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：开发直接评估神经活动的非侵入性成像方法或技术。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还需要用于回路激活的非侵入性方法。最近，通道视紫红质，光门控离子通道，我们在2002年发现的趋光性受体介导的光诱导的膜去极化的衣原体藻类，已被广泛用于非侵入性的，高度的时间和空间分辨的神经元刺激，包括实验相结合的光学刺激的特定目标的神经元和神经元活动成像。然而，迄今为止识别的仅有的四种通道视紫红质的低的和非特异性的离子电导，以及它们的非最佳吸收光谱，其需要用在活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效用。使用一个敏感的和快速的电生理在体内测量系统，我们开发了完整的细胞中的光诱导电流进行测定，我们已经发现通道视紫红质活性在几个远亲的衣原体，表明通道视紫红质介导的趋光性是无处不在的趋光性鞭毛藻的叶绿体内柱头。我们的初步筛选表明，在不同环境中的藻类中的通道视紫红质受体在其吸收最大值，动力学，和光循环特性。因此，自然界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上级目前使用的两种的特性。目前的挑战是找到最有效和最有前途的候选人用于神经生物学。我们的电生理测量方法的独特优势是，它允许探测通道视紫红质产生的光电流在悬浮液中的完整的微生物独立于它们的大小和细胞壁结构。我们提出了三个步骤来开发新的光谱调谐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，以识别具有各种光谱最大值的天然高效和高导电的通道视紫红质。我们的方法是快速的，并允许筛选菌株收藏中的数百种藻类。基于我们对通道视紫红质在体内的功能和趋光性信号机制的理解，我们将从研究适应极低离子强度和/或碱性环境的藻类和嗜冷性的藻类开始我们的研究。（喜冷）北极物种，我们预测其在生理盐水中和在用于脑电路分析的环境温度至37 ℃下可能含有高得多的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选择待分析藻类的策略。(ii)最有希望的新视蛋白基因的同源克隆。一般而言，微生物视蛋白的强保守区域以及四种已知视蛋白基因序列中的长段相同序列给予PCR引物将有效的信心。我们预计，最容易获得的通道视紫红质基因，从藻类最密切相关的原始发现的物种，如雪居物种的衣原体。随着越来越多的基因被获得，不断增长的数据库应该使我们能够从更远的亲戚那里克隆。如果需要的话，在我们多年的微生物视紫红质结构-功能、光化学和光谱调谐经验的指导下，将进行位点特异性诱变和嵌合体构建，以进一步优化新通道视紫红质的所需特性。(iii)新型通道视紫红质在动物细胞中的表达及特性研究。我们将建立表达水平，这对神经生物学应用至关重要，以及HEK 293细胞中最有希望的候选物的功能特征，之后新的光敏工具将提供给神经科学家用于视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.