An N‐Terminally Elongated Peptide From <i>Conus rolani</i> Defines a New Class of Ribbon α‐Conotoxins Targeting Muscle <scp>nAChRs</scp>

Conotoxins, peptides found in cone snail venoms, selectively target ion channels and receptors to incapacitate prey. α-Conotoxins are extensively investigated for their potent modulation of nicotinic acetylcholine receptors (nAChRs). This study describes the discovery and characterization of RoIA, a novel α-conotoxin from Conus rolani. RoIA belongs to the α4/4 conotoxin class and features an atypical N-terminal elongation of 18 amino acids. The biological activity of RoIA is assessed through both in vivo and in vitro assays using the three potential folding isoforms (globular, ribbon, and bead) of the full-length and truncated (lacking the N-terminal elongation) analogs. The full-length RoIA analog exhibits delayed but potent paralytic activity when administered to mice and fish; the ribbon isoform shows the highest potency. Notably, only the ribbon isoform of the truncated peptide is active on heterologously expressed muscle nAChRs, suggesting that the N-terminal elongation may be released in vivo or form interactions that are not recapitulated in vitro. This discovery challenges the prevailing understanding that native α-conotoxins adopt a globular conformation and illustrates that Conus can explore novel chemical spaces using an alternative disulfide bond connectivity. This research enhances our knowledge of the complex mechanisms by which toxins manifest their physiological effects.