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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：
8337047
负责人：
LAWRENCE B COHEN
金额：
$ 2万
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/8337047
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 Fluorescence Generations Goals 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 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 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 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）：大脑回路中的活动模式及其经验依赖性变化是感觉信息、感知和运动控制处理的基础。关于大脑回路的解剖学布线和完整大脑中单个神经元的特性，我们已经了解了很多，但是关于单个神经元的特性如何从它们的连接中显现出来，以及多组神经元如何在行为过程中被激活，仍然存在相当大的谜团。部分问题在于，高精度的电记录通常一次只能从一个或几个神经元中获得，而显著事件实际上是由大型神经元集合处理的。为了提供来自哺乳动物神经元的快速高分辨率记录，该提议寻求改进基于荧光蛋白（FP）的电压传感器。这些探针将是独立的，不需要任何外源因子来发挥作用，因此将是遗传编码的。我们正在寻找这样的探针，其易于在细胞表面上表达，随着膜电位改变而显示出强度的最大变化，对膜电位的变化迅速响应，并且对细胞的破坏性最小。该项目的成员参与了第一代FP-电压传感器的开发，包括荧光振荡器（FlaSh），电压敏感荧光蛋白（VSFP）和钠通道蛋白活性报告构建体（SSCP）。这些构建体已经证明了产生随着细胞膜电位的变化而改变荧光强度的通道-FP构建体的可行性。响应特性的显著改善可能来自离子通道/转运蛋白和荧光蛋白空间的伪饱和检查。该提议将创建膜蛋白/ FP融合构建体的大型文库，所述膜蛋白/ FP融合构建体改变膜蛋白、插入的FP的位置和插入的FP的同种型。这些文库将通过一种新的基于转座子的FP插入过程来创建。将筛选构建体在海马神经元中的表面表达，并使用快速荧光测量结合电压钳电生理学测试电压依赖性荧光变化。我们将表达最有前途的FP-电压传感器在脑切片和体内使用病毒感染，然后生产转基因小鼠。

项目成果

期刊论文数量（16）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Genetically encoded fluorescent sensors of membrane potential.

DOI：
10.1007/s11068-008-9026-7
发表时间：
2008-08
期刊：
Brain cell biology
影响因子：
0
作者：
Baker BJ;Mutoh H;Dimitrov D;Akemann W;Perron A;Iwamoto Y;Jin L;Cohen LB;Isacoff EY;Pieribone VA;Hughes T;Knöpfel T
通讯作者：
Knöpfel T

Mechanistic studies of the genetically encoded fluorescent protein voltage probe ArcLight.