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  • Resistance to avermectins has been associated

    2020-05-28

    Resistance to avermectins has been associated with point mutations of GluCls in C. elegans, D. melanogaster, T. urticae and P. xylostella (Dent et al., 2000; Dermauw et al., 2012; Kane et al., 2000; Kwon et al., 2010; Wang et al., 2016a, 2017). Our electrophysiological study found a 8.2-fold reduction in abamectin sensitivity with the exon 9c 12-aa deletion isoform when compared to 9a, which is the most common type of PxGluCl (described in Wang et al., 2016a and Wang et al., 2017). In addition, the exon 9b 9-aa deletion of PxGluCl also reduced the sensitivity to abamectin by 2.2-fold compared with 9a. Another study of P. xylostella (Liu et al., 2014) found that the expression of exon 9c produced a shift in the EC50 of abamectin from 118 nM to 1146 nM, which was about 10-fold higher than that of wild type channels. In contrast, with B. mori a partial deletion of exon 9 named 9pΔ (corresponding to the exon 9c of PxGluCl) had almost no impact on ivermectin action (Furutani et al., 2014). The inhibitory potencies of fipronil differed among the three variants, with 9b showing more sensitivity (2.7- to 3.4-fold) than 9a or 9c isoforms. Furthermore, all heteromeric Vitamin D3 mg showed lower sensitivity to fipronil than homomeric channels. We found that fipronil inhibited glutamate-induced currents in PxGluCls with micromolar IC50s (Table 2). These IC50s values are significantly higher than found with other insects and nemotodes, where IC50s range from 10 nM to 2930 nM with the exception of C. elegans glc-3 (IC50 of 11.5 μM). These findings suggest that substantial variations in sensitivity to fipronil can occur between different species. Although PxGluCls are considered the secondary target of fipronil after GABACls, it is possible that the upregulated constitutive expression of 9a, 9c or heteromeric channels may enhance synergistic resistance to fipronil in the field. The individual 9a, 9b and 9c splice variants appear to have a similar sensitivity towards glutamate as we found no significant difference in the EC50s and nHs between homomeric channels. Similarly in L. striatellus there were no significant differences in the response of LsGluCl-AL (a 21-bp stretch observed immediately downstream of intron 8) channel to glutamate, when compared with the LsGluCl-AS (the normal sequence) channel (Wu et al., 2017). However, other studies did find differences in glutamate sensitivity depending on the exon 9 transcript. With the B. mori GluCl the 9pΔ deletion (corresponding to PxGluCl exon 9c) had a small effect on glutamate action (Furutani et al., 2014). An even greater difference was found in an earlier study of P. xylostella GluCl where exon 9c variant was found to be about 13-fold less sensitive to glutamate compared to the wild-type (exon 9a) receptor (Liu et al., 2014). The intracellular domain remains the most poorly characterized region of LGIC receptors. Only the structures of acetylcholine and serotonin receptors have part of their TM3-TM4 linkers resolved (Unwin, 2005; Basak et al., 2018). These structures have an alpha-helical region of about 20 residues termed the ‘MA’ helix that precedes TM4 and extends into the cytoplasm where MAs meet to form a pentameric helix bundle. Although this structural feature may be conserved in GluCl receptors, the region encoded by the exon 9 variants is further than 20 residues from TM4 and is therefore predicted to lie just beyond the putative MA helix. We speculate that variation in this region may affect inter-monomer packing by the MA helices. As each MA helix is contiguous with TM4, a change in MA packing could be propagated via contact with TM4 to effect the conformation of the transmembrane domain and reposition residues that are ligand-binding determinants. Therefore an allosteric modification of the avermectin and fipronil binding sites may explain the effect of splice variants on GluCl sensitivity towards these two compounds. The structural effects exerted by the splice variants may not be propagated as far as the extracellular domain, which could account for the similar glutamate sensitivity of all the GluCls investigated in our study. However, a previous study with PxGluGl exon 9a and 9c variants did find a significant difference in glutamate sensitivity (Liu et al., 2014), which suggests the involvement of another mechanism. It is possible that post-translational modifications mediated by the TM3-TM4 linker can effect GluCl glutamate sensitivity. We note that the TM3-TM4 linkers of nAChR and 5-HT3A receptors provide sites for post-translational modifications, which may affect channel trafficking, kinetics and desensitization (McKinnon et al., 2012; Tsetlin et al., 2011).