The long term goal in this project remains the elucidation of the structure and function of nicotinic acetylcholine receptors (nAChRs). Classical approaches to discern functionally significant neuronal nAChR subtypes in the central nervous system (CNS) have been frustrated by the limited number of selective pharmacological agents. Structure-based information will be used in this project to drive the development of new research tools to investigate nAChR subunit-specific functionality in the nervous system. The first aim focuses on the characterization of a "Knock-In" mouse, Chrna3tm1(Hwrt), created through homologous recombination-mediated targeted gene replacement. Targeted DMAencoding five muscle-type a1-derived amino acid substitutions confers functional sensitivity of receptors to nanomolar a-bungarotoxin (Bgtx) even in those cases where only 1 of the 2 receptor a3 subunits is mutant. Heterozygous mice express hybrid nAChRs containing one mutant and one wild-type a3 subunit, but are otherwise normal phenotypically. Consistent with a stochastic expression of hybrid receptors, -2/3 of the nicotinic response in sympathetic neurons from Met mice can be blocked by Bgtx. Biochemical and electrophysiological methods will be used to fully assess the functional consequences of this mutation in heterozygous mice backcrossed into the C57BI/6/J background. The expression of the mutant a3 subunit in the CNS will be investigated by fluorescence, autoradiography, and micro-injection of Bgtx into discrete brain regions rich in a3. In the second aim, Bgtx-sensitive P2, (33,04 and a5 subunits will be prepared and characterized electro- physiologically following heterologous expression in oocytes and in adenovirus-transfected neurons. These results will determine the future feasibility of generating Bgtx-sensitive knock-in mice in these 4 subunits. Relevance: Nicotine is an extremely addictive drug responsible for up to 20% of all preventable mortality in the western world. It also significantly enhances cognitive performance, and some inherited forms of epilepsy involve nicotinic receptors. Loss of cholinergic neurons is implicated in Alzheimer's disease, a disorder with no effective treatment. Understanding the functional role of nicotinic receptors in the CNS therefore has significant potential to benefit human health. In addition, the results from this study could lead to the development of therapeutic drugs reproducing some of the beneficial effects of nicotine.