Rhodopsins; Mining; Molecular; Molecular Conformation; Mutagenesis; Neurons; Neurosciences Research; Optics; Parkinson Disease; Pathway interactions; Penetration; Physiological; Proteins; Research; Resources; Retinal Pigments; Rhodopsin; Role; Seminal; Spectrum Analysis; Structure; Tachycardia; Techniques; Technology; Tissues; Visually Impaired Persons; Work; X-Ray Crystallography; brain circuitry; constriction; heart rhythm; in vivo; inhibitor; innovation; light gated; microbial; nervous system disorder; neural circuit; optogenetics; painful neuropathy; photoactivation; programs; receptor; redshift; response; sight restoration; tool

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蛋白的结构，功能和机制，广泛的视觉 具有潜水功能的色素样蛋白质。在过去的十年中，一个亚科，光门控的离子通道 （ChannelRhopopsins），由于其在变革技术中的核心作用而产生了出色的影响 光遗传学。我们最初在叶绿素藻类的衣原体中找到它们，作为光的 通过响应光的阳离子电流来定义细胞膜的接收器。随后 神经科学家发现，这些在神经元产生中表达的光门控阳离子通道（CCR） 向色电流推出，使光线触发动作电位。启用神经元的目标光活化 通过在神经回路中表达CCR，事实证明是一种强大的技术，改变了许多方面 神经科学研究。然而，他们的光门控通道活动是最不理会的视紫红蛋白之一 根据分子机制的功能。在过去的5年中，我们的工作有几个进步，与我们的 数十年来有关微生物视紫红蛋白的知识和专业知识，指导我们当前的研究策略。 在2015年，我们发现了独家的阴离子传导（生理上cl-）通道Ropopsins（ACR） 隐藻藻的远处。光遗传学的突破，ACR可实现有效的光诱导 超极化，因此是神经输血的潜在抑制剂。也是我们的研究计划的半氛围 光遗传学中最常用的ACR的最新晶体结构（来自Guillardia theta）的晶体结构显示了一个 我们提出的封闭的黑暗状态下的隧道是由3个定义明确的收缩封闭的通道。 GTACR1隧道是唯一成像的候选离子途径，并提供一个值 阐明光线通道门控神秘的资源。从我们的研究中学到的原则可能会 也增强我们对其他微生物视紫红蛋白的理解。我们目前的研究调查了多样性 以及通道旋转的分子机制，作者：（i）正在进行的基因组开采以扩大我们的知识和 还可以推进光遗传学，重点是ACR，但包括CCR（例如可能的K+和Ca ++通道）。 最近，我们确定了两个新的ACR家族和长期追求的红移ACR（“ RubyAcrs”）。 组织渗透长波长，对于光遗传学很有价值，并为阐明颜色调整开辟道路 通道旋转的机理； （ii）阐明通道功能中电步骤的关系与 通过基于结构的诱变，体内的光电性生理学和动力学光学的光化学转变 体外振动光谱； （iii）通过X射线晶体学确定原子结构和 冷冻，包括创新的方法来形象瞬态开放通道构象。阐明 ChannelRhopopsins的机制将提高基础科学，并促进工程以优化和 为他们定制新的光遗传应用。