DEVELOPMENT OF VOLTAGE-SENSING FLUORESCENT PROTEIN BASED ON GFP

膜电压测量是了解可兴奋细胞功能的重要技术之一。本课题旨在利用绿色荧光蛋白（Green fluorescent protein， GFP）开发能感知膜电压的荧光蛋白探针。利用其中一个电压门控钾通道作为碱基结构，利用分子生物学技术将GFP融合到该蛋白S4片段附近的区域，构建电压感应荧光探针。将cdna转染到HEK293细胞中，在荧光显微镜上安装CCD摄像系统，检测电压传感探针的荧光素是否随膜电压变化而变化。不幸的是，在S4段c端附近有GFP的探针没有荧光。然后在S1的n端或S3的n端插入GFP。这些蛋白质变成荧光。部分钾通道（S1-S4， S3-S4和S4-S6）也用于融合蛋白。所有这些被截断的通道都变成了荧光。使用S3-S4区域的探针一个留在细胞质中，其他探针被运输到膜上。为了提高蛋白质到质膜的运输，探针被融合到另一个属于氨基酸转运蛋白的蛋白质上。融合蛋白比原始蛋白更容易被转运到质膜上。目前，探测器发出的信号很小，不能用作探测器。我们试图通过改变连接顺序和GFP的位置来提高信噪比。

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.