Advanced Naturally Designed Channelrhodopsins for Photocontrol of Neural Activity

描述(由申请人提供)：本申请涉及广泛的挑战领域(06)使能技术，06-AG-101*：神经科学蓝图：开发非侵入性成像方法或直接评估神经活动的技术。对神经元回路的研究需要记录电活动和特定神经元的精确兴奋。活动的非侵入性成像也需要非侵入性的电路激活方法。最近，我们在2002年发现的光门离子通道通道视紫红质，是衣藻中介导光诱导膜去极化的趋光性受体，已被广泛用于非侵入性、高时间和空间分辨率的神经元刺激，包括结合特定靶向神经元的光刺激和神经元活动成像的实验。然而，到目前为止仅有的四种通道视紫红质的低离子电导和非特异性离子电导，以及它们的非最佳吸收光谱需要在活体组织中强烈散射的短波长光激活，极大地限制了它们的应用。利用我们开发的灵敏而快速的体内电生理测量系统，我们在衣藻的几个远亲中发现了通道视紫红质活性，这表明通道视紫红质介导的趋光性在具有叶绿体内柱头的趋光性鞭毛藻中普遍存在。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在吸收峰值、动力学和光循环特性方面存在差异。因此，大自然已经开发出数千种不同的通道视紫红质，其中许多很可能具有比目前使用的两种更好的特性。挑战是找到最有效和最有希望用于神经生物学的候选者。我们的电生理测量方法的独特优势是，它允许探测完整微生物悬浮液中的通道视紫质产生的光电流，而不受其大小和细胞壁结构的影响。我们提出了三个步骤来开发新的光谱可调和高效的通道视紫红质，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，以识别自然高效和高导电性的具有不同光谱峰值的通道视紫红质。我们的方法是快速的，可以对菌株集合中可用的数百种藻类进行筛选。基于我们对通道视紫红质在体内的功能和趋光性信号机制的了解，我们将首先研究适应极低离子强度和/或碱性环境的藻类，以及嗜冷(嗜寒)的北极物种，我们预测这些藻类可能含有在生理盐水中和在常温至37摄氏度下用于大脑电路分析的离子电导更高的通道视紫红质。 我们不断增长的实验数据将使我们的策略进一步完善，以选择要分析的藻类物种。(Ii)最有希望的新视蛋白基因的同源克隆。微生物视蛋白具有很强的保守性，在已知的四个视蛋白基因序列中有很长一段相同的序列，这使我们有信心聚合酶链式反应将是有效的。我们预计，最容易从与最初发现的物种关系最密切的藻类中获得通道视紫红质基因，例如雪地衣藻。随着更多基因的获得，不断增长的数据库应该能够让我们从更远的亲戚那里克隆。如果需要，我们将根据我们在微生物视紫红质结构功能、光化学和光谱调谐方面的多年经验，应用定点突变和嵌合体构建，以进一步优化新通道视紫红质的所需特性。(Iii)新通道视紫红质在动物细胞中的表达和特性。我们将建立HEK293细胞中对神经生物学应用至关重要的表达水平和最有希望的候选细胞的功能特征，随后将向神经科学家提供新的光活性工具，用于光神经电路分析和其他生物医学应用。

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.