Identification and Functional Expression of a Glutamate- and Avermectin-Gated Chloride Channel from Caligus rogercresseyi, a Southern Hemisphere Sea Louse Affecting Farmed Fish

Abstract

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 EC50 value of around 200 nM, being cooperative (nH = 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.

Distinct molecular targets for macrocyclic lactone and isoxazoline insecticides in the human louse: new prospects for the treatment of pediculosis

“…4C-D). As previously shown in the literature, RDL and GluCls are permeable to chloride anions [35,37,[48][49][50][51]. Here, the TM2 amino acid sequences of Phh-RDL and Phh-GluCl are respectively 100% identical with RDL and GluCls from other insect species such as A. mellifera, D. melanogaster, T. castaneum (Fig.…”

“…Here, we report that both Phh-GluCl-1 and Phh-RDL-1 can be robustly expressed in Xenopus oocytes, whereas neither Phh-GluCl-2 nor Phh-RDL-2 subunits formed functional receptors in our conditions. Interestingly, Phh-GluCl had one of the highest affinity for glutamate compared to other GluCls of arthropod species, the reported EC 50 of which varied between 6.89 and 384.2 µM [35,43,[48][49][50][58][59][60][61][62]. Likewise, Phh-RDL had one of the lowest EC 50 for GABA among described insect RDL receptors showing EC 50 varying between 5.43 +/-0.02 µM in the brown planthopper Nilaparvata lugens [63] and 445 +/-47 µM in the carmine spider mite Tetranychus cinnabarinus [61] with the exception of the RDL-2 subunit (2.4 +/-0.3 µM) from the honey bee parasite mite Varroa destructor [51].…”

Emamectin is a non-selective allosteric activator of nicotinic acetylcholine receptors and GABA A/C receptors

“…Emamectin is known to activate invertebrate glutamate gated chloride channels [9]. However, it is unknown whether it can activate cation selective channels.…”

“…Structurally, it is also similar to ivermectin, a widely used anti-parasite agent. Emamectin exerts its action by irreversibly activating chloride channels in insects [9]. However, less is known about its effect on mammalian channels in the pentameric ligand-gated ion channel family.…”

Mutational Analysis at Intersubunit Interfaces of an Anionic Glutamate Receptor Reveals a Key Interaction Important for Channel Gating by Ivermectin

“…Later, additional genes encoding subunits of Glu-gated chloride channels were cloned from C. elegans (Yates et al, 2003) and other invertebrates (Lynagh et al, 2015) like, parasitic worms (Delany et al, 1998; Jagannathan et al, 1999; Dufour et al, 2013; Lynagh et al, 2014), insects (Eguchi et al, 2006; Dong et al, 2013; Furutani et al, 2014; Kita et al, 2014; Meyers et al, 2015; Wu et al, 2017), crustaceans (Cornejo et al, 2014), and mollusk (Kehoe et al, 2009). In several cases, a single subunit was found to form a functional homomeric receptor–channel that can be gated by both Glu and IVM independently.…”