Realtime Single Molecular Motion Analysis of Nicotinic Acetylcholine Receptor Alpha 7 by Diffracted X-Ray Tracking Method

Abstract

The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel. Binding of ligands (e.g. acetylcholine, nicotine) to nAChR induces ion flux through the channel formed by pentameric subunits, which leads to the changes of cell excitability, and then triggers the following signaling cascades in the peripheral and the central nervous system. In the previous study we applied Diffracted X-ray Tracking (DXT) method to acetylcholine binding protein (AChBP) and Torpedo nAChR. DXT is a method to track the X-ray diffraction spots from the gold nanocrystal labeled on an individual single protein in real time and real space. We observed molecular fluctuations of the proteins even without ligand, and enhancement of tilting and twisting motions with ligand [Sekiguchi, et al. (2014) Sci. Rep. 4: 6384].Here, to acquire more precise information on molecular motions of nAChR in the three sub-states (close, open, and desensitization) and their sate-to-state transitions, we applied the DXT for the neuronal nAChR alpha 7. The alpha 7 receptor in the central nervous system plays essential roles in the cholinergic neuro-signal transduction, and it is one of the drug targets for therapeutics of Alzheimer diseases and cognitive impairment associated with schizophrenia (CIAS). Alpha 7 receptor was recombinantly prepared from the cDNA expressed in Xenopus oocytes. To orderly configure the receptors defined tags were introduced at the defined positions; e.g. “Met tag” for labeling with gold nanocrystal in the N-terminal, and “His tag” for absorption on mica substrate in the second cytoplasmic loop of the receptor. Characteristic molecular motion(s) during the three states of the nAChR alpha 7 were revealed by the DXT analysis. Possible transition modeling will be discussed.