α-conotoxin BuIA, a novel peptide from Conus bullatus, distinguishes among neuronal nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels. alpha Subunits, together with beta2 and/or beta4 subunits, form ligand-binding sites at alpha/beta subunit interfaces. Predatory marine snails of the genus Conus are a rich source of nAChR-targeted peptides. Using conserved features of the alpha-conotoxin signal sequence and 3'-untranslated sequence region, we have cloned a novel gene from the fish-eating snail, Conus bullatus; the gene codes for a previously unreported alpha-conotoxin with unusual 4/4 spacing of amino acids in the two disulfide loops. Chemical synthesis of the predicted mature toxin was performed. The resulting peptide, alpha-conotoxin BuIA, was tested on cloned nAChRs expressed in Xenopus oocytes. The peptide potently blocks numerous rat nAChR subtypes, with highest potency for alpha3- and chimeric alpha6-containing nAChRs; BuIA blocks alpha6/alpha3beta2 nAChRs with a 40,000-fold lower IC50 than alpha4beta2 nAChRs. The kinetics of toxin unblock are dependent on the beta subunit. nAChRs with a beta4 subunit have very slow off-times, compared with the corresponding beta2 subunit-containing nAChR. In each instance, rat alphaxbeta4 may be distinguished from rat alphaxbeta2 by the large difference in time to recover from toxin block. Similar results are obtained when comparing mouse alpha3beta2 to mouse alpha3beta4, and human alpha3beta2 to human alpha3beta4, indicating that the beta subunit dependence extends across species. Thus, alpha-conotoxin BuIA also represents a novel probe for distinguishing between beta2- and beta4-containing nAChRs.