Isolation and partial characterization of three acetylcholine-gated chloride channels in Haemonchus contortus

Abstract

Haemonchus contortus is a parasitic nematode infecting ruminants causing anemia and poor health due to the parasite’s blood-feeding nature. The ability to manage infection has been confounded by the rapid development of antiparasitc drug resistance in H. contortus populations. The Cys-loop superfamily of ligand gated ion channels are well recognized as critical drug targets for many invertebrate specific compounds. With the rise in resistance seen worldwide to existing anthelmintics, novel drug targets must be identified so new treatments can be developed. This thesis describes the identification of three acetylcholine-gated chloride channelss (hco-acc-1, hco-acc-2, and hco-acc-4) genes from the nematode parasite Haemonchus contortus and provides evidence suggesting that they could be developed as a future anthelmintic targets. While these genes appear similar in sequence to the previously characterized C. elegans acc-1, acc-2 and acc-4 genes, Hco-ACC-2 is about 2-fold less sensitive to acetylcholine, perhaps indicating a different in vivo function within the parasite. Further pharmacological analysis of ACC-2 via two-electrode voltage-clamp electrophysiology demonstrated activity of several agonists including carbachol, which elicited a similar response as acetylcholine. Furthermore, in silico protein modelling and agonist docking of all three channels has revealed a unique agonist binding site with several novel residues that appear to be important for binding to cholinergic agonists. Antibodies were generated against the Hco-ACC-1 protein for use in immunolocaliztion studies. Hco-ACC-1 localizes to the anterior half of the pharynx, specifically in pharyngeal muscle tissue in H. contortus.