Neuronal nicotinic acetylcholine receptors (nAChRs) with putative alpha3 beta4-subunits have been implicated in the mediation of signaling in various systems, including ganglionic transmission peripherally and nicotine-evoked neurotransmitter release centrally. However, progress in the characterization of these receptors has been hampered by a lack of alpha3 beta4-selective ligands. In this report, we describe the purification and characterization of an alpha3 beta4 nAChR antagonist, alpha-conotoxin AuIB, from the venom of the "court cone," Conus aulicus. We also describe the total chemical synthesis of this and two related peptides that were also isolated from the venom. alpha-Conotoxin AuIB blocks alpha3 beta4 nAChRs expressed in Xenopus oocytes with an IC50 of 0.75 microM, a kon of 1.4 x 10(6) min-1 M-1, a koff of 0.48 min-1, and a Kd of 0.5 microM. Furthermore, alpha-conotoxin AuIB blocks the alpha3 beta4 receptor with >100-fold higher potency than other receptor subunit combinations, including alpha2 beta2, alpha2 beta4, alpha3 beta2, alpha4 beta2, alpha4 beta4, and alpha1 beta1 gamma delta. Thus, AuIB is a novel, selective probe for alpha3 beta4 nAChRs. AuIB (1-5 microM) blocks 20-35% of the nicotine-stimulated norepinephrine release from rat hippocampal synaptosomes, whereas nicotine-evoked dopamine release from striatal synaptosomes is not affected. Conversely, the alpha3 beta2-specific alpha-conotoxin MII (100 nM) blocks 33% of striatal dopamine release but not hippocampal norepinephrine release. This suggests that in the respective systems, alpha3 beta4-containing nAChRs mediate norepinephrine release, whereas alpha3 beta2-containing receptors mediate dopamine release.

Activation of presynaptic nicotinic acetylcholine receptors (nAChRs) can induce the release of neurotransmitters such as dopamine and norepinephrine in the CNS. Accumulating evidence suggests that distinct nAChR subtypes are involved; however, it has been difficult to determine the subunit composition of these receptors, in part because of the lack of a sufficient variety of selective nAChR ligands. We present experimental data that at least two different nAChR complexes are involved in dopamine release, one of which has an ␣3/␤2 subunit interface.The recently discovered peptide ␣-conotoxin MII is a potent and selective inhibitor of rat nAChRs containing an interface formed by ␣3 and ␤2 subunits. We used this peptide to examine nicotine-stimulated release of dopamine from rat striatal synaptosomes and of norepinephrine from hippocampal synaptosomes. MII (100 nM) blocks 34-49% of the nicotinestimulated dopamine release, but not dopamine release evoked by elevated [K ϩ ]. Furthermore, two peptides structurally related to ␣-conotoxin MII, namely ␣-conotoxin MI (selective for ␣1␤1␥␦ nAChRs) and ␣-conotoxin ImI (selective for ␣7-containing nAChRs), have no effect on nicotine-stimulated dopamine release. The results indicate that one third to half of the dopamine release in the striatal preparation is mediated by nAChRs with an ␣3/␤2 subunit interface. In contrast, Յ10% of nicotine-stimulated release of norepinephrine from hippocampal synaptosomes is modulated by nAChRs with ␣3/␤2 subunit interfaces.

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Parkinson's disease, a neurodegenerative movement disorder characterized by selective degeneration of nigrostriatal dopaminergic neurons, affects approximately 1% of the population over 50. Because nicotinic acetylcholine receptors (nAChRs) may represent an important therapeutic target for this disorder, we performed experiments to elucidate the subtypes altered with nigrostriatal damage in parkinsonian monkeys. For this purpose we used (125)I-alpha-conotoxin MII (CtxMII), a relatively new ligand that identifies alpha3 and/or alpha6 subunits containing nAChR subtypes. In brain from untreated monkeys, there was saturable (125)I-alpha-CtxMII binding to a single population of high-affinity nicotinic sites (K(d) = 0.9 nm), primarily localized in the visual, habenula-interpeduncular, and nigrostriatal-mesolimbic pathways. Administration of the selective dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine resulted in damage to the nigrostriatal system and parkinsonism. Autoradiographic analysis showed that (125)I-alpha-CtxMII sites were selectively reduced (>/=99%) in the basal ganglia and that the lesion-induced decreases correlated well with declines in the dopamine transporter, a marker of dopaminergic neuron integrity. These findings may indicate that most or all of (125)I-alpha-CtxMII-labeled nAChR subtypes in the basal ganglia are present on nigrostriatal dopaminergic neurons, in contrast to (125)I-epibatidine sites. These data suggest that the development of ligands directed to nAChR subtypes containing alpha3 and/or alpha6 subunits may yield a novel treatment strategy for parkinsonian patients with nigrostriatal dopaminergic degeneration.

Nicotinic acetylcholine receptors (nAChRs) represent an important modulator of striatal function both under normal conditions and in pathological states such as Parkinson's disease. Because different nAChR subtypes may have unique functions, immunoprecipitation and ligand binding studies were done to identify their subunit composition. As in the rodent, ␣2, ␣4, ␣6, ␤2, and ␤3 nAChR subunit immunoreactivity was identified in monkey striatum. However, distinct from the rodent, the present results also revealed the novel presence of ␣3 nAChR subunit-immunoreactivity in this same region, but not that for ␣5 and ␤4. Relatively high levels of ␣2 and ␣3 subunits were also identified in monkey cortex, in addition to ␣4 and ␤2. Experiments were next done to determine whether striatal subunit expression was changed with nigrostriatal damage. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment decreased ␣6 and ␤3 subunit immunoreactivity by ϳ80% in parallel with the dopamine transporter, suggesting that they are predominantly expressed on nigrostriatal dopaminergic projections. In contrast, ␣3, ␣4, and ␤2 subunit immunoreactivity was decreased ϳ50%, whereas ␣2 was not changed. These data, together with those from dual immunoprecipitation and radioligand binding studies ([ 3 H]cytisine, 125 I-␣-bungarotoxin, and 125 I-␣-conotoxin MII) suggest the following: that ␣6␤2␤3, ␣6␣4␤2␤3, and ␣3␤2* nAChR subtypes are present on dopaminergic terminals and that the ␣4␤2 subtype is localized on both dopaminergic and nondopaminergic neurons, whereas ␣2␤2* and ␣7 receptors are localized on nondopaminergic cells in monkey striatum. Overall, these results suggest that drugs targeting non-␣7 nicotinic receptors may be useful in the treatment of disorders characterized by nigrostriatal dopaminergic damage, such as Parkinson's disease.Parkinson's disease is a neurodegenerative disorder characterized by severe movement disability (Olanow, 2004;Samii et al., 2004). Although the underlying cause seems to be a loss of nigrostriatal dopaminergic neurons, other neurotransmitter systems are also affected. This includes the cholinergic system, in which declines have been observed in several cholinergic measures, including nicotinic acetylcholine receptors (nAChRs ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; A85380, 3-(2(S)-azetidinylmethoxy)pyridine dihydrochloride; RTI-121, 3␤-(4-iodophenyl)tropane-2␤-carboxylic acid isopropyl ester; BSA, bovine serum albumin; CI, confidence interval; *, nicotinic receptors containing the indicated ␣ and/or ␤ subunit and also additional undefined subunits.