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，感知和电动机控制的处理。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，但 关于单个神经元的特性如何从它们的连通性中出现 以及在行为过程中如何激活多组神经元。问题的一部分是很高 精密电记录通常仅从一个或几个神经元中获得 事件实际上是由大型神经元组件处理的。提供快速的高分辨率 该提案旨在改善基于荧光蛋白（FP）电压，从哺乳动物神经元记录 传感器。这些探针将是独立的，不需要任何外源性因素才能起作用，因此 在遗传上可以编码。我们正在寻找在细胞表面上很容易表达的探针，显示 随着膜电位变化的强度最大变化，对变化的变化迅速响应 膜电位，对细胞的破坏性极小。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 （SPARC）。这些结构已经证明了创建改变渠道fp构造的可行性 荧光强度随细胞膜电位变化而变化。反应的重大改善 特征可能来自对离子通道/转运蛋白的伪饱和检查和 荧光蛋白空间。该建议将创建大量的膜蛋白 / FP融合图书馆 构造改变膜蛋白，插入的FP的位置和插入的FP的同工型的位置。 这些库将通过新型的基于转座的FP插入过程创建。结构将是 筛选在海马神经元中的表面表达，并测试了电压依赖性荧光 使用快速荧光测量结果结合了电压钳电生理学的变化。我们将 使用病毒感染，然后在体内表达最有希望的FP电压传感器，然后体内 转基因小鼠的产生。