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.

这项提议旨在开发更好的工具来分析脑细胞和脑回路，并 大脑活动的大规模记录。就灵敏度而言，目前可用的工具相对原始 和速度。神经元的一个主要功能是处理电信号。因此，一种特殊的工具 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‘s)是一种明显的战略方法，用于“使大脑在行动中可视化”。基因编码 神经学家对传感器特别感兴趣，因为它们可以作为蛋白质在个体中表达。 哺乳动物大脑中的细胞类型。因为每个大脑区域有多达100种不同的细胞类型，传感器 在特定细胞类型中的表达对于成像该细胞类型的活动是必不可少的。 最近，GEVI的信号大小有了很大的改善(即ArcLight，40%/100 Mv)，但ArcLight的响应时间常数相对较慢(τ=10毫秒)。现在有几种速度更快的GEVI (τ=0.3msec至2.0msec)，但它们的信号大小较小(~10%/100 mv)。这项提议的一个目标是开发一种 GEVI对膜电位变化的反应既大又快。 除了大小和速度之外，其他几个探头特性也至关重要。一个是 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绿色荧光蛋白及其 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。一个目标是开发有用的红色地理信息系统。其次，许多GEV都有一个乙字形 荧光-电压关系。该关系沿电压轴的位置可以通过 GEVI的电压敏感域的突变。因此，GEVI可以成为不同范围的选择性记者 神经元膜电位的变化，从而对动作电位活动与阈值下活动具有选择性。 这种选择性既取决于半最大激活时的电压，也取决于乙状波的陡度。 曲线。最后，将GEVI定位于神经元的特定区域，包括细胞体，这一点非常重要。 树突状突触后区和突触前终末。 建议中的所有探针都基于膜蛋白(A)的电压敏感域 斑马鱼或斑马鱼的磷酸酶)，N-端或C-端插入一个或两个荧光蛋白 区域。改进的GEVI的开发将涉及用于产生新的探针的分子生物学(更大， 更快、红色、在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 分离的神经元和斑马鱼胚胎。在这些初始筛查中运行良好的探测器将被 被掺入病毒颗粒中，用于在哺乳动物的大脑中进行体内转染和测量。改进 而选择性的GEVI将有助于社区进行大脑活动的光学测量。