Structural and functional dynamics of the KcsA channel : Single molecule measurements of the conformational change during gating.

为了检测离子通道在门控过程中的构象变化，建立了衍射x射线跟踪方法。改进了跟踪衍射斑精细运动的方法，具有较高的空间和时间分辨率。对于高通量白色x射线源，我们使用了两个同步加速器设备（ESRF[欧洲同步辐射设施，格勒诺布尔，法国]；SLS[瑞士光源，瑞士维利根]）。在光束科学家的帮助下，通过对白x射线光谱的优化，大大降低了背景噪声，实现了高达5000帧/秒的高速记录。在这些实验设置下，在单分子水平上记录了KcsA钾通道打开和关闭时的构象变化。KcsA通道是一个pH敏感通道，通过将溶液pH从中性跳到酸性，预计将记录初始门控过程。pH值的跃迁是利用笼状质子，用355nm的紫外激光照射。测量了pH值在腔内跳跃的时间过程，发现pH值的跳跃在100ms内完成。pH跃迁前后构象变化差异显著。对这些视频数据进行图像处理，检测衍射斑点，并在一系列帧上跟踪轨迹。自动检索点的坐标是这个程序的目标。

To detect conformational changes of ion channels upon gating, the diffracted X-ray tracking method has been established. We improved the method to trace fine movements of the diffraction spots with the high spatial and temporal resolutions. For the high flux white X-ray source, we used two synchrotron facilities (ESRF [European Synchrotron Radiation Facility, Grenoble, France] ; SLS [Swiss Light Source, Villigen, Switzerland]). By optimizing the spectrum of white X-rays with the aid of the beam scientists, the background noise was reduced substantially, and we attained the high speed recording as high as 5000 frame/s. Under these experimental setups, the conformational changes of the KcsA potassium channel upon opening and closing of the gate were recorded in the single molecule level. The KcsA channel is a pH-sensitive channel, and by jumping the solution pH from neutral to acidic, the initial gating processes were expected to be recorded. The pH jump was attained by utilizing a caged proton, to which the UV lazer of 355 nm was irradiated. The time course of the pH jump in the chamber was measured and it was revealed that the pH jump was completed within 100 ms. The conformational changes before and after the pH jump differed significantly. These video data were under the image processing for detecting the diffraction spots and tracing of the trajectories on the series of the frames. Automatic retrieval of the coordinate of the spots is the goal of this program.