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  • Cys loop GABA receptors are

    2022-05-23

    Cys-loop GABA receptors are well-studied targets for insecticides and also play an important role in nematode biology. These receptors are also targets for the anthelmintic piperizine (Accardi et al., 2012). The nematode GABA receptor most studied is the UNC-49 receptor which plays a role in muscle contraction essential for locomotion (Bamber et al., 1999). The importance of this receptor to the biology of nematode parasites is evident from the fact that genes encoding the various subunits of the UNC-49 receptor family are found in many parasitic nematode genomes (Accardi et al., 2012). Moreover, the UNC-49 receptor is not analogous to GABA receptors found in mammals and exhibits a unique pharmacology suggesting structural differences in the binding pocket (Bamber et al., 2003; Kaji et al., 2015). In several cases, differences in binding site residues between the UNC-49 receptor and mammalian GABA receptors have partially explained differences in function (Kaji et al., 2015; Kwaka et al., 2018). However, there is much to be learned about how various residues in and around the binding pocket contribute to the overall function of the nematode UNC-49 receptor as well as other nematode GABA receptors and GABA receptors in general. Such knowledge is important for our understanding of the TTNPB and function of GABA neurotransmission and the potential of these receptors as targets for future nematocides. The crystal structure of the Caenorhabditis elegans GluCl (Hibbs and Gouaux, 2011) has provided the means to begin to examine the structure of the UNC-49 receptor from H. contortus which has been key to understanding the residue requirements for receptor function and pharmacology (Kaji et al., 2015). There are many residue types that play key roles in overall receptor function. However, charged residues are particularly important in that they play essential roles in the structure and function of the various binding loops and they have been shown to TTNPB interact directly with GABA (Ashby et al., 2012; Newell et al., 2004). While many of these residues have been studied in other GABA receptors there is little information on their role in GABA receptors from nematodes. Molecular dynamic (MD) simulations have been extremely useful in visualizing and quantifying inter- and intramolecular interactions between residues (Ashby et al., 2012; Kwaka et al., 2018). In addition, when coupled with mutational analysis this approach can provide a deeper understanding of receptor function, particularly receptors from parasitic nematodes where there has been limited study. Here we have investigated the function of various charged residues in the UNC-49 receptor from the parasitic nematode H. contortus using mutational analysis, homology modelling and molecular dynamic simulations. We show that this approach can provide novel insight into the function of cys-loop receptors from parasitic organisms which may aid research that focuses on the development of novel anthelmintics. In addition, using the results from this study along with knowledge of human GABAA receptors, we have examined other nematode GABA receptor subunits for the presence or absence of key residues that can be used to explain or predict function.
    Materials and methods
    Results
    Discussion This study has confirmed that a glutamic acid residue in loop B (E185) is absolutely essential for GABA receptor activation in nematode UNC-49 GABA receptor. The analogous residues of E185 are E155 in the mammalian GABAA receptor and E204 in the Drosophila RDL. In both case these residues have been shown to be crucial channel function (Newell et al., 2004; Ashby et al., 2012; Miller et al., 2014). In the Drosophila RDL receptor, like the UNC-49 receptor, all mutations to E204 resulted in channels that were unresponsive. It has been suggested that this loop B glutamic acid acts as a control element where it interacts with the positive amine group of GABA coupling binding to the channel activation (Newell et al., 2004). Not only does E185 form a critical interaction with GABA but it also interacts with an arginine in loop C (R241). Mutations of R241 showed drastically impaired function of the channel, with R241A showing an approximately 80 fold decrease in sensitivity and R241K showing an approximately 59 fold decrease in sensitivity. The crucial role of R241 is also supported by our MD simulations which revealed an ionic bond between R241 and E185. This interaction between analogous residues has also been observed in the human GABAA receptor (Bergmann et al., 2013; Miller et al., 2014) and it may function in the closure of the channel (Miller et al., 2014). It appears therefore that a glutamic acid in loop B and a loop C arginine and their potential interaction are crucial for GABA receptor function across phyla.