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. Xib<inf>1</inf> and xib<inf>13</inf> 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 xib<inf>1</inf> and xib<inf>13</inf> increase PKA-II and Erk1/2 sensitization signaling in nociceptive neurons, which may initiate pain sensitization. Our phylogenetic analysis suggests that xib<inf>13</inf> either originates from the common ancestor of pancrustaceans or earlier while xib<inf>1</inf> is more restricted to remipedes. The ten-cysteine scaffolded xib<inf>2</inf> emerged from xib<inf>1</inf>, a result that is supported by our phylogenetic and machine learning-based analyses. Conclusions: Our functional characterization of synthesized variants of xib<inf>1</inf>, xib<inf>2</inf>, and xib<inf>13</inf> elucidates their potential as inhibitors of potassium channels in mammalian systems. The specific interaction of xib<inf>2</inf> with Kv1.6 channels, which are relevant to treating variants of epilepsy, shows potential for further studies. At higher concentrations, xib<inf>1</inf> and xib<inf>13</inf> 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 xib<inf>1</inf>, xib<inf>2,</inf> and xib<inf>13</inf> requires further elucidation. A novel finding on their evolutionary origin is the apparent emergence of X. tulumensis-specific xib<inf>2</inf> from xib<inf>1</inf>. 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.