Effects of cofactors RIC-3, TMX3 and UNC-50, together with distinct subunit ratios on the agonist actions of imidacloprid on Drosophila melanogaster Dα1/Dβ1 nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes

Insect nicotinic acetylcholine receptors (nAChRs) require cofactors for functional heterologous expression. A previous study revealed that TMX3 was crucial for the functional expression of Drosophila melanogaster Dα1/Dβ1 nAChRs in Xenopus laevis oocytes, while UNC-50 and RIC-3 enhanced the acetylcholine (ACh)-induced responses of the nAChRs. However, it is unclear whether the coexpression of UNC-50 and RIC-3 with TMX3 and the subunit stoichiometry affect pharmacology of Dα1/Dβ1 nAChRs when expressed in X. laevis oocytes. We have investigated the effects of coexpressing UNC-50 and RIC-3 with TMX3 as well as changing the subunit stoichiometry on the agonist activity of ACh and imidacloprid on the Dα1/Dβ1 nAChRs. UNC-50 and RIC-3 hardly affected the agonist affinity of ACh and imidacloprid for the Dα1/Dβ1 nAChRs formed by injecting into X. laevis oocytes with an equal amount mixture of the subunit cRNAs, but enhanced current amplitude of the ACh-induced response. Imidacloprid showed higher affinity for the Dβ1 subunit-excess Dα1/Dβ1 (Dα1/Dβ1 = 1/5) nAChRs than the Dα1 subunit-excess Dα1/Dβ1 (Dα1/Dβ1 = 5/1) nAChRs, suggesting that imidacloprid prefers the Dα1-Dβ1 orthosteric site over the Dα1-Dα1 orthosteric site.