The avermectins (ivermectin, doramectin etc) and milbemycins are effective anthelmintics used widely in animal and human medicine for the past twenty years. The actual site of action of the avermectins on the GluCl is unclear, but binding studies have concluded that it does not share a binding site with glutamate. The GluCl channels have been well characterised in Caenorhabditis elegans and are beginning to be characterised in parasitic nematode species such as Haemonchus contortus, Dirofilaria immitis and Cooperia oncophora. The aim of this project was to characterise the H. contortus GluClα3B subunit and its interactions with agonists, glutamate and ivermectin using electrophysiology to study Xenopus oocytes expressing GluClα3B homomeric channels and ligand binding studies on COS-7 cells expressing the subunits. Site–directed mutagenesis was used to introduce resistance associated candidate polymorphisms into the H. contortus GluClα3B subunit. The effects of these changes on the response to glutamate and ivermectin were assessed. One mutation found in IVR C. oncophora, L256F, confers a 3-fold loss of sensitivity to glutamate and a 6.5 fold loss of sensitivity to IVM. This mutation is found in the C-terminal area of the extracellular region of the channel and, from homology modelling, we know it lies in close proximity and possibly interferes with another candidate mutation V235A, and the Cysteine residue at position 192 which forms one side of the structurally significant disulphide bridge. Further introduction of different mutations at this position showed the larger the substituted amino acid, the greater the effect on IVM sensitivity. Another amino acid substitution (T300S) results in the prohibition of a functional channel. The protein is produced and is able to bind IVM with high affinity but does not create a functional channel. These data show that polymorphisms found in field isolates of parasites can have a significant effect on GluCl channels and may contribute to drug resistance.

Les récepteurs pentamériques canaux (pLGICs) sont des récepteurs neuronaux impliqués dans la neurotransmission rapide et qui comprennent les récepteurs suivants : nAchR, GABAR, GlyR or 5HT3R. Lorsqu’ils ne fonctionnent pas correctement ils sont impliqués dans des pathologies comme Alzheimer ou Parkinson. Dans cette étude, nous avons réalisé des simulations de dynamique moléculaire d’un homologue procaryote des pLGICs. Grâce à l’analyse de 2.5 us de simulation nous avons pu capturer la fermeture complète dudit récepteur et décrire un mécanisme de gating. Ce mécanisme en deux étapes, 1) twisting puis 2) blooming semble compatible avec tous les pLGICs. Dans un second temps, nous avons utilisé notre connaissance du mécanisme de gating afin de faire des calculs d’énergie libre le long du twisting, pour différents complexes protéine/ligands. De cette façon, nous avons pu discriminer entre des ligands actifs et inactifs et ainsi fournir des pistes pour le design de nouveaux traitements. / Pentameric ligand gated ion channels (pLGICs) are brain receptors involved in fast neurotransmission and include nAchR, GABAR, GlyR or 5HT3R. When dysfunctioning, they are involved in diseases such as Alzheimer’s and Parkinson’s. In this study we have performed molecular dynamic simulations of an eukaryotic homologue of the pLGICs (GluCl) to understand the gating mechanism of pLGICs. Thanks to the analysis of two 2.5 us long simulations in which we could capture the full closing of the receptor we described in great details a gating mechanism in two steps, first twisting then blooming, that we believe applicable to the whole pLGICs family. In a second time we used our description of the gating mechanism to perform free energy calculations along the twisting reaction coordinate, for various ligands in complex with GluCl. Doing so we could show a significant difference between IVM-bound and non-bound states and provide hints for the design of new treatments.