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Neurotoxicology
2025 Mar 11;107:1-10. doi: 10.1016/j.neuro.2025.01.001.
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The stoichiometry of the α4β2 neuronal nicotinic acetylcholine receptors determines the pharmacological properties of the neonicotinoids, and recently introduced butenolide and sulfoximine.
Kaaki S
,
Cartereau A
,
Boussaine K
,
Taillebois E
,
Thany SH
.
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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 butenolide, 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)2(β2)3 and low sensitivity (α4)3(β2)2 nAChRs. Using the two-electrode voltage-clamp technique, we found that the low sensitivity (α4)3(β2)2 nAChRs were activated by all tested insecticides, whereas the high sensitivity (α4)2(β2)3 nAChR was only activated by ACh. Imidacloprid, sulfoxaflor and flupyradifurone confirmed their agonist effects by reducing the responses to the ACh EC80 concentrations, for both low (α4)3(β2)2 and high sensitivity (α4)2(β2)3 stoichiometries. Clothianidin only inhibited ACh responses of the low sensitivity (α4)3(β2)2 stoichiometry. Mutation E226P in the α4 subunit of the low sensitivity (α4)3(β2)2 receptors inhibits the agonist potency of imidacloprid and flupyradifurone, whereas mutation L273T (in the β2 subunit) in the high sensitivity (α4)2(β2)3 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)2(β2)3 stoichiometry.
Fig. 1. (A) Chemical structures of acetylcholine (ACh) and the four insecticides, imidacloprid (IMI), clothianidin (CLT), sulfoxaflor (SFX), and flupyradifurone (FLU). (B) High sensitivity (α4)2(β2)3 and low sensitivity (α4)3(β2)2 nicotinic acetylcholine receptor stoichiometries used in the present study. As indicated, low sensitivity (α4)3(β2)2 receptors are characterised by the presence of an additional α/α interface. (C) Concentration-response relationships for acetylcholine (ACh) using α4β2 wild-type, the low sensitivity (α4)3(β2)2 and the high sensitivity (α4)2(β2)3, respectively. (D, E, F, and G) Concentration-response relationships for imidacloprid (IMI), clothianidin (CLT), sulfoxaflor (SFX) and flupyradifurone (FLU). Each point plotted in the concentration response curves represents mean ± S.E.M of 5–7 oocytes. For each experimental condition, we used 3–5 different batches.
Fig. 2. Inhibitory effect of the four insecticides on the ACh responses of low sensitivity (α4)3(β2)2 nAChR stoichiometry. The figures present the current responses induced by 300 µM ACh (EC80 value for ACh in the low sensitivity (α4)3(β2)2) co-applied with increasing concentrations (1 pM, 1 nM, 1 µM, 100 µM, 300 µM, and 1 mM) of IMI (A), CLT (B), SFX (C), and FLU (D). ACh control responses are represented in black, and successive responses are shown using a color gradient, where darker shades represent higher concentrations, and lighter shades represent lower concentrations. The black bar represents 10 s co-application of 300 µM ACh with each insecticide. (E) illustrates the inhibitory curves. The pIC50 values are indicated in the Table 3. All currents were normalized using 300 µM ACh. Each point represents mean ± SEM of n = 5–6 different recordings based on 3–5 batches.
Fig. 3. Inhibitory effect of the four insecticides on the ACh responses of high sensitivity (α4)2(β2)3 nAChR stoichiometry. The figures present the current responses induced by 30 µM ACh (EC80 value for ACh in the high sensitivity (α4)2(β2)3) co-applied with increasing concentrations (1 pM, 1 nM, 1 µM, 100 µM, 300 µM, and 1 mM) of IMI (A), CLT (B), SFX (C), and FLU (D). ACh control responses are represented in black, and successive responses are shown using a color gradient, where darker shades represent higher concentrations, and lighter shades represent lower concentrations. The black bar represents 10 s co-application of 30 µM ACh with each insecticide. (E) illustrates the inhibitory curves. The pIC50 values are indicated in the Table 2. All currents were normalized using 30 µM ACh. Each point represents mean ± SEM of n = 5–6 different recordings based on 3–5 batches.
Fig. 4. (A) Protein alignment showing the mutations of the glutamic acid to proline at position 226 in the loop C of the α4 subunit, and lysine to threonine at position 273 in the second transmembrane domain of the β2 subunit. Note that, the mutation of a glutamic acid to a proline at position 226 corresponds to the proline at position 242 in the YXCC motif in loop C of drosophila Dα2 subunit. This mutation resulted in enhanced responses to IMI of the human α4β2 receptor (Toshima et al., 2009). (B, C and D) Concentration-response relationships for acetylcholine (ACh) using the 1:1 ratio (B), 10:1 ratio (C), and 1:10 ratio (D) of α4 and β2 mRNAs injected in the oocytes. Responses are normalised using 100 µM ACh and each point represents the mean ± SEM of 5–7 oocytes. For each experimental condition, we used 3–5 different batches.
Fig. 5. Examples of currents induced by 100 µM ACh and the four insecticides (IMI, CLT, SFX and FLU) on the wild-type α4β2 nAChRs (A to C), nine α4β2 mutated receptors, and stoichiometries (D to L). Note that at the tested concentration, 100 µM FLU did not activate α4β2 (A), LS (α4)3(β2)2 (B), HS (α4)2(β2)3 (C), and (α4E226P)2(β2)3 (I) receptors. It induces a very low activation on (α4)2(β2L273T)3 (F), α4E226Pβ2 (G), and (α4E226P)3(β2)2 (H). Black bars represent 10 s bath application of each compound. Note that the corresponding histograms and statistical analyses are illustrated in the supplementary data fig. S1.
