Advanced Naturally Designed Channelrhodopsins for Photocontrol of Neural Activity

用于神经活动光控制的先进自然设计通道视紫红质

• 批准号: 7937966
• 负责人: JOHN LEE SPUDICH
• 金额: $ 40.04万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7937966
• 关键词: Address; Algae; Animals; Arctic Regions; Area; Biological Assay; Brain; Breathing; Cell Wall; Cells; Characteristics; Chimera organism; Chlamydomonas; Chlamydomonas reinhardtii; Cloning; Collection; Data; Databases; Development; Distant; Environment; Gated Ion Channel; Genes; Image; Investigation; Ionic Strengths; Kinetics; Life; Light; Love; Maps; Mastigophora; Measurement; Mediating; Membrane; Methods; Microbial Rhodopsins; Mus; Muscle Contraction; Nature; Neurobiology; Neurons; Neurosciences; Opsin; Optics; Photochemistry; Physiological; Property; Proteins; Relative (related person); Research; Saline; Screening procedure; Signal Transduction; Site-Directed Mutagenesis; Snow; Spinal cord injury; Stigmata; Stretching; Structure; Suspension substance; Suspensions; System; Technology; Temperature; Therapeutic; Tissues; absorption; base; blind; brain tissue; design; experience; high throughput screening; improved; in vivo; light gated; microbial; microorganism; neuronal circuitry; next generation; photoactivation; receptor; relating to nervous system; research study; restoration; social stigma; tool

Description (provided by applicant): This application addresses broad Challenge Area (06) Enabling Technologies, 06-AG-101*: Neuroscience Blueprint: Development of non-invasive imaging approaches or technologies that directly assess neural activity. Investigation of neuronal circuitry requires both registration of electrical activity and precise excitation of specific neurons. Non-invasive imaging of activity requires also non-invasive methods for circuit activation. Very recently channelrhodopsins, light-gated ion channels we found in 2002 to be phototaxis receptors that mediate light-induced membrane depolarization in Chlamydomonas algae, have been intensively used for non-invasive, highly temporally and spatially resolved neuronal stimulation, including experiments combining optical stimulation of specifically targeted neurons and neuronal activity imaging. However, the low and nonspecific ionic conductance of the only four channelrhodopsins identified so far, and their nonoptimal absorption spectra requiring photoactivation with short-wavelength light that strongly scatters in living tissue, greatly limit their utility. Using a sensitive and rapid electrophysiological in vivo measurement system we developed for assaying light-induced currents in intact cells, we have found channelrhodopsin activity in several distant relatives of Chlamydomonas, indicating channelrhodopsin-mediated phototaxis is ubiquitous in phototactic flagellated algae with an intrachloroplast stigma. Our preliminary screening shows that the channelrhodopsin receptors in algae from various environments vary in their absorption maxima, kinetics, and photocycling properties. Therefore, nature has already developed thousands of different channelrhodopsins, many of which are very likely to have properties superior to the two currently used. The challenge is to find the most effective and promising candidates for use in neurobiology. The unique advantage of our electrophysiological measurement method is that it allows probing of channelrhodopsin-generated photocurrents in suspension of intact microorganisms independently of their size and cell-wall structure. We propose three steps to develop new spectrally tuned and highly efficient channelrhodopsins for research in neuronal circuitry and other biomedical applications: (i) High-throughput screening to identify naturally highly efficient and highly conductive channelrhodopsins with various spectral maxima. Our method is fast and permits screening of hundreds of algal species available in strain collections. Based on our understanding of the functioning of channelrhodopsins in vivo and the phototaxis signaling mechanism, we will start our study by examining algae adapted to exceedingly low ionic strength, and/or alkaline environments, and psychrophilic (coldloving) arctic species, which we predict are likely to contain channelrhodopsins of much higher ionic conductance in physiological saline and at ambient to 37C temperatures used in brain circuitry analysis. Our growing experimental data will enable further refinement in our strategy to choose the algal species to be analyzed.(ii) Homology cloning of the most promising new opsin genes. Regions of strong conservation of microbial opsins in general as well as long stretches of identical sequence in the four known opsin gene sequences give confidence that PCR primers will be effective. We expect channelrhodopsin genes from algae most closely related to the original discovery species, such as the snow-dwelling species of Chlamydomonas, to be most easily obtained. As more genes are obtained, the growing database should enable us to clone from more distant relatives. If needed, site-specific mutagenesis and chimera construction guided by our years of experience in microbial rhodopsin structure-function, photochemistry, and spectral tuning will be applied to further optimize the desired properties to the new channelrhodopsins. (iii) Expression and characterization of the new channelrhodopsins in animal cells. We will establish expression levels, crucial for neurobiological applications, and functional characteristics of the most promising candidates in HEK293 cells, following which the new photoactive tools will be made available to neuroscientists for their use in optoneurocircuitry analysis and other biomedical applications.

