A Protein Based Optical Probe of Membrane Potential

描述（由申请人提供）：了解神经系统功能需要同时监测大量功能神经元的电活动。理解网络神经元大群之间的时间相互作用对于充分理解中枢系统如何处理信息是很重要的。光学记录方法的优势在于，它们提供了在有限的系统扰动下记录显著神经元事件所需的空间和时间分辨率。目前可用的电压敏感染料很难在实验中应用，对于给定的电位变化会产生微小的荧光变化，并且不能针对特定的神经元子集，从而降低了所获得信号的复杂性。目前的应用程序旨在生产一种基于蛋白质的、电压敏感的、用于神经元的光学探针。我们开发了一种探针（称为SPARC），它基于大鼠mu1骨骼肌钠通道与荧光蛋白（即GFP）的融合。这种结构体现了我们在膜电位光学探针中寻找的许多特征。注射SPARC cRNA后，非洲爪蟾卵母细胞在细胞膜去极化过程中表现出快速（<1 ms）、可重复（测试时为1000脉冲/小时）和可逆（0.1-0.5% δ F/F/100mV）的荧光信号。这种变化可以很容易地记录在动作电位大小的去极化脉冲（<2 ms）中。本应用程序旨在增加该探针产生的荧光信号的δ F/F和信噪比，并验证该探针在神经元中的使用。这将需要进行以下研究：(I)充分表征目前开发的SPARC探针。（二）修改荧光蛋白插入位点的性质和位置。（III）改变在这些位点插入的荧光蛋白的类型和数量。（IV）最后，我们将尝试开发一种FRET版本的探针，以增强随着电压阶跃所看到的荧光变化。

DESCRIPTION (provided by applicant): Understanding nervous system function will require the simultaneous monitoring of the electrical activity of a large number of functioning neurons. It will be important to understand the temporal interactions between large groups in networked neurons to fully understand how the central system processes information. Optical recording methods offer advantages in that they provide the spatial and temporal resolution necessary to record salient neuronal events with limited perturbations of the system. Currently available voltage-sensitive dyes are very difficult to apply experimentally, produce small changes in fluorescence for a given potential change and cannot be targeted to a specific subset of neurons, thereby reducing the complexity of the signal obtained. The present application seeks to produce a protein-based, voltage-sensitive, optical probe for use in neurons. We have developed a probe (termed SPARC), which is based on the fusion of the rat mu1 skeletal muscle sodium channel with a fluorescent protein (i.e., GFP). This construct embodies many of the features we are seeking in an optical probe of membrane potential. Xenopus laevis oocytes, injected with the cRNA for SPARC, exhibit a fluorescent signal that undergoes a rapid (<1 ms), reproducible (>1000 pulses/hour tested) and reversible change (0.1-0.5% delta F/F/100mV) in intensity during depolarization of the cell membrane. This change can be easily recorded during action potential sized depolarization pulses (<2 ms). The present application seeks to increase the delta F/F and signal-to-noise ratio of the fluorescent signal arising from this probe and validate the use of this probe in neurons. This will entail the following studies: (I) fully characterize the presently developed SPARC probe. (II) Modify the nature and location of the fluorescent protein insertion site. (III) Alter the type and number of fluorescent proteins inserted at these sites. (IV) Finally, we will attempt to develop a FRET version of the probe to enhance the fluorescence change seen with voltage steps.