Opsineering: Engineering Novel ChannelRhodospins for Optogenetics Applications

描述（由申请人提供）：光遗传学是通过在哺乳动物神经元中转基因表达微生物视蛋白来进行遗传编码、光诱导和细胞控制。当这些视蛋白以特定的细胞类型表达并被光激活时，它们在时间和空间上分别刺激活体动物神经元的独立超极化和去极化通道。通道视紫红质（ChRs）是光遗传学中用于触发光诱导去极化的微生物视蛋白。ChRs是光门控离子通道，其工作时间为毫秒级，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大脑区域中以高时间精度在不同细胞类型的膜中表达。这与较差的时间动态或缺乏特异性的化学或电刺激方法形成对比。然而，目前可用于神经元回路询问的光遗传学工具是有限的，基于表达，光波长激活，动力学和离子特异性。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一种完善的方法，用于修饰和优化蛋白质的所需功能。目前的文献和加州理工学院Gradinaru和Arnold实验室的初步合作表明，通道视紫红质可以在功能上适应实验室的进化和操作。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。目的是设计通道视紫红质的最佳离子选择性，动力学，可逆性和移位的光激发波长。这些新的通道蛋白将用于探测大脑回路，以更好地理解和模拟健康和非健康的大脑功能，作为控制和诊断成瘾、抑郁和帕金森病等神经系统疾病的基础。

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.