Cofactor-enabled functional expression of fruit fly, honeybee, and bumblebee nicotinic receptors reveals picomolar neonicotinoid actions

Significance Neonicotinoids acting on insect nicotinic acetylcholine receptors (nAChRs) are deployed for crop protection, but growing evidence for adverse effects on insect pollinators has led to restricted use of some neonicotinoids in the EU. It is therefore vital to understand the target site actions of neonicotinoids in pollinators, but to date the difficulties of heterologous expression of insect nAChRs have hampered progress. We have found that a thioredoxin (TMX3) enables robust functional expression of honeybee, bumblebee, and fruit fly nAChRs in Xenopus laevis oocytes. With this advance, we show that expressed bee nAChRs are more neonicotinoid-sensitive than those of fruit fly, and clothianidin can modulate both honeybee and bumblebee nAChRs at a concentration below that commonly observed in agricultural fields.
The difficulty of achieving robust functional expression of insect nicotinic acetylcholine receptors (nAChRs) has hampered our understanding of these important molecular targets of globally deployed neonicotinoid insecticides at a time when concerns have grown regarding the toxicity of this chemotype to insect pollinators. We show that thioredoxin-related transmembrane protein 3 (TMX3) is essential to enable robust expression in Xenopus laevis oocytes of honeybee (Apis mellifera) and bumblebee (Bombus terrestris) as well as fruit fly (Drosophila melanogaster) nAChR heteromers targeted by neonicotinoids and not hitherto robustly expressed. This has enabled the characterization of picomolar target site actions of neonicotinoids, findings important in understanding their toxicity.