Diversely evolved xibalbin variants from remipede venom inhibit potassium channels and activate PKA-II and Erk1/2 signaling.

Background: The identification of novel toxins from overlooked and taxonomically exceptional species bears potential for various pharmacological applications. The remipede Xibalbanus tulumensis, an underwater cave-dwelling crustacean, is the only crustacean for which a venom system has been described. Its venom contains several xibalbin peptides that have an inhibitor cysteine knot (ICK) scaffold.

Results: Our screenings revealed that all tested xibalbin variants particularly inhibit potassium channels. Xib₁ and xib₁₃ with their eight-cysteine domain similar to spider knottins also inhibit voltage-gated sodium channels. No activity was noted on calcium channels. Expanding the functional testing, we demonstrate that xib₁ and xib₁₃ increase PKA-II and Erk1/2 sensitization signaling in nociceptive neurons, which may initiate pain sensitization. Our phylogenetic analysis suggests that xib₁₃ either originates from the common ancestor of pancrustaceans or earlier while xib₁ is more restricted to remipedes. The ten-cysteine scaffolded xib₂ emerged from xib₁, a result that is supported by our phylogenetic and machine learning-based analyses.

Conclusions: Our functional characterization of synthesized variants of xib₁, xib₂, and xib₁₃ elucidates their potential as inhibitors of potassium channels in mammalian systems. The specific interaction of xib₂ with Kv1.6 channels, which are relevant to treating variants of epilepsy, shows potential for further studies. At higher concentrations, xib₁ and xib₁₃ activate the kinases PKA-II and ERK1/2 in mammalian sensory neurons, suggesting pain sensitization and potential applications related to pain research and therapy. While tested insect channels suggest that all probably act as neurotoxins, the biological function of xib₁, xib_2, and xib₁₃ requires further elucidation. A novel finding on their evolutionary origin is the apparent emergence of X. tulumensis-specific xib₂ from xib₁. Our study is an important cornerstone for future studies to untangle the origin and function of these enigmatic proteins as important components of remipede but also other pancrustacean and arthropod venoms.