Functional co-expression of two insect nicotinic receptor subunits (Nlα3 and Nlα8) reveals the effects of a resistance-associated mutation (Nlα3Y151S) on neonicotinoid insecticides

Neonicotinoid insecticides, such as imidacloprid, are selective agonists of insect nicotinic acetylcholine receptors (nAChRs) and are used extensively to control a variety of insect pest species. Previously, we have identified a nAChR point mutation (Y151S) associated with insecticide resistance in the brown planthopper Nilaparvata lugens . Although this mutation has been identified in two different N. lugens nAChR subunits (Nlα1 and Nlα3) because of difficulties in heterologous expression of Nlα3; its influence on agonist potency has been examined only in Nlα1-containing nAChRs. Here we describe the cloning of a novel nAChR subunit from N. lugens (Nlα8), together with evidence for its co-assembly with Nlα3 in native and recombinant nAChRs. This has, for the first time, enabled the functional effects of the Nlα3^Y151S mutation to be examined. The Nlα3^Y151S mutation has little effect on agonist potency of acetylcholine but has a dramatic effect on neonicotinoid insecticides (reducing I _max values and increasing EC₅₀ values). The apparent affinity of neonicotinoids was higher and the effect of the Y151S mutation on neonicotinoid agonist potency was more profound in Nlα3-containing, rather than Nlα1-containing nAChR. We conclude that Nlα3- and Nlα1-containing nAChRs may be representative of two distinct insect nAChR populations.     

Heteromeric co-assembly of two insect nicotinic acetylcholine receptor α subunits: influence on sensitivity to neonicotinoid insecticides

Neonicotinoid insecticides, such as imidacloprid, are selective agonists of insect nicotinic acetylcholine receptors (nAChRs) and are used extensively in areas of crop protection and animal health to control a variety of insect pest species. Here, we describe studies performed with nAChR subunits Nlα1 and Nlα2 cloned from the brown planthopper Nilaparvata  lugens , a major insect pest of rice crops in many parts of Asia. The influence of Nlα1 and Nlα2 subunits upon the functional properties of recombinant nAChRs has been examined by expression in Xenopus oocytes. In addition, the influence of a Nlα1 mutation (Y151S), which has been linked to neonicotinoid lab generated resistance in N. lugens , has been examined. As in previous studies of insect α subunits, functional expression has been achieved by co-expression with the mammalian β2 subunit. This approach has revealed a significantly higher apparent affinity of imidacloprid for Nlα1/β2 than for Nlα2/β2 nAChRs. In addition, evidence has been obtained for the co-assembly of Nlα1 and Nlα2 subunits into 'triplet' nAChRs of subunit composition Nlα1/Nlα2/β2. Evidence has also been obtained which demonstrates that the resistance-associated Y151S mutation has a significantly reduced effect on neonicotinoid agonist activity when Nlα1 is co-assembled with Nlα2 than when expressed as the sole α subunit in a heteromeric nAChR. These findings may be of importance in assessing the likely impact of the target-site mutations such as Y151S upon neonicotinoid insecticide resistance in insect field populations.     

Amino acids within loops D, E and F of insect nicotinic acetylcholine receptor beta subunits influence neonicotinoid selectivity

