Channelrhodopsin-Calcium Channel Complexes for Ultrasensitive Optogenetics

描述（由申请人提供）：藻类通道视紫红质是广泛用于神经元活性的靶向光控制的光门控通道。在过去的六年里，他们的光激活的时间和空间精度已被证明是精辟和强大的研究 最近，在神经系统疾病的动物模型中进行的通道视紫红质实验疗法已经产生了有希望的结果。然而，通道视紫红质极低的离子电导限制了它们的使用，并且是人类临床试验的重要障碍，因为它需要高水平表达和高光强度。外源膜蛋白的过度表达对长期细胞健康有害，也会产生免疫反应风险。与神经元的光敏性差形成鲜明对比的是，天然藻类细胞中的通道视紫红质是低丰度的感光受体，其在极低的光下以接近单光子的灵敏度介导趋光性。这种精致的光敏性来自于藻细胞质膜中高传导性Ca 2+通道的二次激活导致通道视紫红质电流放大约103倍。我们建议开发一类新的超灵敏的光遗传学工具，通过与天然在莱茵衣藻中执行此功能的Ca 2+通道共表达来放大通道视紫红质作用，从而具有约103倍的光灵敏度。我们的方法是确定负责放大的通道视紫红质介导的光电流在藻类的分子组成部分，并构建一个组合的通道视紫红质-Ca 2+通道工具。我们将应用全基因组转录组分析加上电生理检测的扩增过程中，以确定扩增所需的Ca 2+通道和其他组件，如果它们存在。我们的证据强烈支持直接相互作用机制，因此我们希望确定一个或多个能够通过蛋白质-蛋白质相互作用直接扩增的Ca 2+通道。我们将通过microRNA沉默进一步分析它们的作用，并利用这些信息在哺乳动物细胞中重建藻类用于昏暗光趋光性的超敏感通道视紫红质-Ca 2+通道复合物。我们希望这种新的光遗传学工具能够在质膜中以远低于人类细胞应激水平的蛋白质水平表达时有效。这些超灵敏的分子复合物将消除其临床应用的重大障碍，并加强对神经回路的研究，使目前技术无法进行的大脑研究成为可能。 公共卫生相关性：视紫红质是光活性蛋白质，其能够通过组合的光学和遗传技术（“光遗传学”）光激活神经元。通道视紫红质的光激活已被用于绘制哺乳动物脑组织中的电路，以及用于实验治疗，包括恢复失明小鼠的视力。该项目旨在开发新型的光遗传学工具，其灵敏度比目前可用的工具高约1000倍，基于通道视紫红质与特定Ca 2+通道的相互作用。这种超灵敏的光激活通道复合物将使生物医学研究成为可能，并克服了光遗传学在人类临床应用的重大障碍。

DESCRIPTION (provided by applicant): Algal channelrhodopsins are light-gated channels widely used for targeted photocontrol of neuron activity. Over the past six years, the temporal and spatial precision of their light-activation has proven to be incisive and powerful for research on brain circuitry, and more recently channelrhodopsin experimental therapy in animal models of neurological diseases has produced promising results. However, the extremely low ion conductance of channelrhodopsins limits their use and is a significant barrier for human clinical trials because it necessitates high level expression and high light intensities. Overexpression of a foreign membrane protein is detrimental to long-term cellular health and also creates an immune response risk. In stark contrast to the poor photosensitivity in neurons, channelrhodopsins in their native algal cells are low abundance photosensory receptors that mediate phototaxis in extremely low light with near single-photon sensitivity. This exquisite photosensitivity derives from the ~103-fold amplification of channelrhodopsin currents by secondary activation of highly conductive Ca2+ channels in the algal plasma membrane. We propose to develop a new class of ultrasensitive optogenetic tools with the ~103-fold greater light sensitivity by amplification of channelrhodopsin action by co-expression with the Ca2+ channels that naturally perform this function in the alga Chlamydomonas reinhardtii. Our approach is to identify molecular components responsible for amplification of channelrhodopsin-mediated photocurrents in the algae and to construct a combined channelrhodopsin-Ca2+ channel tool. We will apply genome-wide transcriptome profiling coupled to electrophysiological assay of the amplification process to identify the Ca2+ channels and other components necessary for amplification, if they exist. Our evidence strongly favors a direct interaction mechanism, so we expect to identify one or more Ca2+ channels capable of direct amplification by protein-protein interaction. We will further analyze their action by microRNA silencing, and use this information to re-create in mammalian cells an ultrasensitive channelrhodopsin-Ca2+ channel complex used by algae for dim-light phototaxis. We expect this new optogenetic tool to be able to be effective when expressed in the plasma membrane at protein levels well below those that stress human cells. These ultrasensitive molecular complexes will remove a significant barrier to their clinical use, as well as enhance research on neurocircuitry, enabling brain studies not possible with current technology. PUBLIC HEALTH RELEVANCE: Channelrhodopsins are photoactive proteins enabling light activation of neurons by combined optical and genetic techniques ("optogenetics"). Light-activation of channelrhodopsins has been used to map circuitry in mammalian brain tissue, as well as for experimental therapeutics, including restoration of vision in blind mice. This project seeks to develop new types of optogenetic tools with ~1000-fold higher sensitivity than the currently available ones, based on interaction of channelrhodopsins with specific Ca2+ channels. Such ultrasensitive light-activated channel complexes will enable biomedical research not possible with current technology and overcome a significant barrier to clinical use of optogenetics in humans.