Abstract
Nematode cys-loop ligand-gated ion channels (LGICs) have been shown to be attractive targets for the development of novel anti-parasitic drugs. The ACC-1 family of receptors are a unique group of acetylcholine-gated chloride channels present only in invertebrates, and sequence analysis suggests that they contain a novel binding site for acetylcholine. We have isolated a novel member of this family, Hco-ACC-2, from the parasitic nematode Haemonchus contortus and using site-directed mutagenesis, electrophysiology and molecular modelling examined how two aromatic amino acids in the binding site contributed to agonist recognition. It was found that instead of a tryptophan residue in binding loop B, which essential for ligand binding in mammalian nAChRs, there is a phenylalanine (F200) in Hco-ACC-2. Amino acid changes at F200 to either a tyrosine or tryptophan were fairly well tolerated, where a F200Y mutation resulted in a channel hypersensitive to ACh and nicotine as well as other cholinergic agonists such as carbachol and methacholine. In addition, both pyrantel and levamisole were partial agonists at the wild-type receptor and like the other agonists showed an increase in sensitivity at F200Y. On the other hand, in Hco-ACC-2 there is a tryptophan residue at position 248 in loop C that appears to be essential for receptor function, as mutations to either phenylalanine or tyrosine resulted in a marked decrease in agonist sensitivity. Moreover, mutations that swapped the residues F200 and W248 (ie. F200W/W248F) produced non-functional receptors. Overall, Hco-ACC-2 appears to have a novel cholinergic binding site that could have implications for the design of specific anthelmintics that target this family of receptors in parasitic nematodes.