BPS2026 – X-ray single-molecule detection of activated α7 nicotinic acetylcholine receptors using caged-acetylcholine

The α7 nicotinic acetylcholine receptor (nAChR α7) is composed of five α7 subunits arranged symmetrically around a central cation pore. Acetylcholine (ACh) is an agonist ligand to open the channel, whereas prolonged application results in a decrease of the response, desensitization. Although biochemical and structure-function studies on the nAChR have been accumulated, the dynamic changes occurring at the moment nAChR α7 binds to ACh, particularly during the desensitized state have not been exhaustively studied. We performed diffracted X-ray tracking method (DXT) in SPring-8 BL40XU beamline to monitor the twisting motion of nAChR α7 using photo-caged ACh, which releases ACh at specific times and locations under visible light irradiation (390 nm) with a time-resolution of 100 μs. The X-ray diffraction spots from ZnO nanocrystals labeled with nAChR α7 were recorded by an X-ray image intensifier and a CMOS camera. The tilting precision is 0.18 mrad, and the twisting precision is 0.74 mrad. The diffusion constant of twisting motion is 1.47 mrad 2 /ms in ligand-free condition. The binding of a ligand reduces the freedom of movement of nAChR α7. The results showed that nAChR α7 twist in a counterclockwise (CCW) direction during channel opening and possibly during desensitization. Before desensitization, it exhibits a faster clockwise (CW) rotational motion. Interestingly, in the desensitized state, the motion remains in the CW direction, accompanied by an 11% opening component in the CCW direction.