Changes in the structure and function of the Nicotinic Acetylcholine Receptor (AChR) are linked to pathogenic responses on human muscle and brain. The long-range goal of this research proposal is to define the functional role of lipid-protein interactions in the conformational transitions of the AChR. The objective of this project is to define the modes by which lipid protein interactions are linked to the gating machinery of the AChR. The central hypothesis of this research is that AChR channel kinetics is modulated allosterically by specific sites on the receptor that are in direct contact with the membrane lipids. This hypothesis has been formulated on the basis of strong preliminary data, which suggest that single amino acid replacements at discrete lipid-exposed positions on the M4 transmembrane segments of the AChR greatly enhanced the macroscopic response to acetylcholine. The rationale for the proposed research is that lipid-protein interactions could represent a very important mechanism for the regulation of AChR channel function in cholinergic synapses of the muscle and brain and understanding the molecular basis of these mechanisms would help to identify conditions in which the impairment of AChR function can lead to disease. The central hypothesis will be tested and the objective of the application will be accomplished by pursuing three specific aims: (1) to define helix-helix contacts, structural constraint positions and additional allosteric sites of the muscle-type AChR, using periodic tryptophan substitutions along the M4 transmembrane domains, (2) Define the role of 11 lipid exposed positions on the functional differences between the Torpedo and muscle-type AChR. (3) Perform Fluorescence Photobleaching Recovery (FRAP) to estimate translational diffusion measurements of AChRs of the Torpedo and muscle type AChRs in the plasmatic membrane of Xenopus oocytes. The proposed work is innovative because it capitalizes on new approaches developed by the applicants to estimate AChR translational diffusion constant in oocytes in vivo. It is our expectation that these approaches will define new mechanisms by which lipid-protein interactions that modulate the allosteric transitions of AChR. The outcomes will be significant because it is expected that the new knowledge will disclose new mechanisms for AChR regulation in the intact synapse. This research will be of additional significance, because it will also provide information on the secondary structure and the spatial organization of the M4 transmembrane segment, as well as to the structure-function relationships of the AChR. Another biological significance of this project is that it will provide new understanding of the role of lipid-exposed domains within the AChR and how such lipid-protein interactions can contribute to functional differences between AChR species. Analysis of the lipid-protein interactions of the AChR are not only important for understanding the functioning of ligand-gated ion channels, but may also provide insight into the mechanisms of action of certain therapeutic drugs, such as alcohols and general anesthetics.
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