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选通过程中的构象变化，建立了衍射X射线跟踪方法。我们改进了跟踪衍射点精细运动的方法，使其具有较高的时空分辨率。对于高通量白X射线源，我们使用了两个同步加速器设施(ESRF[欧洲同步辐射设施，法国格勒诺布尔]；SLS[瑞士光源，维利根，瑞士])。借助于束流科学家对白X射线光谱的优化，大大降低了背景噪声，获得了高达5000帧/S的高速记录。在这些实验装置下，在单分子水平上记录了KCsA钾通道在门打开和关闭时的构象变化。KCSA通道是一个对pH敏感的通道，通过将溶液的pH从中性跃升到酸性，预计将记录初始的门控过程。利用355 nm紫外光照射笼状质子实现了pH跃升。测量了反应室内pH跃升的时间进程，发现pH跃升在100ms内完成。PH跃升前后的构象变化明显不同。这些视频数据正在进行图像处理，以检测衍射点并跟踪帧序列上的轨迹。自动获取光点的坐标是本程序的目标。

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.