The stoichiometry of the α4β2 neuronal nicotinic acetylcholine receptors determines the pharmacological properties of the neonicotinoids, and recently introduced butenolide and sulfoximine.

Although neonicotinoids were considered safe for mammals for many decades, recent research has proven that these insecticides can alter cholinergic functions by interacting with neuronal nicotinic acetylcholine (ACh) receptors (nAChRs). One such receptor is the heteromeric α4β2 nAChR, which exists under two different stoichiometries: high sensitivity and low sensitivity α4β2 nAChRs. To replace these insecticides, new classes of insecticides have been developed, such as, sulfoximine, sulfoxaflor, and the butanolide, flupyradifurone. In this study, we injected Xenopus laevis oocytes with 1:10 and 10:1 α4:β2 subunit RNA ratios, in order to express the high (α4)(β2) and low sensitivity (α4)(β2) nAChRs. Using the two-electrode voltage-clamp technique, we found that the low sensitivity (α4)(β2) nAChRs were activated by all tested insecticides, whereas the high sensitivity (α4)(β2) nAChR was only activated by ACh. Imidacloprid, sulfoxaflor and flupyradifurone confirmed their agonist effects by reducing the responses to the ACh EC concentrations, for both low (α4)(β2) and high sensitivity (α4)(β2) stoichiometries. Clothianidin only inhibited ACh responses of the low sensitivity (α4)(β2) stoichiometry. Mutation E226P in the α4 subunit of the low sensitivity (α4)(β2) receptors inhibits the agonist potency of imidacloprid and flupyradifurone, whereas mutation L273T in the high sensitivity (α4)(β2) nAChR leads to activation by all insecticides. Major agonist effects were found with the double mutation of the E226P in the α4 subunit, and the L273T in the β2 subunit of the high sensitivity (α4)(β2) stoichiometry.