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 ...

We identified a previously unidentified conotoxin gene from Conus generalis whose precursor signal sequence has high similarity to the O1-gene conotoxin superfamily. The predicted mature peptide, αO-conotoxin GeXIVA (GeXIVA), has four Cys residues, and its three disulfide isomers were synthesized. Previously pharmacologically characterized O1-superfamily peptides, exemplified by the US Food and Drug Administration-approved pain medication, ziconotide, contain six Cys residues and are calcium, sodium, or potassium channel antagonists. However, GeXIVA did not inhibit calcium channels but antagonized nicotinic AChRs (nAChRs), most potently on the α9α10 nAChR subtype (IC50 = 4.6 nM). Toxin blockade was voltage-dependent, and kinetic analysis of toxin dissociation indicated that the binding site of GeXIVA does not overlap with the binding site of the competitive antagonist α-conotoxin RgIA. Surprisingly, the most active disulfide isomer of GeXIVA is the bead isomer, comprising, according to NMR analysis, two well-resolved but uncoupled disulfide-restrained loops. The ribbon isomer is almost as potent but has a more rigid structure built around a short 310-helix. In contrast to most α-conotoxins, the globular isomer is the least potent and has a flexible, multiconformational nature. GeXIVA reduced mechanical hyperalgesia in the rat chronic constriction injury model of neuropathic pain but had no effect on motor performance, warranting its further investigation as a possible therapeutic agent.