The results from these studies will help in better understanding of nicotinic regulation of brain catecholaminergic systems, one of which is implicated in addictive properties of nicotine, as well as aid in synthesis of ligands that can discriminate among specific nAChR subtypes. This study will use newly discovered alpha-conotoxins to examine properties of nAChRs mediating catecholaminergic neurotransmission and to analyze alpha-conotoxin/nAChR interactions. Examination of nAChRs mediating nicotinic-regulation of catecholaminergic neurotransmission will be carried out by a neurotransmitter release assay, in which nicotine-stimulated [3H]dopamine and [3H]norepinephrine release from striatal and hippocampal preparations, respectively, will be examined. Another aim of this proposal is to use novel alpha-conotoxins that are 10-400 fold more selective for nAChRs containing the alpha6 vs. alpha3 subunit molecular analysis of alpha-conotoxin/nAChR interaction. This goal will be achieved by, first, construction of chimeras which incorporate segments of the extracellular region of the alpha3 nAChR subunit into alpha6 subunit and expressing the receptors in oocytes and examining their affinity for the alpha-conotoxins. Subsequently, using site-directed mutagenesis, heterologous amino acids in the region conferring selectivity will be exchanged between alpha3 and alpha6 subunits to determine amino acid residues responsible for conferring the high alpha6 affinity.
