Nicotine exerts many of its behavioral effects through a diverse family of nicotinic acetylcholine receptors. In particular, nicotine evoked dopamine release is thought to play an important role in the establishment and maintenance of nicotine dependence. alpha-Conotoxin MII is a recently-discovered toxin, isolated from the predatory cone snail Conus magus, which blocks alpha3-beta2subtype nicotinic receptors with high potency and selectivity. Alpha-Conotoxin MII also selectively and potently blocks measures of nicotine evoked dopamine release in vitro, and may thus be a tool with which to explore a behaviorally significant nicotinic receptor subtype. Both native and radiolabeled ([125I]-alphaCtx MII) alpha-conotoxin MII are available and will be used to characterize a-conotoxin MII bindings sites. We intend to test the native toxin against [3H]epibatidine binding (at alpha3beta2-transfected cell and mouse brain preparations) and measures of nicotinic activation in vitro. [125]alphaCtx MII binding will be used to characterize both transfected cell and centrally expressed receptors, using biochemical and autoradiographic techniques. These studies are intended to define the numbers, distribution and function of alpha-conotoxin MII sites in the mammalian (mouse) CNS. Comparison of the sites expressed in the cell line with those in brain will determine how similar the native and model sites are. In addition, we will attempt to develop a further set of probes based on alpha-conotoxin MII incorporating photoactivatable and biotinyl tags into the toxin's primary peptide sequence. These probes are designed to allow the exploration of alpha-conotoxin MII bindings sites' composition and structure. The proposed studies will provide insights into the nature and importance of a novel, native nicotinic receptor. Hopefully, this knowledge will lead to a better understanding of the basis (of) nicotine's centrally mediated effects, and may lead to the development of improved nicotinic drugs and/or treatments for nicotine dependence.
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