Investigation of Acetylcholine Receptor Diversity in a Nematode Parasite Leads to Characterization of Tribendimidine-and Derquantel-Sensitive nAChRs

Nicotinic acetylcholine receptors (nAChRs) of parasitic nematodes are required for body movement and are targets of important classical anthelmintics like levamisole and pyrantel, as well as novel anthelmintics like tribendimidine and derquantel. Four biophysical subtypes of nAChR have been observed electrophysiologically in body muscle of the nematode parasite Oesophagostomum dentatum, but their molecular basis was not understood. Additionally, loss of one of these subtypes (G 35 pS) was found to be associated with levamisole resistance. In the present study, we identified and expressed in Xenopus oocytes, four O. dentatum nAChR subunit genes, Ode-unc-38, Ode-unc-63, Ode-unc-29 and Ode-acr-8, to explore the origin of the receptor diversity. When different combinations of subunits were injected in Xenopus oocytes, we reconstituted and characterized four pharmacologically different types of nAChRs with different sensitivities to the cholinergic anthelmintics. Moreover, we demonstrate that the receptor diversity may be affected by the stoichiometric arrangement of the subunits. We show, for the first time, different combinations of subunits from a parasitic nematode that make up receptors sensitive to tribendimidine and derquantel. In addition, we report that the recombinant levamisole-sensitive receptor made up of Ode-UNC-29, Ode-UNC-63, Ode-UNC-38 and Ode-ACR-8 subunits has the same single-channel conductance, 35 pS and 2.4 ms mean open-time properties, as the levamisole-AChR (G35) subtype previously identified in vivo. These data highlight the flexible arrangements of the receptor subunits and their effects on sensitivity and resistance to the cholinergic anthelmintics; pyrantel, tribendimidine and/or derquantel may still be effective on levamisole-resistant worms. Author Summary Parasitic nematode infections of humans and animals are world-wide. In humans, they cause disease and perpetuate a cycle of poverty. In animals, the parasites cause welfare problems and production loss. In developing countries, the debilitating effect of nematode parasites in school children limits their education, and in adults reduces productivity. These two factors, along with the production loss associated with nematode infections of animals that affects human nutrition, sustain poverty. Treatment and prophylaxis of these parasites requires the use of anthelmintic drugs. Here, we employ the Xenopus oocyte expression system to investigate the diversity of anthelmintic receptors in the parasitic nematode Oesophagostomum dentatum. We demonstrate effects of the novel' anthelmintics tribendimidine and derquantel on these reconstituted receptors, revealing, for the first time, the subunits that make up a tribendimidine- and derquantel-sensitive receptor from a parasitic nematode. We show that the receptor structure and pharmacology can be plastic, and depends on subunit composition and stoichiometry. The factors that affect the diversity of the receptor may contribute to anthelmintic resistance. Our results demonstrate the presence of acetylcholine receptor subtypes that may serve as anthelmintic targets and suggest that pyrantel, tribendimidine and/or derquantel may still be effective on levamisole-resistant worms