Characterization of the Ca2+-gated and voltage-dependent K+-channel Slo-1 of nematodes and its interaction with emodepside.
Characterization of the Ca2+-gated and voltage-dependent K+-channel Slo-1 of nematodes and its interaction with emodepside.
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DOI：
10.1371/journal.pntd.0003401
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发表时间：
2014-12
影响因子：
3.8
通讯作者：
Krücken J
中科院分区：
文献类型：
--
作者：
Kulke D;von Samson-Himmelstjerna G;Miltsch SM;Wolstenholme AJ;Jex AR;Gasser RB;Ballesteros C;Geary TG;Keiser J;Townson S;Harder A;Krücken J
环辛缩酚酸肽emodepside及其母体化合物PF 1022 A是广谱杀线虫药物，能够消除对其他驱虫剂耐药的线虫。环辛缩酚酸肽的作用模式仅部分了解，但涉及latrophilin Lat-1受体和电压和钙激活钾通道Slo-1。遗传学证据表明，emodepside发挥其驱虫活性主要通过Slo-1。事实上，E111 -1缺陷型秀丽隐杆线虫菌株是完全对依莫地昔酯具有抗性的。然而，还没有报道过依莫地昔对Slo-1的直接作用，并且这些通道仅针对C.线虫和相关圆线虫。分子和生物信息学分析鉴定了猪蛔虫、马副蛔虫、犬弓蛔虫、犬恶丝虫、马来丝虫、口尾丝虫和类圆线虫的全长Slo-1 cDNA。两个旁系同源物被确定在绦虫鞭虫，猪鞭虫和旋毛虫。在鞭虫属中鉴定了几种编码截短通道的剪接变体。Slo-1通道形成一个单系群，表明Enoplea和Chromadorea分化后发生了重复。为了探讨一种具有代表性的蛋白质C. elegans Slo-1a在非洲爪蟾卵母细胞中表达，并在电生理学（电压钳）实验中进行研究。卵母细胞与1-10 μM emodepside孵育，在实验性增加的细胞内Ca 2+不存在的情况下，在宽范围的阶跃电位上引起电流显著增加，表明emodepside直接打开C。秀丽隐杆线虫Slo-1a.依莫地塞洗脱没有逆转这种作用，Slo-1抑制剂verruculogen仅在依莫地塞之前应用时有效，而在依莫地塞之后无效。在一些寄生线虫的几个剪接变异体和旁系同源物的鉴定表明，有很大的差异，物种之间的通道特性。最重要的是，这项研究首次表明emodepside直接打开了Slo-1通道，显著提高了对这类药物作用方式的理解。苯缩酚酸是作为猫和狗的不同驱虫剂的成分引入市场的驱虫剂，并且是已商业化的环八缩酚酸肽类的唯一成员。电压门控和钙激活钾通道Slo-1与依莫地昔的作用方式有关，但依莫地昔直接诱导Slo-1通道性质变化的证据尚未报道。依地普酯对广谱寄生线虫有活性，但推定的靶标Slo-1仅被描述用于模式线虫秀丽隐杆线虫和相对密切相关的圆线虫。在这里，SLO-1渠道的蛔虫，丝虫和蛔虫进行了描述。出乎意料的是，在后一组中，两个不同的Slo-1通道在其基因组中编码。梭elegans Slo-1a通道在非洲爪蟾卵母细胞中表达。在膜去极化后，没有实验增加细胞内Ca 2+，没有观察到Slo-1特异性电流。与此相反，在卵母细胞与依莫得甙预孵育后观察到非常大的Slo-1特异性电流，表明该药物打开了C。elegans Slo-1a通道不可逆。这是第一份报告表明直接相互作用的cyclocatepsipeptide与SLO-1通道，这大大提高了我们的理解这种药物的作用模式。
The cyclooctadepsipeptide emodepside and its parent compound PF1022A are broad-spectrum nematicidal drugs which are able to eliminate nematodes resistant to other anthelmintics. The mode of action of cyclooctadepsipeptides is only partially understood, but involves the latrophilin Lat-1 receptor and the voltage- and calcium-activated potassium channel Slo-1. Genetic evidence suggests that emodepside exerts its anthelmintic activity predominantly through Slo-1. Indeed, slo-1 deficient Caenorhabditis elegans strains are completely emodepside resistant. However, direct effects of emodepside on Slo-1 have not been reported and these channels have only been characterized for C. elegans and related Strongylida. Molecular and bioinformatic analyses identified full-length Slo-1 cDNAs of Ascaris suum, Parascaris equorum, Toxocara canis, Dirofilaria immitis, Brugia malayi, Onchocerca gutturosa and Strongyloides ratti. Two paralogs were identified in the trichocephalids Trichuris muris, Trichuris suis and Trichinella spiralis. Several splice variants encoding truncated channels were identified in Trichuris spp. Slo-1 channels of trichocephalids form a monophyletic group, showing that duplication occurred after the divergence of Enoplea and Chromadorea. To explore the function of a representative protein, C. elegans Slo-1a was expressed in Xenopus laevis oocytes and studied in electrophysiological (voltage-clamp) experiments. Incubation of oocytes with 1-10 µM emodepside caused significantly increased currents over a wide range of step potentials in the absence of experimentally increased intracellular Ca2+, suggesting that emodepside directly opens C. elegans Slo-1a. Emodepside wash-out did not reverse the effect and the Slo-1 inhibitor verruculogen was only effective when applied before, but not after, emodepside. The identification of several splice variants and paralogs in some parasitic nematodes suggests that there are substantial differences in channel properties among species. Most importantly, this study showed for the first time that emodepside directly opens a Slo-1 channel, significantly improving the understanding of the mode of action of this drug class. Emodepside is an anthelmintic introduced into the market as an ingredient of different dewormers for cats and dogs, and is the only member of the cyclooctadepsipeptide class which has been commercialized. The voltage-gated and calcium-activated potassium channel Slo-1 has been implicated in the mode of action of emodepside, but evidence for direct emodepside-induced changes on the properties of Slo-1 channels has not been reported. Emodepside is active against a broad spectrum of parasitic nematodes, but the putative target Slo-1 has only been described for the model nematode Caenorhabditis elegans, and for relatively closely related strongylids. Here, Slo-1 channels of ascarids, filariae and trichocephalids are described. Unexpectedly, in the latter group two distinct Slo-1 channels are encoded in their genomes. The C. elegans Slo-1a channel was expressed in Xenopus laevis oocytes. After membrane depolarization without experimentally increasing intracellular Ca2+, no Slo-1 specific currents were observed. In contrast, very large Slo-1-specific currents were observed after preincubation of oocytes with emodepside, suggesting that the drug opens the C. elegans Slo-1a channel irreversibly. This is the first report demonstrating direct interaction of a cyclooctadepsipeptide with a Slo-1 channel, which substantially enhances our understanding of the mode of action of this drug class.
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