Neuronal nicotinic acetylcholine receptors (nAChRs) both mediate direct cholinergic synaptic transmission and modulate synaptic transmission by other neurotransmitters. Novel ligands are needed as probes to discriminate among structurally related nAChR subtypes. ␣-Conotoxin MII, a selective ligand that discriminates among a variety of nAChR subtypes, fails to discriminate well between some subtypes containing the closely related ␣3 and ␣6 subunits. Structure-function analysis of ␣-conotoxin MII was performed in an attempt to generate analogs with preference for ␣6-containing [␣6* (asterisks indicate the possible presence of additional subunits)] nAChRs. Alanine substitution resulted in several analogs with decreased activity at ␣3* versus ␣6* nAChRs heterologously expressed in Xenopus laevis oocytes. From the initial analogs, a series of mutations with two alanine substitutions was synthesized. 125 I]␣-conotoxin MII binding to putative ␣6␤2* nAChRs in mouse brain homogenates (K i ϭ 3.3 nM). Thus, structure-function analysis of ␣-conotoxin MII enabled the creation of novel selective antagonists for discriminating among nAChRs containing ␣3 and ␣6 subunits.nAChRs activated by the endogenous neurotransmitter acetylcholine belong to the superfamily of ligand-gated ion channels that also includes GABA A , 5-hydroxytryptamine-3, and glycine receptors (Changeux, 1993). These different ligand-gated ion channels show considerable sequence and structural homology. Each of the subunits has a relatively hydrophilic amino terminal half (ϳ200 amino acids) that constitutes an extracellular domain. This is followed by three hydrophobic transmembrane domains, a large intracellular loop, and then a fourth hydrophobic transmembrane span.A large number of genes have been cloned that encode subunits of nAChRs. It has been proposed that these subunits may be divided into subfamilies on the basis of both gene structure and mature protein sequence. The subunits ␣2, ␣3, ␣4, and ␣6 belong to subfamily III, tribe 1; ␤2 and ␤4 belong to tribe III-2; and the putative structural subunits ␣5 and ␤3 belong to tribe III-3 (Corringer et al., 2000). Within tribe III-1, subunits ␣3 and ␣6 show considerable sequence identity (ϳ80% in the ligand-binding extracellular domain). Thus, designing ligands to distinguish between ␣3* 1 and ␣6* is particularly challenging.␣-Conotoxin MII is a 16 amino acid peptide originally isolated from the venom of the marine snail Conus magus. This peptide potently targets neuronal in preference to the muscle subtype of nicotinic receptor with high affinity for both ␣3␤2 and ␣6* nAChRs. Unfortunately, ␣-conotoxin MII may not distinguish well between ␣3* and ␣6* nAChRs (Kuryatov et al., 2000). In an effort to remedy this situation and produce a selective ligand for ␣6* nAChRs, we have generated a series of ␣-conotoxin MII analogs.The ␣6 subunit is expressed in catecholaminergic neurons and in retina (Le Novère et al., 1996Vailati et al., 1999). In striatum, ␣6* nAChRs seem to play a central role in the modulation ...

Until now, there have been no antagonists to discriminate between heteromeric nicotinic acetylcholine receptors (nAChRs) containing the very closely related alpha6 and alpha3 subunits. nAChRs containing alpha3, alpha4, or alpha6 subunits in combination with beta2, occasionally beta4, and sometimes beta3 or alpha5 subunits, are thought to play important roles in cognitive function, pain perception, and the reinforcing properties of nicotine. We cloned a novel gene from the predatory marine snail Conus purpurascens. The predicted peptide, alpha-conotoxin PIA, potently blocks the chimeric alpha6/alpha3beta2beta3 subunit combination as expressed in oocytes but neither the muscle nor the major neuronal nAChR alpha4beta2. Additionally, this toxin is the first described ligand to discriminate between nAChRs containing alpha6 and alpha3 subunits. Exploiting the unusual intron conservation of conotoxin genes may represent a more general approach for defining conotoxin ligand scaffolds to discriminate among closely related receptor populations.