This project studies the relationship between structure and function for two classes of membrane protein that are important for synaptic transmission. It employs functional assays and expression techniques. A first set of goals concern neurotransmitter-gated channels, particularly the nicotinic AChR family. Studies will continue to localize domains in the putative extracellular region that are important for the gating of the channel. These studies have begin with chimeric clones expressed in oocytes and will proceed to site-directed mutagenesis. Tests will continue with synthetic peptides corresponding to hydrophilic sequences within the receptors, on receptors expressed in oocytes. If these sequences correspond to functional domains, they may affect channel gating. Chimeric cDNA clones (nAChR/GABA) will be employed to localize the region(s) important for charge selectivity. Non-natural amino acids will be introduced into nAChRs using synthetic aminoacyl amber suppressor tRNAs. Functional measurements on these receptors may reveal positions that are important in ligand binding and gating. An incremental approach will be taken to high-resolution structural studies. The approach relies upon characterizing important soluble domains of the membrane proteins. A second set of goals concerns neurotransmitter transporters, particularly for GABA and other amino acids. Cloning and characterization of new neurotransmitter transporter family members will continue. Electrophysiological measurements will continue in mechanistic studies on the cloned and expressed transporters. Studies will continue on modulation and regulation of the transporters expressed in mammalian cells. Important functional domains on the transporters will be localized by measuring the function of chimeric transporters in heterologous expression systems. Site-directed mutagenesis will follow. The receptor and transporter molecules studied are crucial for healthy neuronal function and constitute the targets for many clinically important drugs.
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