Heterologous formation of neonicotinoid-sensitive nAChRs containing UNC-38 and UNC-29 subunits from Bursaphelenchus xylophilus.

Nicotinic acetylcholine receptor (nAChR) subunits are encoded by a large multigene family and generate a large number of pentameric receptors with various properties. At present, nematode species, such as Caenorhabditis elegans, have the largest number of nAChR subunits. In this study, two nAChR subunits (Bxy-Unc-38 and Bxy-Unc-29) were cloned from Bursaphelenchus xylophilus, a fatal nematode pest on pine trees causing pine wilt disease. When Bxy-Unc-38 and Bxy-Unc-29 were co-expressed in Xenopus oocytes, constructed functional nAChRs showed agonist responses to acetylcholine and imidacloprid, a neonicotinoid insecticide. When complementary RNAs (cRNAs) of Bxy-Unc-38 and Bxy-Unc-29 were injected at different ratios, the assembled nAChRs showed different pharmacological subtypes, especially in terms of the sensitivity to imidacloprid and another two neonicotinoids. At cRNA ratios 1:1 and 1:5 (Bxy-Unc-38: Bxy-Unc-29), nAChRs showed low sensitivity to test neonicotinoids, which were partial agonists on the receptors. In contrast, at cRNA ratio 5:1, the three test neonicotinoids were full agonists and showed much higher potency compared to that on the receptors with cRNA ratio 1:1 and 1:5. For example, EC values of the three neonicotinoids on the receptors with cRNA ratio 1:5 were 170-222 times of those of receptors with cRNA ratio 5:1. The results showed that the subunit stoichiometry of Bxy-Unc-38/Bxy-Unc-29 receptor dramatically affected the agonist potency of neonicotinoids, and even altered the action property. Due to the high sensitivity of the constructed nAChRs at cRNA ratio 5:1, the construct would serve as an important model to study the interaction between invertebrate nAChRs and neonicotinoids.