Identification of a novel class of nicotinic receptor antagonists - Dimeric conotoxins VxXIIA, VxXIIB, and VxXIIC from Conus vexillum

The venoms of predatory marine snails ( Conus spp.) contain diverse mixtures of peptide toxins with high potency and selectivity for a variety of voltage-gated and ligand-gated ion channels. Here we describe the chemical and functional characterization of three novel conotoxins, alpha D-VxXIIA, alpha D-VxXIIB, and alpha D-VxXIIC, purified from the venom of Conus vexillum. Each toxin was observed as an similar to 11-kDa protein by LC/MS, size exclusion chromatography, and SDS-PAGE. After reduction, the peptide sequences were determined by Edman degradation chemistry and tandem MS. Combining the sequence data together with LC/MS and NMR data revealed that in solution these toxins are pseudo-homodimers of paired 47-50-residue peptides. The toxin subunitsexhibited a novel arrangement of 10 conserved cystine residues, and additional post-translational modifications contributed heterogeneity to the proteins. Binding assays and two-electrode voltage clamp analyses showed that alpha D-VxXIIA, alpha D-VxXIIB, and alpha D-VxXIIC are potent inhibitors of nicotinic acetylcholine receptors (nAChRs) with selectivity for alpha 7 and alpha 2 containing neuronal nAChR subtypes. These dimeric conotoxins represent a fifth and highly divergent structural class of conotoxins targeting nAChRs.