Binding site loops D and G make a stronger contribution than loop C to the actions of neonicotinoids on the NACHO-assisted, robustly expressed Drosophila melanogaster Dα1/Dβ1 nicotinic acetylcholine receptor.

TMX3 is essential for the functional expression of insect nicotinic acetylcholine receptors (nAChRs) and RIC-3 and UNC-50 modulate it. In addition to these cofactors, NACHO has been shown to enhance functional expression of certain vertebrate nAChRs and an insect homomeric nAChR. Here, we have examined the impact of Drosophila melanogaster NACHO (DmNACHO) on the ACh-induced response amplitude of the fruit fly Dα1/Dβ1 nAChRs coexpressed in Xenopus laevis oocytes with DmRIC-3, DmTMX3 and DmUNC-50 and examined the actions of neonicotinoid insecticides. DmNACHO markedly enhanced the ACh and neonicotinoid-induced response amplitude of Dα1/Dβ1 nAChRs coexpressed with the three cofactors DmRIC-3, DmTMX3 and DmUNC-50, while scarcely influencing ligand affinity. Given the robust Dα1/Dβ1 nAChR expression with the aid of the four cofactors, we investigated the impact of mutations in loops C, D and G of the orthosteric ligand binding domain (LBD) on the actions of the neonicotinoids imidacloprid and thiacloprid. Both the R81T mutation in loop D and the A60S mutation in loop G significantly reduced the agonist actions of the neonicotinoids, whereas the S221E mutation in loop C had no significant effect on agonist actions on this nAChR. Further, no greater affinity and efficacy reducing effects were observed even when the S221E mutation in loop C was combined with the R81T mutation in loop D, the A60S mutation in loop G, or both, demonstrating that loop D and loop G are more critical than loop C in determining the target-site actions of imidacloprid and thiacloprid.