Diversely evolved xibalbin variants from remipede venom inhibit 3 potassium channels and activate PKA-II and Erk1/2 signalling

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. Result: 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, xib2, 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.研究背景：从被忽视且分类学上具有特殊性的物种中识别新型毒素，在诸多药理学应用中具备可观潜力。桨足类（remipede）Xibalbanus tulumensis是一种栖息于水下洞穴的甲壳动物，也是目前已知唯一被报道具有毒液系统的甲壳类动物。其毒液中含有多种带有抑制剂半胱氨酸结（inhibitor cysteine knot，ICK）结构域的雷比宾（xibalbin）肽类。 实验结果：我们的筛选实验显示，所有被测的xibalbin变体均能特异性抑制钾离子通道。Xib1与xib13拥有与蜘蛛结蛋白相似的八半胱氨酸结构域，同时还可抑制电压门控钠离子通道。未观察到其对钙离子通道的活性。在扩展功能测试中，我们证实xib1与xib13能够在伤害性感觉神经元中增强PKA-II与Erk1/2的敏化信号通路，这一过程可能引发疼痛敏化。我们的系统发育分析表明，xib13的起源可追溯至泛甲壳类动物的共同祖先或更早时期，而xib1的分布则更局限于桨足类。拥有十半胱氨酸结构域的xib2由xib1演化而来，这一结论得到了系统发育分析与基于机器学习的分析结果的支持。 研究结论：我们对xib1、xib2与xib13的合成变体开展的功能表征研究，阐明了它们在哺乳动物系统中作为钾离子通道抑制剂的潜力。xib2与癫痫亚型治疗相关的Kv1.6通道的特异性相互作用，为后续相关研究提供了潜在方向。在较高浓度下，xib1与xib13可激活哺乳动物感觉神经元内的PKA-II与ERK1/2激酶，提示其具备引发疼痛敏化的作用，在疼痛研究与治疗领域存在应用潜力。尽管针对昆虫通道的测试显示所有变体均可能作为神经毒素，但xib1、xib2与xib13的生物学功能仍需进一步阐明。本研究的一项新发现是，X. tulumensis特异性的xib2显然由xib1演化而来。本研究为未来厘清这些神秘蛋白的起源与功能奠定了重要基础——这些蛋白不仅是桨足类毒液的重要组成部分，同时也是泛甲壳类与节肢动物毒液的关键组分。