Early studies from the mollusk Aplysia reported the presence
Early studies from the mollusk, Aplysia, reported the presence of a unique class of 10058-F4 receptors in neurons, the acetylcholine-gated chloride channels (ACCs) (Kehoe and McIntosh, 1998). Later, studies identified these receptors in the model nematode Caenorhabditis elegans (Putrenko et al., 2005; Wever et al., 2015). In C. elegans the ACC-1 family is made up of eight receptor subunit genes, acc-1, -2, -3, and -4, and lgc-46, -47, -48, and -49 (Jones and Sattelle, 2008). The sheep parasite, Haemonchus contortus, contains homologues for seven of the ACC-1 gene members, with the absence of lgc-48 (Laing et al., 2013). As a whole, this family of receptors has potential to be novel antiparasitic drug targets. This is primarily due to the fact that they appear to be expressed in tissues that are sensitive to anthelmintic action, and as sequence analysis suggests, they are not present in mammals and exhibit a unique acetylcholine binding site (Putrenko et al., 2005; Wever et al., 2015). However, the structural components that are important for agonist recognition of this class of cholinergic receptors has not been explored.
Here we have isolated a novel member if the ACC-1 family (Hco-ACC-2) from the parasitic nematode H. contortus and investigated the binding site through site-directed mutagenesis and pharmacological analysis. Several introduced point mutations that changed key aromatic residues at the binding site revealed some interesting pharmacological properties. Molecular modelling was used to visualize the structure of the binding pocket and the interaction of key residues with a variety of agonists.
Discussion We have identified a member of the ACC-1 family in the parasitic nematode H. contortus and investigated its binding site using site-directed mutagenesis, homology modelling and pharmacological analysis. This family of receptors appear to be unique to invertebrates and not present in mammals, making them attractive targets for novel nematocides. This has been validated by research demonstrating that this family of receptors are expressed in tissues that are sensitive to anthelmintic action (Wever et al., 2015). The research described here has examined in detail the structure of the ACC binding site which is important for future research focused on the discovery of novel therapeutics. One of the key features of ACh binding to mammalian nAChR is the presence of a tryptophan residue that participates in π-cationic interactions with the quaternary amine on ACh. In mammalian nAChRs this tryptophan residue is present in loop B (Beene et al., 2002). However, in ACC receptors there is no tryptophan residue in loop B. Instead, sequence analysis and homology modelling reveal a tryptophan residue present in loop C (W248). W248 is facing into the binding pocket and appears to be within 5 Å of the quaternary amine of the agonists that were docked in this study. While we did not confirm that W248 is participating in a π-cationic interaction with agonists, it is clear that it is essential for the function of Hco-ACC-2. Evidence for the importance of W248 in the function of ACC receptors is highlighted by mutational analysis, which showed that changing W248 to either W248Y or W248F resulted in a severely impacted receptor that did not respond to any agonists other than ACh. This could be partially explained by the removal of the indole nitrogen which is found in tryptophan. The presence of an indole nitrogen causes a large negative electrostatic cloud around this residue, allowing for increased potential of π-cationic interactions (Mecozzi et al., 1996). Since this indole is not present in tyrosine or phenylalanine, it could explain why these mutations were detrimental to receptor function. In addition, the positioning of this tryptophan in loop C could be important in ACC receptor function, as swapping the residues from loop C to B (F200W/W248F) produced a non-functional channel. This phenomenon is reminiscent of the novel nematode 5-HT receptor MOD-1 which also has a tryptophan residue in loop C instead of loop B, as seen in 5-HT3 receptors (Ranganathan et al., 2000). The loop C tryptophan in the MOD-1 receptor was shown to participate in the essential π-cationic interaction with serotonin which is key for receptor binding (Mu et al., 2003). Interestingly, this loop C tryptophan is not only present in ACC and MOD-1 receptors but other amine-gated chloride channels such as LGC-55 (tyramine) (Rao et al., 2010) and LGC-53 (dopamine) (Beech et al., 2013) as well as the nematode DEG-3 receptor (Fig. 3). From, an evolutionary perspective it appears that the presence of a tryptophan in loop C is an essential requirement for a diverse array of nematode cys-loop receptors, and their ability to bind to wide range of ligands.