Cornejo Isabel, Andrini Olga, Niemeyer María Isabel, Marabolí Vanessa, González-Nilo F Danilo, Teulon Jacques, Sepúlveda Francisco V, Cid L Pablo
Centro de Estudios Científicos (CECs), Valdivia, Chile.
UPMC Université Paris 06, UMR_S 1138, Team 3, Paris, France; INSERM, UMR_S 872, Paris, France.
PLoS Pathog. 2014 Sep 25;10(9):e1004402. doi: 10.1371/journal.ppat.1004402. eCollection 2014 Sep.
Parasitic sea lice represent a major sanitary threat to marine salmonid aquaculture, an industry accounting for 7% of world fish production. Caligus rogercresseyi is the principal sea louse species infesting farmed salmon and trout in the southern hemisphere. Most effective control of Caligus has been obtained with macrocyclic lactones (MLs) ivermectin and emamectin. These drugs target glutamate-gated chloride channels (GluCl) and act as irreversible non-competitive agonists causing neuronal inhibition, paralysis and death of the parasite. Here we report the cloning of a full-length CrGluClα receptor from Caligus rogercresseyi. Expression in Xenopus oocytes and electrophysiological assays show that CrGluClα is activated by glutamate and mediates chloride currents blocked by the ligand-gated anion channel inhibitor picrotoxin. Both ivermectin and emamectin activate CrGluClα in the absence of glutamate. The effects are irreversible and occur with an EC(50) value of around 200 nM, being cooperative (n(H) = 2) for ivermectin but not for emamectin. Using the three-dimensional structure of a GluClα from Caenorabditis elegans, the only available for any eukaryotic ligand-gated anion channel, we have constructed a homology model for CrGluClα. Docking and molecular dynamics calculations reveal the way in which ivermectin and emamectin interact with CrGluClα. Both drugs intercalate between transmembrane domains M1 and M3 of neighbouring subunits of a pentameric structure. The structure displays three H-bonds involved in this interaction, but despite similarity in structure only of two these are conserved from the C. elegans crystal binding site. Our data strongly suggest that CrGluClα is an important target for avermectins used in the treatment of sea louse infestation in farmed salmonids and open the way for ascertaining a possible mechanism of increasing resistance to MLs in aquaculture industry. Molecular modeling could help in the design of new, more efficient drugs whilst functional expression of the receptor allows a first stage of testing of their efficacy.
寄生性海虱对海洋鲑科鱼类养殖构成了重大的卫生威胁，该产业占世界鱼类产量的7%。罗氏海盘虫是侵扰南半球养殖鲑鱼和鳟鱼的主要海虱种类。大环内酯类药物伊维菌素和阿维菌素对罗氏海盘虫的控制最为有效。这些药物作用于谷氨酸门控氯离子通道（GluCl），作为不可逆的非竞争性激动剂，导致神经元抑制、麻痹并使寄生虫死亡。在此，我们报告了从罗氏海盘虫中克隆出全长CrGluClα受体。在非洲爪蟾卵母细胞中的表达及电生理分析表明，CrGluClα可被谷氨酸激活，并介导被配体门控阴离子通道抑制剂印防己毒素阻断的氯离子电流。在没有谷氨酸的情况下，伊维菌素和阿维菌素均可激活CrGluClα。这些作用是不可逆的，EC(50)值约为200 nM，伊维菌素呈协同作用（n(H) = 2），而阿维菌素则不然。利用秀丽隐杆线虫的GluClα三维结构（这是唯一可获得的真核生物配体门控阴离子通道结构），我们构建了CrGluClα的同源模型。对接和分子动力学计算揭示了伊维菌素和阿维菌素与CrGluClα相互作用的方式。两种药物都插入到五聚体结构相邻亚基的跨膜结构域M1和M3之间。该结构显示出参与这种相互作用的三个氢键，但尽管结构相似，其中只有两个与秀丽隐杆线虫晶体结合位点保守。我们的数据强烈表明，CrGluClα是用于治疗养殖鲑科鱼类海虱感染的阿维菌素的重要靶点，并为确定水产养殖业中对大环内酯类药物耐药性增加的可能机制开辟了道路。分子建模有助于设计新的、更有效的药物，而受体的功能表达则允许对其疗效进行第一阶段的测试。
