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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

基本信息

批准号：
8510730
负责人：
JOHN LEE SPUDICH
金额：
$ 18.24万
依托单位：
UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-07-16 至 2015-01-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8510730
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

项目摘要

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.

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。