DEVELOPMENT OF VOLTAGE-SENSING FLUORESCENT PROTEIN BASED ON GFP

膜电压的测定是了解可兴奋细胞功能的重要手段之一。本研究的目的是利用绿色荧光蛋白（GFP），开发一种能够检测细胞膜电压的荧光蛋白探针。为了开发电压敏感荧光探针，使用电压门控钾通道之一作为基础结构，并且通过分子生物学技术将GFP融合到该蛋白质的S4片段附近的区域。将cDNA转染到HEK293细胞中，用荧光显微镜上配备的CCD照相机系统测试来自电压敏感探针的荧光是否响应于膜电压而改变。不幸的是，在S4片段的C-末端侧附近具有GFP的探针不发荧光。然后，将GFP插入S1的N末端或S3的N末端。这些蛋白质变成了荧光。部分钾通道（S1-S4、S3-S4和S4-S6）也用于融合蛋白。所有这些截断的通道变成荧光。其中一个使用S3-S4区的探针留在细胞质中，其他探针被转运到细胞膜上。为了改善蛋白质向质膜的转运，将探针与属于氨基酸转运蛋白的另一种蛋白质融合。融合蛋白比原始蛋白更好地运输到质膜。此时，来自探针的信号非常小，无法用作探针。我们试图通过改变GFP的连接序列和位置来提高信噪比。

Measurement of membrane voltage is one of the most important techniques to understand function of excitable cells. The purpose of this project is developing fluorescent protein probes which can sense membrane voltage, using Green Fluorescent Protein (GFP). To develop voltage-sensing fluorescent probes, one of the voltage-gated potassium channels were used as a base structure, and GFP was fused to the region near the S4 segment of this protein by means of molecular biological techniques. cDNAs were transfected to HEK293 cells and it was tested whether the fluorescenee from voltage-sensing probes change in response to the membrane voltage with the CCD camera system equipped on the fluorescent microscope. Unfortunately, the probes which had GFP near the C-terminal side of the S4 segment did not become fluorescent. Then, GFP was inserted at the N-terminus of S1, or the N-terminus of S3. These proteins became fluorescent. Parts of potassium channel (S1-S4, S3-S4, and S4-S6) were also used for fusion protein. All these truncated channels became fluorescent. One of the probes which used the S3-S4 region remained in cytoplasm and others was transported to the membrane. To improve the transportation of the proteins to the plasma membrane, the probes were fused to another protein which belongs to the ammo-acid transporters. The fused protein is transported to the plasma membrane better than the original protein. At this moment, the signals from the probes are so small to use as probes. We are trying to improve the signal/noise ration by changing the connecting sequences and the position of the GFP.