Channelrhodopsin-Calcium Channel Complexes for Ultrasensitive Optogenetics

用于超灵敏光遗传学的视紫红质通道-钙通道复合物

• 批准号: 8359246
• 负责人: JOHN LEE SPUDICH
• 金额: $ 22.8万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-16 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8359246
• 关键词: Algae; Anabolism; Animal Model; Appearance; Biological Assay; Biomedical Research; Brain; Calcium Channel; Candidate Disease Gene; Cations; Cell membrane; Cell physiology; Cells; Chlamydomonas reinhardtii; Clinical; Clinical Trials; Complementary DNA; Complex; Coupled; Data; Drosophila genus; Figs - dietary; Gene Expression Profile; Gene Silencing; Genetic Techniques; Health; Human; Immune response; Investigational Therapies; Ion Channel; Ions; Light; Mammalian Cell; Maps; Mastigophora; Mediating; Membrane; Membrane Potentials; Membrane Proteins; MicroRNAs; Molecular; Monitor; Mus; Neurons; Optics; Photons; Photophobia; Photosensitivity; Phototransduction; Procedures; Process; Proteins; Research; Resolution; Risk; Stimulus; Stress; System; TRP channel; Technology; Testing; Therapeutic; Therapeutic Uses; Time; Variant; Vision; base; blind; brain tissue; genome-wide; light gated; light intensity; millisecond; nervous system disorder; new technology; optogenetics; overexpression; patch clamp; protein protein interaction; receptor; restoration; sensor; tool

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.

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动的靶向光控制的光门控通道。在过去的六年里，它们的光激活的时间和空间精度已被证明对于研究来说是深刻而强大的 神经系统疾病动物模型中的通道视紫红质实验疗法已经产生了有希望的结果。然而，视紫红质通道极低的离子电导限制了它们的使用，并且是人体临床试验的一个重大障碍，因为它需要高水平的表达和高光强度。外源膜蛋白的过度表达不利于长期细胞健康，还会产生免疫反应风险。与神经元中较差的光敏感性形成鲜明对比的是，其天然藻类细胞中的通道视紫红质是低丰度的光感觉受体，在极低的光照下以接近单光子的敏感性介导趋光性。这种精致的光敏性源自藻类质膜中高导电性 Ca2+ 通道的二次激活，视紫红质通道电流放大了约 103 倍。我们建议开发一类新型超灵敏光遗传学工具，通过与天然在藻类莱茵衣藻中执行此功能的 Ca2+ 通道共表达来放大视紫红质通道的作用，从而将光灵敏度提高约 103 倍。我们的方法是确定藻类中负责放大视紫红质通道介导的光电流的分子成分，并构建组合的视紫红质通道-Ca2+通道工具。我们将应用全基因组转录组分析与扩增过程的电生理分析相结合，以确定 Ca2+ 通道和扩增所需的其他组件（如果存在）。我们的证据强烈支持直接相互作用机制，因此我们期望识别出一个或多个能够通过蛋白质-蛋白质相互作用直接放大的 Ca2+ 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新创建藻类用于微光趋光性的超敏感通道视紫红质-Ca2+通道复合物。我们期望这种新的光遗传学工具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能够有效。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，使当前技术不可能进行的大脑研究成为可能。 公共健康相关性：视紫红质通道蛋白是光敏蛋白，通过结合光学和遗传技术（“光遗传学”）能够光激活神经元。视紫红质通道的光激活已被用来绘制哺乳动物脑组织中的电路图，以及用于实验治疗，包括恢复失明小鼠的视力。该项目旨在基于视紫红质通道与特定 Ca2+ 通道的相互作用，开发新型光遗传学工具，其灵敏度比现有工具高约 1000 倍。这种超灵敏的光激活通道复合物将使现有技术无法进行的生物医学研究成为可能，并克服光遗传学在人类临床应用的重大障碍。