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

Abstract

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels. α Subunits, together with β2 and/or β4 subunits, form ligand-binding sites at α/β subunit interfaces. Predatory marine snails of the genus Conus are a rich soarce of nAChR-targeted peptides. Using conserved features of the α-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 α-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, α-conotoxin BuIA, was tested on cloned nAChRs expressed in Xenopus oocytes. The peptide potently blocks numerous rat nAChR subtypes, with highest potency for α3- and chimeric α6-containing nAChRs; BuIA blocks α6/α3β2 nAChRs with a 40,000-fold lower IC50 than α4β2 nAChRs. The kinetics of toxin unblock are dependent on the β subunit. nAChRs with a β4 subunit have very slow off-times, compared with the corresponding β2 subunit-containing nAChR. In each instance, rat αxβ4 may be distinguished from rat αxβ2 by the large difference in time to recover front toxin block. Similar results are obtained when comparing mouse α3β2 to mouse α3β4, and human α3β2 to human α3β4, indicating that the β subunit dependence extends across species. Thus, α-conotoxin BuIA also represents a novel probe for distinguishing between β2- and β4-containing nAChRs.