Scan of Protein Space for Optical Voltage Probes

光学电压探针的蛋白质空间扫描

• 批准号: 7912355
• 负责人: LAWRENCE B COHEN
• 金额: $ 24.02万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2011-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7912355
• 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

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.

大脑回路中的活动模式及其依赖于经验的变化是大脑活动的基础。 感觉信息、知觉和运动控制的处理。我们已经了解了很多关于 大脑回路的解剖布线和完整大脑中单个神经元的特性，但 关于单个神经元的特性是如何从它们的连接中显现出来的，仍然存在相当大的谜团 以及在行为过程中多组神经元是如何被激活的。问题的一部分是如此之高 精确的电记录通常一次只从一个或几个神经元获得，当显着时， 事件实际上是由大量的神经元处理的。提供快速高分辨率 从哺乳动物神经元的记录，该提议寻求改善基于荧光蛋白（FP）的电压 传感器.这些探针将是独立的，不需要任何外源因子发挥作用，因此将 是基因编码的我们正在寻找易于在细胞表面表达的探针，显示 最大的变化强度与膜电位的改变，迅速作出反应的变化， 膜电位，并且对细胞的破坏性最小。该项目的成员参与了 开发第一代FP电压传感器，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 （二）。这些构建体已经证明了创建通道-FP构建体的可行性，所述通道-FP构建体改变了 荧光强度与细胞膜电位的变化。应对措施的重大改进 特征可以来自离子通道/转运蛋白的假饱和检查， 荧光蛋白空间。该提议将创建膜蛋白/ FP融合的大型文库 改变膜蛋白、插入的FP的位置和插入的FP的同种型的构建体。 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。建筑将是 筛选海马神经元中的表面表达并测试电压依赖性荧光 使用快速荧光测量结合电压钳电生理学的变化。我们将 在脑切片和体内使用病毒感染表达最有前途的FP-电压传感器， 转基因小鼠的生产。