The proposed study is designed to examine gene expression, ligand recognition and structure of two major post-synaptic proteins in the cholinergic nervous system, acetylcholinesterase and the nicotinic acetylcholine receptor. These proteins work in concert in many cholinergic synapses to achieve the appropriate fidelity of neurotransmission in the brain and periphery and are the targets of several pharmacologic agents. Our approach relies on expression of these proteins from their cloned genes, site-specific mutagenesis and analysis of ligand binding and structure through studying specificity of peptide toxins, fluorescence spectroscopy and X-ray crystallography. In particular, we plan to study the interaction of the three fingered peptide, fasciculin with acetylcholinesterase to correlate the residues contributing to the binding energy with the structure of the wild-type and mutant complexes determined by X-ray crystallography. We also plan to examine the portals of entry of small substrates into fasciculin complexed acetylcholinesterase. Since the enzyme functions at diffusion-controlled rates, despite having its active center deep in a gorge, we plan to use decay of fluorescence anisotropy to examine whether flap-like motions of a domain or more rapid breathing motions govern ligand entry into the gorge. Other structural investigations with cholinesterase involve its comparison with the homologous a/P hydrolase-fold protein, neuroligin, in order to delineate regions of non-catalytic functions in the two molecules. Studies with the nicotinicreceptors rely on toxins selective for particular binding interfaces (cc-conotoxinM- 1, 8 subunit; waglerin, s subunit; N. mossctmbica mossambica a-neurotoxin, y and 8 but not s subunits). From site- specific mutagenesis on the a-toxin and receptor, we will employ therniodynamic mutant cycle analysis to ascertain proximities of amino acid residues in the complex. This information will be used to model toxin binding to receptors. We will also attempt to develop a x-ray crystal structure based template of the extracellular domain of the receptor to enhance structural information for these studies. Finally, studies on acetylcholinesterase gene expression will be continued with particular emphasis on tissue specific expression and splicing in differentiating muscle. The first intron, 5' of the translation start site, appears critical for the enhanced expression associated with muscle specific expression, and we plan to delineate how this region serves as an enhancer or represser of transcriptional activity and whether it influences the transport of mRNA from the nucleus.
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