Expression of recombinant α-toxin BmKM9 from scorpion Buthus martensii Karsch and its functional characterization on sodium channels
Expression of recombinant α-toxin BmKM9 from scorpion Buthus martensii Karsch and its functional characterization on sodium channels
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蝎子重组α毒素BmKM9的表达及其钠通道功能表征
DOI：
10.1016/j.peptides.2017.09.017
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发表时间：
2018-01-01
期刊：
影响因子：
3
通讯作者：
Wu, Yingliang
中科院分区：
文献类型：
--
作者：
Yang, Fan;Liu, Shuang;Wu, Yingliang
蝎毒素是研究离子通道的宝贵药理学工具，也是治疗离子通道病的潜在药物。蝎毒中具有四个二硫键的长链毒素通过作用于钠通道而表现出不同寻常的生物活性或生物毒性。然而，大多数毒素的功能特性仍然不清楚，因为它们在粗毒液中的含量很小，并且它们的生产具有挑战性，通过化学和基因工程技术。在这里，我们表达了一种长链α-毒素，BmKM 9，发现在蝎Karsch的毒液，并表征其对钠通道的药理学特性。与以前的毒素生产不同，重组BmKM 9（rBmKM 9）没有额外的氨基酸残基，如His标签和凝血酶切割位点。重折叠毒素可抑制rNa（v）1.4、hNa（v）1.5和hNa（v）1.7钠通道的失活。在这些通道上进一步进行了剂量反应实验。rNa（v）1.4、hNa（v）1.5和hNa（v）1.7的EC 50计算值分别为131.7 +/- 6.6 nM、454.2 +/- 50.1 nM和30.9 +/- 10.3 μ M。通道激活实验表明，rBmKM 9毒素可使rNa（v）1.4和hNa（v）1.5通道激活曲线向负方向移动，呈现典型的β毒素特征。然而，rBmKM 9毒素对钠通道的激活特性不同，它对rNa（v）1.4和hNa（v）1.5通道的失活移位能力不同。稳态失活的V-1/2值发生变化，rNa（v）1.4的V-1/2值变得更加正值，hNa（v）1.5的V-1/2值变得更加负值。此外，hNa（v）1.5通道从失活中的恢复比rNa（v）1.4通道暴露于rBmKM 9后的恢复更显著延迟。总之，这些发现强调了rBmKM 9毒素具有α-和β-毒素的药理学特性，这将增加对蝎毒素经典分类的挑战。此外，钠通道的表达方法和功能信息将促进毒素的潜在应用，并有助于进一步的通道结构和功能的研究。
Scorpion toxins are invaluable pharmacological tools for studying ion channels and potential drugs for channelopathies. The long-chain toxins from scorpion venom with four disulfide bridges exhibit their unusual bioactivity or biotoxicity by acting on the sodium channels. However, the functional properties of most toxins are still unclear due to their tiny amounts in crude venom and their challenging production by chemical and gene engineering techniques. Here, we expressed one of the long-chain a-toxins, BmKM9, found in the venom of the scorpion Buthus martensii Karsch and characterized its pharmacological properties on sodium channels. Unlike previous toxin production, the recombinant BmKM9 (rBmKM9) possessed no additional amino acid residues such as the His-tag and thrombin cleavage site. The refolded toxin could inhibit the inactivation of rNa(v)1.4, hNa(v)1.5 and hNa(v)1.7 sodium channels. Dose-response experiments were further conducted on these channels. The calculated EC50 values were 131.7 +/- 6.6 nM for rNa(v)1.4, 454.2 +/- 50.1 nM for hNa(v)1.5 and 30.9 +/- 10.3 mu M for hNa(v)1.7. The channel activation experiments indicated that the rBmKM9 toxin could shift the activation curves of rNa(v)1.4 and hNa(v)1.5 channels toward a more negative direction and present the typical features of a beta-toxin. However, instead of the same activation property on sodium channels, the rBmKM9 toxin could result in different inactivation shift capabilities on rNa(v)1.4 and hNa(v)1.5 channels. The V-1/2 values of the steady-state inactivation were altered to be more positive for rNa(v)1.4 and more negative for hNa(v)1.5. Moreover, the recovery of the hNa(v)1.5 channel from inactivation was more significantly delayed than that of the rNa(v)1.4 channel by exposure to rBmKM9. Together, these findings highlighted that the rBmKM9 toxin presents the pharmacological properties of both alpha- and beta-toxins, which would increase the challenge to the classical classification of scorpion toxins. Furthermore, the expression method and functional information on sodium channels would promote the potential application of toxins and contribute to further channel structural and functional studies.