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We describe the identification of a conopeptide sequence in venom duct mRNA from Conus victoriae that suppresses a vascular response to pain in the rat. PCR-RACE was used to screen venom duct cDNAs for those transcripts that encode specific antagonists of vertebrate neuronal nicotinic acetylcholine receptors (nAChRs). One of these peptides, Vc1.1, was active as an antagonist of neuronal nAChRs in receptor binding and functional studies in bovine chromaffin cells. It also suppressed the vascular responses to unmyelinated sensory nerve C-fiber activation in rats. Such vascular responses are involved in pain transmission. Furthermore, its ability to suppress C-fiber function was greater than that of MVIIA, an omega-conotoxin with known analgesic activity in rats and humans. Vc1.1 has a high degree of sequence similarity to the alpha-conotoxin family of peptides and has the 4,7 loop structure characteristic of the subfamily of peptides that act on neuronal-type nAChRs. The results suggest that neuronal alpha-conotoxins should be further investigated with respect to their potential to suppress pain.

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A combination of cDNA cloning and detailed mass spectrometric analyses was employed to identify novel conotoxins from Conus victoriae. Eleven conotoxin sequences were determined using molecular methods: one belonging to the A superfamily (Vc1.1), six belonging to the O superfamily (Vc6.1-Vc6.6) and four members of the T superfamily (Vc5.1-Vc5.4). In order to verify the sequences and identify the post-translational modifications (excluding the disulfide connectivity) of three Conus victoriae conotoxins, vc1a, vc5a and vc6a, deduced from sequences Vc1.1, Vc5.1, and Vc6.1, respectively, liquid chromatography/electrospray ionization ion trap mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nanospray ionization ion trap mass spectrometry with collisionally induced dissociation were performed on reduced and alkylated venom fractions. We report that vc1a, the native form of alpha-conotoxin Vc1.1 (an unmodified 16 amino acid residue peptide that has notable pain-relieving capabilities), includes a hydroxyproline and a gamma-carboxyglutamate residue. Conotoxin vc5a is a 10-residue peptide with two disulfide bonds and a hydroxyproline and vc6a is a 25 amino acid peptide with three disulfide bonds.