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）：藻类通道旋转蛋白是明智的通道广泛用于神经元活性的靶向光控制。在过去的六年中，其光激活的时间和空间精度已被证明是敏锐而有力的研究在脑电路上以及最近在神经系统疾病动物模型中的通道Rhopoptin实验疗法上产生了令人鼓舞的结果。然而，通道旋转蛋白的极低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。外膜蛋白的过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相反，其天然藻类细胞中的通道旋转是低丰度的光感感受器，这些受体在极低的光线下介导光的受体具有近乎单光子的敏感性。这种精致的光敏性源自通道Ropopsin电流的〜103倍扩增，通过在藻类质膜中高电导的Ca2+通道的继发性激活。我们建议通过通过与CA2+通道共表达CACHRHODOPSITAIN作用来开发一种新的超敏化光遗传学工具，并具有〜103倍的光灵敏度，该工具与Ca2+通道进行了表达，这些CA2+通道自然在Reinhardtii的藻类衣原体中执行此功能。我们的方法是鉴定负责扩增藻类中通道旋接介导的光电流的分子成分，并构建一个组合的通道Ropopsin-Ca2+通道工具。我们将应用与扩增过程的电生理测定相结合的全基因组转录组分析，以识别Ca2+通道和其他成分（如果存在）所需的其他成分。我们的证据强烈有利于直接相互作用的机制，因此我们期望鉴定一个或多个CA2+通道能够通过蛋白质 - 蛋白质相互作用直接扩增。我们将通过microRNA沉默进一步分析它们的作用，并使用此信息在哺乳动物细胞中重新创建超敏感的通道Ropsrhopopsin-CA2+通道复合物，该通道由藻类用于昏暗的光触发。我们期望这种新的光遗传学工具能够在质膜中表达的蛋白质水平远低于压力人类细胞的蛋白质水平。这些超敏感的分子复合物将消除其临床使用的重大障碍，并增强对神经循环的研究，从而使当前技术无法进行大脑研究。