The long-term objectives of this application are to understand the structural features of the AChR and to relate these structures to the function of the receptor. The structure of the AChR serves as a paradigm for the structure of ligand gated ion channels and thus provides a basis for an understanding of how this class of proteins function. The structure of the ACh binding sites on the AChR will be investigated by photoaffinity labelling to determine the respective contribution of the subunits to the binding sites. TC will be used as a photoaffinity label; the sites of the gamma- and delta-subunits that react with TC will be defined through isolation of labelled fragments and identification of labelled residues by N-terminal amino acid sequencing. TC will be derivatized with photoreactive groups to enhance photoreactivity and then also used to characterize the binding sites in the same manner. The topology of the AChR subunit primary sequence will be analyzed by labelling of protein exposed on the extracellular or intracellular surface of the AChR. The labelled sites will be identified by isolation of labelled fragments and identification of labelled residues. The location of a putative intracellularly exposed fragment alpha 156-179 will be evaluated specifically. Chemical cross- linking will be used to define the nearest-neighbor relationships of the subunits and of individual amino acids within subunits. Residues near the ACh binding sites will be probed by specifically attaching photoactivated cross-linkers to the disulfide near the ACh binding site.
