Transmembrane Potential Polarization, Calcium Influx, and Receptor Conformational State Modulate the Sensitivity of the Imidacloprid-Insensitive Neuronal Insect Nicotinic Acetylcholine Receptor to Neonicotinoid Insecticides

Neonicotinoid insecticides act selectively on insect nicotinic acetylcholine receptors (nAChRs). Recent studies revealed that their efficiency was altered by phosphorylation/dephosphorylation process and intracellular signalling pathway involved in the regulation of nAChRs. Using whole-cell patch-clamp electrophysiology adapted on dissociated cockroach dorsal unpaired median (DUM) neurons, we demonstrated that intracellular factors involved in the regulation of nAChR function, modulated neonicotinoids sensitivity. DUM neurons were known to express α-bungarotoxin-insensitive nAChR subtypes, nAChR1 and nAChR2. Whereas nAChR1 was sensitive to imidacloprid, nAChR2 was insensitive to this insecticide. Here, we demonstrated that, like nicotine, acetamiprid and clothianidin other type of neonicotinoid insecticides, acted as agonists on nAChR2 subtype. Using acetamiprid, we revealed that both steady-state depolarization and hyperpolarization affected nAChR2 sensitivity. The measurement of the input membrane resistance indicated that change in the acetamiprid-induced agonist activity was related to the receptor conformational state. Using CdCl2, ω-conotoxin GVIA and LOE 908, we reported that inhibition of calcium influx through HVA calcium channels and TRPγ activated by both depolarization and hyperpolarization increased nAChR2 sensitivity to acetamiprid. Finally, using W7, forskolin and cAMP, we demonstrated that adenylyl cyclase sensitive to the calcium/calmodulin complex regulated internal cAMP concentration, which in turns modulated TRPγ function and nAChR2 sensitivity to acetamiprid. Similar TRPγ-induced modulatory effects were also obtained when clothianidin was tested. These findings, bringing new insights on the signalling pathway modulating neonicotinoid efficiency, open novel strategies for optimizing insect pest control.