描述（由申请人提供）：该应用程序解决了广泛的挑战领域（06）使能技术，06-AG-101*：神经科学蓝图：开发直接评估神经活动的非侵入性成像方法或技术。神经元电路的研究需要记录电活动和特定神经元的精确激发。活动的非侵入性成像还需要非侵入性的电路激活方法。最近，视紫红质通道（我们在 2002 年发现的光门控离子通道）是介导衣藻藻类中光诱导膜去极化的趋光性受体，已被广泛用于非侵入性、高度时间和空间分辨率的神经元刺激，包括结合光刺激的实验特定目标神经元和神经元活动成像。然而，迄今为止仅发现的四种通道视紫红质的低且非特异性离子电导，以及它们的非最佳吸收光谱需要用在活体组织中强烈散射的短波长光进行光活化，极大地限制了它们的实用性。使用我们开发的用于分析完整细胞中光诱导电流的灵敏且快速的体内电生理测量系统，我们在衣藻的几种远亲中发现了视紫红质通道蛋白活性，表明视紫红质通道蛋白介导的趋光性在具有叶绿体内柱头的趋光鞭毛藻类中普遍存在。我们的初步筛选表明，来自不同环境的藻类中的通道视紫红质受体的吸收最大值、动力学和光循环特性各不相同。因此，大自然已经开发出了数千种不同的视紫红质通道蛋白，其中许多很可能具有优于目前使用的两种的特性。面临的挑战是找到用于神经生物学的最有效和最有前途的候选者。我们的电生理测量方法的独特优势在于，它可以探测完整微生物悬浮液中视紫红质通道产生的光电流，而与微生物的大小和细胞壁结构无关。我们提出了三个步骤来开发新的光谱调谐和高效通道视紫红质，用于神经元电路和其他生物医学应用的研究：（i）高通量筛选，以识别具有各种光谱最大值的天然高效和高导电通道视紫红质。我们的方法速度快，可以筛选菌株库中的数百种藻类。基于我们对通道视紫红质在体内的功能和趋光性信号传导机制的了解，我们将通过检查适应极低离子强度和/或碱性环境的藻类以及嗜冷（耐冷）的北极物种来开始我们的研究，我们预测这些藻类是在用于脑电路分析的生理盐水中以及在环境温度至 37°C 的温度下，可能含有具有更高离子电导的通道视紫红质。 我们不断增长的实验数据将使我们能够进一步完善我们选择要分析的藻类物种的策略。（ii）最有前途的新视蛋白基因的同源克隆。微生物视蛋白的高度保守区域以及四个已知视蛋白基因序列中的长段相同序列使人们相信PCR引物将是有效的。我们预计与最初发现的物种最密切相关的藻类中的通道视紫红质基因，例如衣藻的雪栖物种，是最容易获得的。随着获得更多基因，不断增长的数据库将使我们能够从更远亲那里进行克隆。如果需要，将应用我们在微生物视紫红质结构功能、光化学和光谱调谐方面多年经验指导的定点诱变和嵌合体构建，以进一步优化新通道视紫红质的所需特性。 (iii)新视紫红质在动物细胞中的表达和表征。我们将建立对神经生物学应用至关重要的表达水平，以及 HEK293 细胞中最有希望的候选者的功能特征，随后新的光活性工具将提供给神经科学家用于光神经电路分析和其他生物医学应用。