Identification of a Novel Class of Nicotinic Receptor Antagonists: DIMERIC CONOTOXINS VxXIIA, VxXIIB, and VxXIIC FROM CONUS VEXILLUM

Marion Loughnan,Annette Nicke,Alun Jones,Christina I Schroeder,Simon T Nevin,David J Adams,Paul F Alewood,Richard J Lewis

doi:10.1074/jbc.m603703200

Marion Loughnan, Annette Nicke + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m603703200

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Abstract

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, alphaD-VxXIIA, alphaD-VxXIIB, and alphaD-VxXIIC, purified from the venom of Conus vexillum. Each toxin was observed as an approximately 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 subunits exhibited 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 alphaD-VxXIIA, alphaD-VxXIIB, and alphaD-VxXIIC are potent inhibitors of nicotinic acetylcholine receptors (nAChRs) with selectivity for alpha7 and beta2 containing neuronal nAChR subtypes. These dimeric conotoxins represent a fifth and highly divergent structural class of conotoxins targeting nAChRs.

Highlights

Nicotinic acetylcholine receptors4 belong to the Cys-loop superfamily of pentameric ligand-gated ion channels
This study describes the discovery and characterization of three novel post-translationally modified conotoxins, VxXIIA, VxXIIB, and VxXIIC, which occur as dimers and produce a slowly reversing block of ␣7 and ␣3␤2 nicotinic acetylcholine receptors (nAChRs)
Of intact and reduced material revealed that these proteins in C. mustelinus, C. miles, C. capitaneus, and C. vexillum occurred as dimers of two peptide subunits

Summary

Identification of a Novel Class of Nicotinic Receptor Antagonists

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. Binding assays and two-electrode voltage clamp analyses showed that ␣D-VxXIIA, ␣D-VxXIIB, and ␣D-VxXIIC are potent inhibitors of nicotinic acetylcholine receptors (nAChRs) with selectivity for ␣7 and ␤2 containing neuronal nAChR subtypes These dimeric conotoxins represent a fifth and highly divergent structural class of conotoxins targeting nAChRs. Nicotinic acetylcholine receptors (nAChRs) belong to the Cys-loop superfamily of pentameric ligand-gated ion channels. Conotoxins are small disulfide-rich peptide toxins found in the venom of predatory marine snails from the genus Conus. These venom peptides generally target a variety of voltage-gated and ligand-gated ion channels [6, 7]. These ␣D-conotoxins contribute to our growing knowledge of ligands interacting at nAChRs

EXPERIMENTAL PROCEDURES

RESULTS

Sequence and cystine framework

DISCUSSION