Functional investigation of conserved glutamate receptor subunits reveals a new mode of action of macrocyclic lactones in nematodes

Abstract

Glutamate-gated chloride channels receptors (GluCls) are involved in the inhibition of neurotransmission in invertebrates and represent major molecular targets for therapeutic drugs. Among these drugs, macrocyclic lactones (MLs) are widely used as anthelmintic to treat parasitic nematodes impacting both human and animal health. Despite massive use of MLs since the 80s, the exact molecular targets of these drugs are still unknown in many important parasite species. Among the GluCl subunit encoding genes, avr-14, glc-2, glc-3 and glc-4 are highly conserved throughout the nematode phylum. Using the Xenopus oocyte as an expression system, we pharmacologically characterized these GluCl subunits from the model nematode Caenorhabditis elegans, the human filarial nematode Brugia malayi and the horse parasitic nematode Parascaris univalens. In contrast with C. elegans, expression of parasitic nematode subunits as homomeric receptors was not reliable and needed glutamate application at the mM range to induce low currents at the nA range. However, the co-expression of GLC-2 and AVR-14B lead to the robust expression of ML-sensitive receptors for the three nematode species. In addition, we demonstrated that for C. elegans and P. univalens, GLC-2 co-assembled with GLC-3 to form a new GluCl subtype with distinct pharmacological properties. Whereas 1M ivermectin, moxidectin and eprinomectin acted as agonist of the GLC-2/GLC-3 receptor from C. elegans, they did not directly activate GLC-2/GLC-3 of P. univalens. In contrast, these MLs potentialized glutamate elicited currents thus representing a unique pharmacological property. Our results highlight the importance of GLC-2 as a key subunit in the composition of heteromeric channels in nematodes and demonstrate that MLs act on novel GluCl subtypes that show unusual pharmacological properties, providing new insights about MLs mode of action. Author summaryThe filarial and ascarid parasitic nematodes include some of the most pathogenic or invalidating species in humans, livestock and companion animals. Whereas the control of these worms is critically dependent on macrocyclic lactones (MLs) such as ivermectin, the mode of action of this anthelmintic class remains largely unknown in these parasites. In the model nematode Caenorhabditis elegans, MLs target GluCl pentameric glutamate-sensitive chloride channels (GluCl). Because MLs are potent anthelmintics on C. elegans, ascarid and filarial nematodes, in the present study we investigated GluCl subunits highly conserved between these distantly related worms. Using the Xenopus oocyte as a heterologous expression system, we identified and performed the pharmacological characterization of novel GluCl receptors from C. elegans, the human filarial parasite Brugia malayi and the horse parasite Parascaris univalens. Our results highlight heteromeric GluCls from parasites as molecular targets for a wide range of MLs. We report an original mode of action of MLs on a new GluCl subtype made of the GLC-2/GLC-3 subunit combination. This study brings new insights about the diversity of GluCl subtypes in nematodes and opens the way for rational drug screening for the identification of next generation anthelmintic compounds.