| The nicotinic acetylcholine receptor (AChR) from Torpedo californica is an allosteric and integral membrane protein composed of five subunits arranged in a stoichiometry of α2βγδ. Each subunit contains a large hydrophilic N-terminal, four transmembrane domains (M1, M2, M3 and M4), a variable intracellular loop between M3 and M4, and a short extracellular C-terminal. The M2 segment contributes to the ion channel pore in the closed conformation, while both the M1 and M2 contribute to the ion channel on its open conformation. The M3 and M4 are lipid-exposed domains. The long-range goal of this research is to understand how lipid protein interactions modulate the AM. The work reported here, first defined allosteric interactions in the lipid-exposed transmembrane segments M3 and M4 of the AChR alpha subunit. Tryptophan-scanning mutagenesis was performed to understand the contribution of these domains to the AChR function. Mutagenic analysis assisted in the construction of a model for the spatial organization of the αM3 and αM4. The mutants were examined in Xenopus laevis oocytes and characterized using electrophysiological techniques. Second, this dissertation examined the functional aspects of cholesterol interactions with the AChR. Finally, it examined the contribution of lipid-exposed residues, with different polarities, on the functional differences of the Torpedo and muscle type AChR. The electrophysiological analysis showed that the lipid interface contributes to the differences in channel function of these receptor subtypes. The results provided a clearer picture of the dynamic properties of the lipid-protein interface and its critical role in the channel gating mechanism of the AChR. |
