ALPHA1 GLYCINE RECEPTOR STRUCTURE AND FUNCTION

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DESCRIPTION: (adapted from Applicant's Abstract) The glycine receptor (GlyR), a glycine-gated Cl- channel, is the major inhibitory neurotransmitter channel of the central nervous system. The overall goal of this project is to determine the secondary structure, topology, and functional role of domains and specific residues of the homomeric recombinant human alpha1 GlyR overexpressed in a baculovirus expression system. These investigations of GlyR structure may provide structure- function information at a molecular level on neurological disorders such as myoclonus, spasticity, familial startle disease or other diseases in which deficiencies of GlyR have been implicated. Additionally, this receptor is a member of the ligand-gated superfamily, which include the homologous nicotinic acetylcholine receptor, the 5-HT3 serotonin receptor, and the GABA receptor, all of which act in rapid mediation of signal transduction at the synapse. These investigations may provide insight into the general conserved mechanisms used in channel design. More specifically, the aims include utilizing the previously developed expression system (Cascio et al., 1993) to produce sufficient quantities of recombinant protein and the amino-terminal domain (NGly) for reconstitution, gross characterization, and subsequent analyses. Crosslinking studies and ultrafiltration studies coupled with gel filtration will be conducted in order to determine the aggregation state of purified recombinantly produced alpha1 GlyR and NGly proteins. The effects of phosphorylation and glycosylation on activity in reconstituted liposomes and single channel measurements in black lipid membranes will be determined. Circular dichroism (CD) spectroscopic studies of the amino-terminal (NGly) domain or on the membrane-bound domain remaining after proteolysis will provide information not only on the folding of this domain, but, by comparison with studies of reconstituted GlyR, will allow assignation of the secondary structure of the remaining portion of the molecule and provide the first quantitation of GlyR secondary structure and a template for subsequent modeling. In addition, the sensitivity of CD to small changes in secondary structure will allow determinations of the effects of ligand binding on protein secondary structure. Site-directed mutagenesis coupled with covalent modification will be used to probe channel topology. In addition, the construction of chimeric channel proteins will allow structure- function maps to be constructed for this ion channel.