Opsineering: Engineering Novel ChannelRhodospins for Optogenetics Applications

描述（由申请人提供）：光遗传学是通过哺乳动物神经元中微生物视蛋白的转基因表达对细胞进行遗传编码、光学诱导和控制。当这些视蛋白以细胞类型特异性的方式表达并且被光激活时，它们在活体动物的神经元中提供独立的超极化和去极化通道的时间和空间上分离的刺激。视紫红质（ChR）是光遗传学中用于触发光诱导去极化的微生物视蛋白。ChR是光门控离子通道，其在毫秒量级上操作，这是与神经元激活相关的时间尺度，并且可以在明确定义的脑区域中以高时间精度在不同细胞类型的膜中表达。这与化学或电刺激方法的不良时间动力学或缺乏特异性形成对比。然而，目前可用于神经元回路询问的光遗传学工具基于表达、光波长激活、动力学和离子特异性而受到限制。我们提出的项目通过蛋白质工程解决了这些限制。 通过定向进化和结构引导重组的蛋白质工程是用于修饰和优化蛋白质以实现所需功能的成熟方法。目前的文献和加州理工学院Gradinaru和Arnold实验室之间的初步合作工作表明，通道视紫红质在功能上是有用的实验室进化和操纵。这项工作将集中在工程改进的通道视紫红质作为光遗传学的生物工具。其目的是工程channelrhodopsins最佳的离子选择性，动力学，可逆性，并转移光激发波长。这些新的通道蛋白将用于探测大脑的电路，以更好地理解和模拟健康和不健康的大脑功能，作为控制和诊断神经系统疾病的基础，如成瘾，抑郁症和帕金森病。

DESCRIPTION (provided by applicant): Optogenetics is genetically encoded, optically induced, control of cells through transgenic expression of microbial opsins in mammalian neurons. When these opsins are expressed in a cell-type specific manner and light activated, they provide temporally and spatially separated stimulation of independent hyperpolarizing and depolarizing channels in neurons in living animals. Channelrhodopsins (ChRs) are the microbial opsins used in optogenetics to trigger light induced depolarization. ChRs are light-gated ion channels that operate on the order of milliseconds, a time scale relevant for neuronal activation, and can be expressed in the membrane of distinct cell types with high temporal precision in well-defined brain regions. This contrasts with the poor temporal dynamics or lack of specificity of chemical or electrical stimulation methods. However, the optogenetics tools currently available for neuronal circuit interrogation are limited based on expression, light-wavelength activation, kinetics and ion specificity. Our proposed project addresses these limitations through protein engineering. Protein engineering through directed evolution and structure-guided recombination are well-established methods for modifying and optimizing proteins for desired functions. Current literature and preliminary collaborative work between the Gradinaru and Arnold labs at Caltech indicate that channelrhodopsins are amenable to functionally useful laboratory evolution and manipulation. This work will be focused toward engineering improved channelrhodopsins for use as biological tools in optogenetics. The aim is to engineer channelrhodopsins for optimal ion selectivity, kinetics, reversibility, and shifted light excitatio wavelengths. These new channel proteins will have applications in probing the brain's circuitry to better understand and model healthy and non-healthy brain function as a foundation for controlling and diagnosing neurological disorders such as addiction, depression and Parkinson's disease.