Neuroactive mini-M conotoxins in Conus tessulatus

Two small peptides were purified from the venom of the vermivorous cone snail, Conus tessulatus, and evaluated for neuroactivity. Structural characterization by Edman sequencing and mass spectrometry revealed that the two peptides, named ts3b and ts3c, are 14 amino acids long and can be classified as mini-M conotoxins under framework III, M-superfamily, M2 branch. Native samples of ts3b and ts3c caused temporary swimming paralysis in fish, yet elicited distinct responses in the dorsal root ganglion (DRG) assay and the Constellation Pharmacology platform. Peptide ts3b caused mild sluggishness and shaking in young mice and irreversible, indirect excitation in a primary culture of DRG neurons. Furthermore, nanomolar concentration of native ts3b affected small diameter peptidergic nociceptors (PNs) and c-low threshold mechanoreceptors (C-LTMRs) in the DRG. Interestingly, ts3c caused an irreversible block in cultured DRG neurons, primarily affecting capsaicin-sensitive PN cells and capsaicin-insensitive non-peptidergic nociceptors (NPNs). The bioactivities suggest that ts3b and ts3c may target distinct ion channels or receptors. Two ts3b toxiforms were also identified as ts3b Asn15 toxiform and ts3b Ser7Arg, Asn15 toxiform. The toxiforms have a similar loop pattern, post-translational modifications, and cysteine framework with ts3b. Native ts3b and the toxiforms caused mild lethargy in mice. Peptide ts3b and ts3c are the first neuroactive mini-M conotoxins described from C. tessulatus. Few neuroactive conotoxins have been reported under the M-2 branch, and these compounds may have novel molecular and pharmacological targets due to their diverse structural conformations.