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The nicotinic acetylcholine receptor (nAChR) mediates fast signal transduction in peripheral and central nervous systems. It is a pentameric ion channel that belongs to the Cys-loop receptor superfamily. nAChR is one of the plausible targets for general anesthetics. The current concern about nAChR is ambivalent structures of the transmembrane (TM) domain, missing information for the intracellular (IC) domain and elusive mechanisms of the action of general anesthetics. Therefore, this thesis focuses on the following two important aspects: first, the structure and dynamics of the nAChR TM and IC domains; and second, general anesthetic effects on various proteins. Part I. The secondary structure of human nAChR α7 TM domain and the monomeric tertiary structure of the water-soluble mutant of nAChR α1 TM domain from the Torpedo electrical ray were determined by NMR. The structures among WSA, nAChR α7, and a bacterial analogue of the pentameric channels (GLIC) are similar, but different from the cryo-EM structure of the nAChR. The backbone dynamics analyses of human nAChR α7 with and without the IC domain suggest that the presence of IC domains dramatically affected the intrinsic dynamics of the TM domains. Part II. The general anesthetic effects on the structure and dynamics of the proteins including two analogues and a real anesthetic target in non-channel and channel-like oligomers were studied. It revealed that general anesthetics prefer amphipathic environments. Compared to the effect on the structures of proteins, general anesthetics show stronger effect on the dynamics. The effect on the dynamics of proteins can manifest directly or allosterically. Part III. The anesthetic effects on the Na+ flux through the reconstituted channels of nAChR TM domains were investigated by fluorescence microscopy. The anesthetic effects on them are the same as on the full-length nAChR. This indicates that the studies of anesthetic effects on the TM domains of nAChR are functional relevant. In summary, this dissertation clarifies the current structural ambiguities, provides additional invaluable dynamic information for the TM and IC domain and reveals the possible mechanism of action of general anesthetics. |
