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

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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.