Nicotine elicits behavioral effects through a diverse family of nicotinic acetyicholine receptors (nAChR) and nicotine evoked dopamine release is thought to play an important role in the establishment and maintenance of nicotine dependence. a-ConotoxinMll (aCtxMII), a toxin isolated from the predatory cone snail Conus magus, potently and selectively blocks a3b2-nAChR expressed in Xenopus oocytes and partially inhibits nicotine-stimulated dopamine release from rat striatal synaptosomes. During the initial funding period for this grant aCtxMII has been used to identify subsets of nAChRs in mouse brain. It was confirmed that aCtxMII inhibits some, but not all, nicotine-stimulated dopamine release in mouse striatum. As expected, b2 null mutants retained virtually no aCtxMII binding or nicotine-stimulated release. Surprisingly, the b3 null mutation eliminated most high affinity aCtxMII binding and aCtxMII-sensitive dopamine release. Equally surprising was the absence of an effect of the a3 null mutation on aCtxMII binding. Therefore, many native nAChRs that interact with aCtxMII are not the a3b2 subtype. Experiments outlined in the current proposal will use ligand binding and functional analyses to further examine the diversity of nAChR. 1) aCtxMII sensitivity of nAChR mediated dopamine release in striatum, nucleus accumbens, frontal cortex and olfactory tubercles of mice that have been mutated to eliminate the expression of specific nAChR subunits (initially a7, a5, b2, b3 and b4) will be evaluated to obtain information about the molecular composition and functional diversity of these important presynaptic nAChRs. 2) Regulation of nAChR binding and function by chronic nicotine treatment using both wild type and null mutant mice, with emphasis on those mutants that affect nAChRs that interact with aCtxMII, will also be examined. 3) Structural properties of the recently identified aCtxPWl, which differs markedly in structure from aCtxMII, but displays a similar pharmacological profile, will be evaluated to determine if this new toxin will prove to be superior or complementary reagent. The proposed studies will provide further insights into the nature of three novel, native nAChRs sensitive to inhibition by aCtxMII and may lead to better understanding of the basis of the effects of nicotine