Alpha-conotoxin MII: A new tool for the study of neuronal nicotinic acetylcholine receptors

The goal of this research is to understand the molecular interactions that determine the affinity and specificity of antagonists of neuronal nicotinic acetylcholine receptors (nAChRs). Neuronal nAChRs are important for several reasons. They have been implicated in several neurological disorders, and they are the site of action of nicotine and mediate its psychoactive and addictive effects. Thus, these receptors are pharmacological targets for potential therapeutic agents.; However, there is one major complication in the development of pharmacological agents targeted toward nAChRs. nAChRs are a large family of very closely related multi-subunit receptors. To date, there have been 12 subunits cloned from brain. These subunits combine to form pentameric receptors resulting in an enormous number of potential subunit combinations. Development of subtype-specific ligands will be critical for the discovery of agents capable of potentially treating these diseases.; We have isolated and characterized a set of small disulfide-rich peptide neurotoxins, named α-conotoxins, that specifically target subtypes of neuronal nAChRs. This work describes the isolation and characterization of α-conotoxin MII (MII). MII competitively inhibits α3β2 nAChRs expressed in Xenopus oocytes with high potency (0.5 nM IC₅₀) and high specificity (>200-fold selectivity). The kinetics of blockade by MII shows that there are two binding sites for the toxin on α3β2 and α3β4 nAChRs and that binding of MII to either site is sufficient for inhibition of receptor function. Further, the kinetics of blockade of these two receptor subtypes suggests a unique model of receptor-discrimination by MII. This model, the dock and lock model, invokes rapid association of MII to a docking site on the β subunit, followed by a shift of toxin to a locking site on the a subunit that results in slow dissociation from the receptor. Structure/activity studies with α-conotoxin MII identified three structural residues (N5, P6 and H12) and three residues (G1, H9 L15) that interact with α3β2 nAChRs. Finally, a series of analogs of MII were synthesized, each containing the photoactivatable amino acid, p-benzoylphenylalanine (BPA). Characterization of these analogs has identified two analogs (N14BPA and 18BPA) that are candidates for use as reagents for cross-linking native receptors.