Scan of Protein Space for Optical Voltage Probes

光学电压探针的蛋白质空间扫描

基本信息

批准号：
7317193
负责人：
LAWRENCE B COHEN
金额：
$ 102.24万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-09-01 至 2012-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7317193
关键词：
Action Potentials Acute Antibodies Bar Codes Behavior Binding Biological Assay Brain Brain imaging Cell membrane Cell surface Cells Characteristics Chimeric Proteins Communities Data Databases Development Digestion Electrophysiology (science)Engineering Environment Epitopes Event Exogenous Factors Extensible Markup Language Fiber Fire - disasters Fluorescence Generations Goals Green Fluorescent Proteins Hippocampus (Brain)Homologous Gene Individual Ion Channel Label Laboratories Learning Length Libraries Life Ligation Location Measurement Measures Membrane Membrane Potentials Membrane Protein Traffic Membrane Proteins Methods Modification Monitor Mus Neurons Noise Numbers Optics Pattern Perception Performance Physiological Plasmids Population Preparation Principal Investigator Process Production Property Protein Isoforms Proteins Recombinant Proteins Reporting Resolution Scanning Screening procedure Sensory Process Signal Transduction Site Slice Structure Subfamily lentivirinae Surface Testing Time Transfection Transgenic Mice Transgenic Organisms Viral Load result Virus Diseases analog base chromophore design experience extracellular improved in vivo member motor control mouse model novel olfactory bulb promoter response sensor size sodium channel proteins trafficking voltage voltage clamp

项目摘要

DESCRIPTION (provided by applicant): The pattern of activity in the circuits of the brain and their experience-dependent changes underlie the processing of sensory information, perception, and motor control. Much has been learned about the anatomical wiring of brain circuits and about the properties of individual neurons in the intact brain, but considerable mystery remains about how the properties of individual neurons emerge from their connectivity and how multiple groups of neurons are activated during behaviors. Part of the problem has been that high precision electrical recording is usually obtained from only one or a few neurons at a time, when salient events are actually processed by large assemblies of neurons. To provide for a fast high-resolution recording from mammalian neurons, this proposal seeks to improve fluorescent protein (FP) based voltage sensors. These probes will be self-contained, not requiring any exogenous factors to function, and thus will be genetically-encodable. We are seeking probes that are readily expressed on the cell's surface, show maximum changes in intensity with membrane potential alterations, respond rapidly to changes in membrane potential, and are minimally disruptive to cells. Members of the project have been involved in the development of first generation FP-voltage sensors, including Fluorescent Shaker (FlaSh), Voltage-Sensitive Fluorescent Protein (VSFP) and Sodium channel Protein Activity Reporting Construct (SPARC). These constructs have demonstrated the feasibility of creating channel-FP constructs that alter fluorescence intensity with changes in cell membrane potential. Significant improvements in the response characteristics may come from a pseudo-saturating examination of the ion channel/transporter and fluorescent protein space. This proposal will create large libraries of membrane protein / FP fusion constructs varying the membrane protein, the location of the inserted FP and the isoform of the inserted FP. These libraries will be created by a novel transposon-based FP insertion process. Constructs will be screened for surface expression in hippocampal neurons and tested for voltage-dependent fluorescence changes using fast fluorescence measurements combined with voltage-clamp electrophysiology. We will express the most promising FP-voltage sensors in brain slices and in vivo using viral infection followed by the production of transgenic mice.

描述（由申请人提供）：大脑电路中的活动模式及其与经验有关的变化是感官信息，感知和运动控制的处理。关于脑电路的解剖接线以及完整大脑中单个神经元的特性已经了解了很多，但是关于单个神经元的特性如何从它们的连接性以及在行为过程中如何激活多组神经元的特性。问题的一部分是，高精度电记录通常一次是从一个或几个神经元中获得的，而当显着事件实际上是由大型神经元组件处理的显着事件时。为了提供哺乳动物神经元的快速高分辨率记录，该提案旨在改善基于荧光蛋白（FP）的电压传感器。这些探针将是独立的，不需要任何外源性因素才能发挥作用，因此可以在遗传上进行调节。我们正在寻找在细胞表面上很容易表达的探针，随着膜电位改变显示强度的最大变化，对膜电位变化的反应迅速，并且对细胞的破坏性极小。该项目的成员已经参与了第一代FP - 电压传感器的开发，包括荧光振动器（Flash），电压敏感的荧光蛋白（VSFP）和钠通道蛋白活性报告构建体（SPARC）。这些构建体证明了创建通道-FP构建体的可行性，该通道FP构建体会随着细胞膜电位的变化改变荧光强度。响应特征的显着改善可能来自对离子通道/转运蛋白和荧光蛋白空间的伪饱和检查。该建议将创建大量的膜蛋白 / FP融合构建体的库，这些膜蛋白改变了膜蛋白，插入的FP的位置和插入的FP的同工型。这些库将通过新型的基于转座的FP插入过程创建。将筛选构建体的海马神经元中的表面表达，并使用快速荧光测量结果与电压钳电生理学结合使用，并测试了电压依赖性荧光变化。我们将使用病毒感染在脑切片和体内表达最有希望的FP - 电压传感器，然后在体内产生转基因小鼠。