The functional impact of mutations in orthosteric binding site of the Drosophila melanogaster Dα2-containing nicotinic receptors points to a greater contribution to neonicotinoid selectivity of an arginine in loop D of Dβ1 than an introduced serine in loop C of Dα2

Studies to date show that certain orthosteric binding sites are involved in the selective actions of neonicotinoids on insect nicotinic acetylcholine receptors (nAChRs). However, few investigations have utilized functional nAChRs consisting only of insect nAChR subunits. For the mechanism of selective neonicotinoid actions, loop C has been shown to play a role, whereas functional studies have advocated roles for loop D and loop G which appear to contribute more strongly to neonicotinoid actions than loop C. Here we have investigated the effects of P242E and P242S mutations in loop C of the Dα2 subunit and R81T mutation in loop D of the Dβ1 subunit on agonist actions of imidacloprid and thiacloprid for Drosophila melanogaster Dα1/Dα2/Dβ1 and Dα1/Dα2/Dβ1/Dβ2 nAChRs coexpressed with cofactors DmNACHO, DmRIC-3, DmTMX3 and DmUNC-50 in Xenopus laevis oocytes. The P242E and P242S mutations in loop C hardly affected the agonist affinity and efficacy of the neonicotinoids. In contrast, the R81T mutation in loop D decreased the affinity and efficacy of imidacloprid while substantially reducing the efficacy of thiacloprid. Combined loop C and loop D mutations resulted in further reduced efficacy of thiacloprid while having no such effect on imidacloprid actions. These results suggest that the proline in loop C and the arginine in loop D underly the binding of neonicotinoids with the greater contribution coming from loop D, and that thiacloprid relies less on the interactions with the arginine in loop D than imidacloprid, and hence is less susceptible to the R81T mutation in the development of resistance.