The overall objective is to understand the structure, function, and regulation of integral membrane proteins that serve as ion channels in excitable cells. Recently all the acetylcholine receptor (AChR) subunits were cloned and the amino acid sequences were determined and used to model the subunits organization into a transmembrane channel structure. Giga-seal patch techniques in conjunction with streaming potential measurements will be used to learn about the internal structure of the AChR channel to provide a basis for judging the four proposed structural models. Streaming potential measurements will determine the number of water molecules coupled to the transport of different size cations. The water coupled to the largest cation that can fit through will indicate the length of the narrowest portion of the channel. The length of the narrow region and ion-water interaction will be related directly to permeation studies. This electrical method of obtaining structure has been used with artificial membranes, but this will be the first time it is extended to study bio-channels in their native membranes. The Na channel and AChR are extensively glycosylated. The carbohydraate contributes 30% of the weight and about 100 negative charges to the Na channel from Electrophorus electricus, and the AChR has a glycosidic linkage near the proposed ACh binding site. However, little is knonw about the function of the carbohydrate. Electrical and standard biochemical methods with radioactive and fluorescent labels will be used to study Na channel and AChR glycosylation. These modification studies address the importance of glycosylation in AChR and Na channel function, ACh binding, channel maturation and lifetime, and events related to synaptogenesis. This should provide a step toward understanding post-translational modifications, which regulate ion channel properties during differentiation, development, and learning. The proposed studies provide insights into molecular mechanisms that are altered by pathological conditions affecting the nervous system and neuromuscular transmission.
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