Channelrhodopsin-Calcium Channel Complexes for Ultrasensitive Optogenetics

描述(申请人提供)：藻类通道视紫红质是一种光门通道，广泛用于神经元活动的靶向光控制。在过去的六年里，它们光激活的时间和空间精度已被证明是尖锐和强大的研究 最近，在神经疾病的动物模型中，视紫红质的实验治疗已经产生了令人振奋的结果。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一个重要障碍，因为它需要高水平的表达和高光强。外来膜蛋白的过度表达对细胞的长期健康有害，还会造成免疫反应风险。与神经元的光敏性差形成鲜明对比的是，它们的天然藻细胞中的通道视紫红质是低丰度的光敏受体，在极弱的光线下调节趋光性，具有接近单光子的敏感性。这种精致的光敏性来自于藻质膜高电导钙通道的二次激活，使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工具，通过在莱茵衣藻中与自然执行这一功能的钙通道共表达来放大通道视紫红质的作用，从而使光敏感度提高约103倍。我们的方法是确定负责放大通道视紫红质介导的藻类光电流的分子成分，并构建一个组合的通道视紫红质-钙通道工具。我们将应用全基因组转录组图谱与扩增过程的电生理分析相结合的方法来识别扩增所需的钙通道和其他成分，如果它们存在的话。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钙通道。我们将进一步分析它们通过microRNA沉默的作用，并利用这些信息在哺乳动物细胞中重建一个超敏感的通道-视紫红质-钙通道复合体，藻类利用它来进行暗光趋光。我们希望这种新的光遗传工具在质膜上以远低于对人类细胞施加压力的蛋白质水平表达时能够有效。这些超灵敏的分子络合物将消除它们临床应用的一个重大障碍，并加强对神经电路的研究，使目前的技术无法进行大脑研究。

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.