New approaches for better protein voltage sensors

更好的蛋白质电压传感器的新方法

• 批准号: 9231604
• 负责人: LAWRENCE B COHEN
• 金额: $ 72.67万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-30 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9231604
• 关键词: Action Potentials; Address; Amino Acid Sequence; Axon; Brain; Brain region; Butterflies; C-terminal; Cardiac; Cells; Characteristics; Communities; Dendrites; Dependence; Dependency; Development; Dyes; Embryo; Energy Transfer; Epilepsy; Fluorescence; Fluorescent Probes; Goals; Image; Individual; Lateral; Light; Location; Measurement; Membrane; Membrane Potentials; Membrane Proteins; Modification; Molecular Biology; Monitor; Movement; Mutate; Mutation; Nerve; Neurons; Noise; Nose; Olfactory Receptor Cells; Optics; Peptide Sequence Determination; Phosphoric Monoester Hydrolases; Population; Positioning Attribute; Presynaptic Terminals; Process; Proteins; Reaction Time; Reporter; Reporting; Screening Result; Side; Signal Transduction; Specificity; Speed; Synapses; Testing; Transfection; Virion; Zebrafish; analog; base; brain cell; cell type; design; dimer; improved; in vivo; interest; millisecond; neuronal cell body; neuronal circuitry; novel; novel strategies; olfactory bulb glomeruli; optical spectra; presynaptic; response; sensor; tool; trafficking; voltage

This proposal aims to develop better tools for analyzing brain cells and circuits and for large-scale recordings of brain activity. The currently available tools are relatively primitive in terms of sensitivity and speed. One major function of a neuron is to process electrical signals. Thus a tool that is of particular significance is high speed membrane potential imaging. Genetically encoded fluorescent protein voltage indicators (GEVI's) are a obvious strategic approach for “visualizing the brain in action”. Genetically encoded sensors are especially interesting to neuroscientists because, as proteins, they can be expressed in individual cell types in the mammalian brain. Because each brain region has up to 100 different cell types, sensor expression in a specific cell type is essential for imaging the activity of that cell type. Recently, there has been a dramatic improvement in the signal size of GEVIs (i.e. ArcLight, 40%/100 mv) but ArcLight has a relatively slow response time constant (τ=10 msec). There are now several faster GEVIs (τ=0.3 to 2.0 msec) but they have smaller signal sizes (~10%/100mv). One goal of this proposal is developing a GEVI with both large and fast responses to membrane potential changes. Several probe characteristics other than size and speed are also critically important. One is the wavelength range of excitation and emission. At present many sensors and activators are based on GFP and its analogues. Thus, probes with red excitation and emission spectra would allow simultaneous dual function measurements. One aim is to develop useful red GEVIs. Second, many GEVIs have a sigmoidal fluorescence-voltage relationship. The position of this relationship along the voltage axis can be adjusted via mutations in the voltage sensitive domain of the GEVI. Thus GEVIs can be selective reporters of different ranges of the neuron membrane potential and thereby selective for action potential activity versus subthreshold activity. This selectivity depends on both the voltage at half-maximal activation as well as the steepness of the sigmoidal curve. Lastly, it will be important to target the GEVIs to specific regions of the neuron including cell body, dendritic post-synaptic zones, and presynaptic terminals. All of the probes in the proposal are based on the voltage sensitive domain of a membrane protein (a phosphatase from Ciona or zebrafish) with one or two fluorescent proteins inserted into the N- or C-terminal region. The development of improved GEVIs will involve molecular biology for generating novel probes (bigger, faster, red, targeted anatomically and physiologically) followed by testing in cultured HEK293 cells, acutely dissociated neurons, and zebrafish embryos. Probes that function well in these initial screens will then be incorporated into virus particles for in vivo transfection and measurements in the mammalian brain. Improved and selective GEVIs would be useful to the community carrying out optical measurements of brain activity.

该提案旨在开发更好的工具来分析脑细胞和电路以及 大脑活动的大规模记录。目前可用的工具在灵敏度方面还比较原始 和速度。神经元的一项主要功能是处理电信号。因此，一个特殊的工具 意义在于高速膜电位成像。基因编码的荧光蛋白电压 指标（GEVI）是“可视化大脑活动”的明显战略方法。基因编码 神经科学家对传感器特别感兴趣，因为作为蛋白质，它们可以在个体中表达 哺乳动物大脑中的细胞类型。由于每个大脑区域有多达 100 种不同的细胞类型，传感器 特定细胞类型中的表达对于对该细胞类型的活性进行成像至关重要。 最近，GEVI 的信号大小有了显着改善（即 ArcLight，40%/100 mv），但 ArcLight 的响应时间常数相对较慢（τ=10 毫秒）。现在有几个更快的 GEVI （τ=0.3 至 2.0 毫秒），但它们的信号大小较小（~10%/100mv）。该提案的一个目标是开发一个 GEVI 对膜电位变化具有大而快速的响应。 除了尺寸和速度之外，几个探头特性也至关重要。其一是 激发和发射的波长范围。目前许多传感器和激活器都是基于GFP及其 类似物。因此，具有红色激发和发射光谱的探针将允许同时具有双重功能 测量。目标之一是开发有用的红色 GEVI。其次，许多 GEVI 具有 s 形曲线 荧光-电压关系。这种关系沿电压轴的位置可以通过调整 GEVI 电压敏感域的突变。因此 GEVI 可以是不同范围的选择性报告基因 神经元膜电位的变化，从而对动作电位活动与阈下活动进行选择性。 这种选择性取决于半最大激活时的电压以及 S 形曲线的陡度 曲线。最后，将 GEVI 靶向神经元的特定区域（包括细胞体）非常重要。 树突突触后区和突触前末梢。 该提案中的所有探针均基于膜蛋白（a 来自 Ciona 或斑马鱼的磷酸酶），N 端或 C 端插入一或两个荧光蛋白 地区。改进的 GEVI 的开发将涉及分子生物学以产生新型探针（更大、 更快，红色，解剖学和生理学上有针对性），然后在培养的 HEK293 细胞中进行测试，急性 分离的神经元和斑马鱼胚胎。在这些初始屏幕中运行良好的探针将被 掺入病毒颗粒中，用于哺乳动物大脑中的体内转染和测量。改进 选择性 GEVI 将有助于社区对大脑活动进行光学测量。