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 阳离子传导通道旋转蛋白（CCR），来自绿色（又称叶绿素）藻类的光触及受体具有 成为最著名的微生物感觉视opsins，因为它们用作光活化的工具 神经射击，这对于开发光遗传学的变革性技术至关重要。 但是，我们对它们的分子机制的理解仍处于早期阶段。令人惊讶的发现 过去一年中，其他两个Channelrhopopsins家族的远距离密码藻藻具有 扩大了研究机会，并能够克服先前的限制到结构/功能研究 渠道机制。首先，我们在照相cryptophyte上的工作揭示了功能上不同的家族 严格执行阴离子的光门控信道。天然阴离子通道旋转（ACR）， 除了他们作为以前未知的现象本质上的兴趣外，还引起了很多兴趣 光遗传学工具是因为它们具有前所未有的光效率，可通过轻门氯化 传导。其次，我们的密码植物研究最近揭示了第三个ChannelRhopopsins，就像 叶绿素CCR，进行阳离子，但具有明显不同的结构。隐形植物CCR显然 已经通过不同的进化途径在阳离子通道函数上融合，与 卤代质质子泵。叶绿素CCR研究的主要局限性是它们非常低 电导及其缺乏对光学和分子的通道功能的体外评估 光谱法。 ACR是已知的最导电轻门控通道，高达50倍的统一通道 电导率比最导电性CCR，为结构/功能研究提供了实用优势。 在过去的一年中，ACRS的强大活动帮助我们建立了许多基本属性，并且 可以使用单层蔬菜（LUV）来监测通道 与相关结构变化的光谱监测并行活性。特定目标1是屏幕 ACR和隐植物CCR同源物及其突变体通过斑块夹在动物细胞中表达 评估保留的电生理学确定通道特性。 AIM 2是深入分析密钥 动物细胞中的突变体和通过光谱法阐明通道机制的突变体 开放和关闭和阴离子的选择性。最初依靠以现有建立的工作结构 微生物视紫红质原子结构，通过分析ACR和类似泵的CCR同源物增强 我们将追求AIM 3，即确定ACR的X射线晶体结构，一个“倒”的ACR突变体开放 在黑暗和类似泵的CCR中。最后，AIM 5是确定CA2+通道的先前目标的延续 参与Chlamydomonas Reinhardtii中的ChannelRhopoptin介导的照片的1000倍扩增 基于一个新机会：可用的C. reinhardtii基因的敲除文库。我们的总体目标是 比较分析阐明并区分三个通道旋转蛋白家族的原理。