Laser-triggered single molecular gating motions of the KcsA potassium channels recorded in a sub-millisecond time Resolution
Résumé
We previously revealed that the KcsA potassium channel exhibited global twisting motions upon gating (Shimizu, H., et al Cell 132, 67-78, 2008). The method we used was the diffracted X-ray tracking (DXT), in which the conformational changes were recorded under equilibrium conditions at video rate. To trace the whole picture of the gating motions, further development of the method is required. We recently have succeeded in developing a laser-triggered measurement system, while the X-ray spectrum was optimized for fast recordings with a high-speed camera. The channel was fixed on a glass plate at the extracellular loop and a gold nanocrystal was attached to the C-terminal end of the channel molecule as a probe. The sample was irradiated with an optimized white-beam from a synchrotron, and the motions of the Laue diffraction spots from the nanocrystal were tracked on a CMOS detector at the speed of 5000 frames/s. During the measurements the pH of the solution was changed from neutral to acidic by laser-photolysis of the caged proton, which triggers the gating motions of the pH-gated KcsA channel. Trajectories of the diffraction spots on the detector plane were translated as movements of the channel in the real space. This modified DXT method now enables us to detect the initial opening processes of the conformational changes in the time resolution comparable to that of the single channel current recordings.