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

Abstract

Neonicotinoid insecticides act selectively on insect nicotinic acetylcholine receptors (nAChRs). Recent studies revealed that their efficiency was altered by the phosphorylation/dephosphorylation process and the intracellular signaling pathway involved in the regulation of nAChRs. Using whole-cell patch-clamp electrophysiology adapted for dissociated cockroach dorsal unpaired median (DUM) neurons, we demonstrated that intracellular factors involved in the regulation of nAChR function modulated neonicotinoid sensitivity. DUM neurons were known to express two 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 types of neonicotinoid insecticides, acted as agonists on the 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 cadmium chloride, ω-conotoxin GVIA, and (R,S)-(3,4-dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl-N,N-di-acetamide (LOE 908), we found that inhibition of calcium influx through high voltage-activated calcium channels and transient receptor potential γ (TRPγ) activated by both depolarization and hyperpolarization increased nAChR2 sensitivity to acetamiprid. Finally, using N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7), forskolin, and cAMP, we demonstrated that adenylyl cyclase sensitive to the calcium/calmodulin complex regulated internal cAMP concentration, which in turn modulated TRPγ function and nAChR2 sensitivity to acetamiprid. Similar TRPγ-induced modulatory effects were also obtained when clothianidin was tested. These findings bring insights into the signaling pathway modulating neonicotinoid efficiency and open novel strategies for optimizing insect pest control.

Subchronic exposure to sublethal dose of imidacloprid changes electrophysiological properties and expression pattern of nicotinic acetylcholine receptor subtypes in insect neurosecretory cells

“…It is interesting to mention that similar results were obtained when DUM neurons were exposed to 50 mM d-TC, as previously reported (n = 4 to 7 cells; Fig. 4B, Bodereau-Dubois et al, 2012). The resulting increase in current density reflecting an inhibition of spontaneous outward potassium conductance indicates that nAChR2 is more open before application of nicotine.…”

“…Although nAChR1 is sensitive to the neonicotinoid imidacloprid, nAChR2 is insensitive to this insecticide, whereas the insect has never been exposed to this insecticide (Courjaret and Lapied, 2001). Furthermore, we have demonstrated that the uncommon conformational state of nAChR2 (i.e., open at the resting state and closed upon cholinergic agonist application) (Courjaret and Lapied, 2001;Courjaret et al, 2003;Bodereau-Dubois et al, 2012) is responsible for the different neonicotinoid sensitivity observed in these two nAChR subtypes. Consequently, because cockroach neuronal preparations together with DUM neurons are commonly used as biological models for vertebrates and invertebrates to study the mode of action of neurotoxic insecticides (Pelhate et al, 1990), these interesting features make DUM neuron nAChR1 and nAChR2 subtypes a suitable model to explore the influence of subchronic exposure to sublethal dose of imidacloprid on both physiological and molecular properties of insect nAChRs.…”

“…As nAChR1 and nAChR2 have been previously reported to be regulated by intracellular calcium rise and calcium-dependent phosphorylation/dephosphorylation process (Courjaret and Lapied, 2001;Courjaret et al, 2003;Bodereau-Dubois et al, 2012;Calas-List et al, 2013) additional experiments using calcium imaging were performed to evaluate a putative evolution of the steady-state resting calcium level after subchronic exposure. As illustrated in Fig.…”

“…Combinations of nAChR subunits result in distinct receptors, with their own electrophysiological and pharmacological properties, which thereby influence sensitivity to neonicotinoids (Lansdell and Millar, 2000;Millar and Lansdell, 2010). In addition, previous studies have shown that neonicotinoid efficacy on nAChR subtypes depends on electropharmacological properties and many cellular and molecular factors such as conformational state, membrane potential, subunit composition and calcium-dependent phosphorylation/dephosphorylation process (Courjaret and Lapied, 2001;Bodereau-Dubois et al, 2012;Calas-List et al, 2013;List et al, 2014;Salgado, 2016;Sun et al, 2016).…”

“…Up to date, there are no data related to the effects of subchronic exposure to sublethal dose of neonicotinoids on both physiological and molecular features of insect nAChRs. For that purpose, cockroach Periplaneta americana neurosecretory cells identified as dorsal unpaired median (DUM) neurons, known to express two distinct a-bgt-insensitive nAChR subtypes named nAChR1 and nAChR2 (Courjaret and Lapied, 2001;Bodereau-Dubois et al, 2012), have been used. Previous findings have reported that nAChR subtypes present different pharmacological properties.…”