Structure/Function of Microbial Sensory Rhodopsins

微生物感觉视紫红质的结构/功能

• 批准号: 9308239
• 负责人: JOHN LEE SPUDICH
• 金额: $ 66.53万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-04-01 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9308239
• 关键词: Algae; Animals; Anions; Bacteriorhodopsins; Biomedical Research; Cations; Cells; Chemicals; Chlamydomonas reinhardtii; Chlorides; Crystallization; Darkness; Development; Distant; Electrophysiology (science); Engineering; Exhibits; Family; Genes; Genetic Techniques; Goals; Human; In Vitro; Investigational Therapies; Ions; Kinetics; Knock-out; Knowledge; Lasers; Libraries; Light; Lighting; Link; Maps; Measurement; Mediating; Methodology; Methods; Microbial Rhodopsins; Modeling; Molecular; Molecular Conformation; Monitor; Movement; Mutagenesis; Nature; Neurons; Optics; Phenotype; Process; Property; Proton Pump; Protons; Pump; Reaction; Research; Retinal; Retinal Pigments; Rhodopsin; Roentgen Rays; Role; Route; Sampling; Schiff Bases; Sensory Receptors; Sensory Rhodopsins; Side; Signal Transduction; Spectrum Analysis; Structure; System; Technology; Testing; Therapeutic Clinical Trial; Vesicle; Vision; Visually Impaired Persons; Work; X-Ray Crystallography; base; brain circuitry; chromophore; comparative; conformer; in vitro Assay; insight; interest; light gated; luminescence resonance energy transfer; microbial; microorganism; mutant; nervous system disorder; optogenetics; patch clamp; photoactivation; receptor; relating to nervous system; sensory rhodopsin I; tool; unilamellar vesicle; voltage clamp

Summary Cation-conducting channelrhodopsins (CCRs), phototaxis receptors from green (aka chlorophyte) algae, have become the best known microbial sensory rhodopsins because of their use as tools for photoactivation of neural firing, which has been essential for development of the transformative technology of optogenetics. However, our understanding of their molecular mechanism is still at an early stage. The surprising discovery in distantly related cryptophyte algae of two additional families of channelrhodopsins in the past year have expanded research opportunities and enable overcoming prior limitations to structure/function studies of channel mechanism. First, our work on a phototactic cryptophyte revealed a functionally different family of light-gated channelrhodopsins that conduct strictly anions. Natural anion channelrhodopsins (ACRs), in addition to their interest as a previously unknown phenomenon in nature, have generated much interest as optogenetic tools because of their unprecedented photoefficiency to silence neurons by light-gated chloride conduction. Second, our cryptophyte studies recently revealed a third family of channelrhodopsins that, like chlorophyte CCRs, conduct cations, but have a distinctly different structure. The cryptophyte CCRs evidently have converged on cation channel function via a different evolutionary route and are closely related to haloarchaeal proton pumps. The main limitations to the study of chlorophyte CCRs has been their very low conductance and their lack of an in vitro assay for their channel function amenable to optical and molecular spectroscopy. ACRs are the most conductive light-gated channels known, having up to 50-fold higher unitary conductance than the most conductive CCRs, providing a practical advantage for structure/function studies. The robust activity of ACRs helped us over this past year to establish many of their basic properties and has made possible developing a purified in vitro system using unilamellar vesicles (LUVs) to monitor channel activity in parallel with spectroscopic monitoring of associated structural changes. Specific Aim 1 is to screen ACR and cryptophyte CCR homologs and their mutants expressed in animal cells by patch clamp electrophysiology to assess residue determinants of channel properties. Aim 2 is to analyze in depth key mutants both in animal cells and in vitro by spectroscopic methods to elucidate the mechanisms of channel opening and closing and anion selectivity. While relying initially on working structures modeled on existing microbial rhodopsin atomic structures and enhanced by analysis of ACRs and the pump-like CCR homologs, we will pursue Aim 3 which is to determine X-ray crystal structures of an ACR, an “inverted” ACR mutant open in the dark, and a pump-like CCR. Finally, Aim 5 is a continuation of a prior aim to identify the Ca2+ channel involved in 1000-fold amplification of channelrhodopsin-mediated photocurrents in Chlamydomonas reinhardtii based on a new opportunity: the availability of a knock-out library of C. reinhardtii genes. Our overall goal is by comparative analysis to elucidate principles that unite and distinguish the three channelrhodopsin families.

概括 阳离子传导视紫红质通道 (CCR) 是来自绿藻（又名叶绿藻）的趋光性受体，具有 因其用作光激活工具而成为最著名的微生物感觉视紫红质 神经放电，这对于光遗传学变革技术的发展至关重要。 然而，我们对其分子机制的理解仍处于早期阶段。这一令人惊讶的发现。 在过去的一年里，另外两个视紫红质通道的远亲隐生藻类已经 扩大了研究机会并能够克服先前结构/功能研究的局限性 首先，我们对趋光隐植物的研究揭示了一个功能不同的家族。 光门控视紫红质，严格传导阴离子。 除了他们对自然界中以前未知的现象的兴趣之外，还引起了很多兴趣 光遗传学工具，因为它们具有前所未有的光效率，可以通过光门控氯化物使神经元沉默 其次，我们的隐植物研究最近揭示了视紫红质的第三个家族，例如 叶绿植物 CCR 传导阳离子，但结构与隐植物 CCR 明显不同。 通过不同的进化路线在阳离子通道功能上趋同，并且与 盐古菌质子泵研究的主要局限性是其非常低。 电导及其缺乏适合光学和分子的通道功能的体外测定 ACR 是已知导电性最强的光门控通道，其单一性高达 50 倍。 电导率高于最具导电性的 CCR，为结构/功能研究提供了实际优势。 ACR 的强劲活动帮助我们在过去的一年中确立了它们的许多基本特性，并已 使得开发使用单层囊泡（LUV）监测通道的纯化体外系统成为可能 与相关结构变化的光谱监测并行的具体目标 1 是筛选。 ACR和隐植物CCR同源物及其突变体在动物细胞中通过膜片钳表达 电生理学评估通道特性的残留决定因素 目标 2 是深入分析的关键。 通过光谱方法在动物细胞和体外研究突变体以阐明通道机制 打开和关闭以及阴离子选择性。同时依赖于以现有模型为模型的初始工作结构。 微生物视紫红质原子结构，并通过分析 ACR 和泵状 CCR 同系物进行增强， 我们将追求目标 3，即确定 ACR（一种“倒置”ACR 突变体）的 X 射线晶体结构 最后，目标 5 是识别 Ca2+ 通道的先前目标的延续。 参与莱茵衣藻中视紫红质通道介导的光电流放大 1000 倍 基于一个新的机会：莱茵衣藻基因敲除文库的可用性，我们的总体目标是 比较分析，以阐明统一和区分三个通道视紫红质家族的原则。