Background. The venom of predatory marine cone snails mainly contains a diverse array of unique bioactive peptides commonly referred to as conopeptides

The venom of each Conus species consists of a diverse array of neurophysiologically active peptides, which are mostly unique to the examined species. In this study, we performed high-throughput transcriptome sequencing to extract and analyze putative conotoxin transcripts from the venom ducts of 3 vermivorous cone snails (C. caracteristicus, C. generalis, and C. quercinus), which are resident in offshore waters of the South China Sea. In total, 118, 61, and 48 putative conotoxins (across 22 superfamilies) were identified from the 3 Conus species, respectively; most of them are novel, and some possess new cysteine patterns. Interestingly, a series of 45 unassigned conotoxins presented with a new framework of C-C-C-C-C-C, and their mature regions were sufficiently distinct from any other known conotoxins, most likely representing a new superfamily. O- and M-superfamily conotoxins were the most abundant in transcript number and transcription level, suggesting their critical roles in the venom functions of these vermivorous cone snails. In addition, we identified numerous functional proteins with potential involvement in the biosynthesis, modification, and delivery process of conotoxins, which may shed light on the fundamental mechanisms for the generation of these important conotoxins within the venom duct of cone snails.

μ-Conotoxin GIIIA, a peptide toxin isolated from Conus geographus, preferentially blocks the skeletal muscle sodium channel Na_V1.4. GIIIA folds compactly to a pyramidal structure stabilized by three disulfide bonds. To assess the contributions of individual disulfide bonds of GIIIA to the blockade of Na_V1.4, seven disulfide-deficient analogues were prepared and characterized, each with one, two, or three pairs of disulfide-bonded Cys residues replaced with Ala. The inhibitory potency of the analogues against Na_V1.4 was assayed by whole cell patch-clamp on rNa_V1.4, heterologously expressed in HEK293 cells. The corresponding IC₅₀ values were 0.069 ± 0.005 μM for GIIIA, 2.1 ± 0.3 μM for GIIIA-1, 3.3 ± 0.2 μM for GIIIA-2, and 15.8 ± 0.8 μM for GIIIA-3 (-1, -2 and -3 represent the removal of disulfide bridges Cys3–Cys15, Cys4–Cys20 and Cys10–Cys21, respectively). Other analogues were not active enough for IC₅₀ measurement. Our results indicate that all three disulfide bonds of GIIIA are required to produce effective inhibition of Na_V1.4, and the removal of any one significantly lowers its sodium channel binding affinity. Cys10–Cys21 is the most important for the Na_V1.4 potency.