Nicotinic acetylcholine (ACh) receptors (nAChRs) are ligand-gated ion channels which mediate fast cholinergic synaptic transmission in insect and vertebrate nervous systems. The nAChR agonist-binding site is present at the interface of adjacent subunits and is formed by loops A-C present in alpha subunits together with loops D-F present in either non-alpha subunits or homomer-forming alpha subunits. To investigate the mechanism of neonicotinoid selectivity, we have examined the effects of altering insect-specific loops D, E and F in hybrid nAChRs containing insect and mammalian subunits (Nlalpha1 from the brown planthopper Nilaparvata lugens and beta2 from rat). Introduction of the insect-specific loops D, E and F, singly or together, into rat beta2 subunit resulted in a leftward shift of the imidacloprid dose-response curves for nAChRs Nlalpha1-beta2 chimeras, reflecting decreases in EC(50), compared to wildtype nAChRs Nlalpha1-beta2. By contrast, the influences on ACh potency were minimal or negligible. The effects of loop D could be interpreted by the earlier findings of Shimomura et al. [2006. Role in the selectivity of neonicotinoids of insect-specific basic residues in loop D of the nicotinic acetylcholine receptor agonist-binding site. Mol. Pharmacol. 70, 1255-1263.], in which T77R and E79V were shown to be responsible for neonicotinoid selectivity. In the present study, S131Y(R) and D133N in loop E and T191W and P192K in loop F were found to contribute to the neonicotinoid selectivity of insect-specific loops E and F. These results indicated the insect-specific loops D, E and F each play important roles in neonicotinoids selectivity. This study contributes to our understanding of the molecular mechanism underlying selectivity of neonicotinoids against insects over vertebrates.     

褐飞虱对吡虫啉的抗性机理和靶标分子毒理学

褐飞虱Nilaparvata lugens是水稻最重要的害虫之一,长期依赖化学防治导致了该害虫对不同类型杀虫剂抗性的产生,对新烟碱类杀虫剂吡虫啉高水平抗性的产生更是造成了巨大的粮食生产损失。近年来在褐飞虱对吡虫啉抗性机理,以及在抗药性机理研究推动下吡虫啉作用靶标褐飞虱神经系统烟碱型乙酰胆碱受体（nicotinic acetylcholine receptors, nAChRs）毒理学等方面取得了许多研究进展。nAChRs是昆虫神经系统中最重要的神经递质受体,是几类重要杀虫剂的作用靶标,其中以新烟碱类杀虫剂为代表。通过对比敏感品系和室内连续筛选获得的高抗吡虫啉品系,在褐飞虱两个nAChRs亚基Nlα1和Nlα3中均发现了抗性相关点突变Y151S,该突变导致了受体与吡虫啉结合亲和力的显著下降,而对内源神经递质乙酰胆碱的亲和力影响很小。Nlα1与褐飞虱另外两个亚基Nlα2和Nlβ1共聚成一个受体,构成吡虫啉低亲和力结合位点;Nlα3与褐飞虱另外两个亚基Nlα8和Nlβ1共聚成一个受体,构成吡虫啉高亲和力结合位点。不仅褐飞虱nAChRs与吡虫啉抗性相关,某些nAChRs附属蛋白也直接影响褐飞虱对吡虫啉的抗性,如Lynx蛋白。关于褐飞虱nAChRs组成、抗药性相关变异、受体附属蛋白对抗药性的影响等方面的研究,均为国内外前沿报道,不仅有助于对新烟碱类杀虫剂抗性机理的理解,对昆虫nAChRs毒理学同样具有很大的推动作用。     

Identification of two Lynx proteins in Nilaparvata lugens and the modulation on insect nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission in the insect brain and are targets for neonicotinoid insecticides. Some proteins, other than nAChRs themselves, might play important roles in insect nAChRs function in vivo and in vitro , such as the chaperone, regulator and modulator. Here we report the identification of two nAChR modulators (Nl-lynx1 and Nl-lynx2) in the brown planthopper, Nilaparvata lugens . Analysis of amino acid sequences of Nl-lynx1 and Nl-lynx2 reveals that they are two members of the Ly-6/neurotoxin superfamily, with a cysteine-rich consensus signature motif. Nl-lynx1 and Nl-lynx2 only increased agonist-evoked macroscopic currents of hybrid receptors Nlα1/β2 expressed in Xenopus oocytes, but not change the agonist sensitivity and desensitization properties. For example, Nl-lynx1 increased I _max of acetylcholine and imidacloprid to 3.56-fold and 1.72-fold of that of Nlα1/β2 alone, and these folds for Nl-lynx2 were 3.25 and 1.51. When the previously identified Nlα1^Y151S mutation was included (Nlα1^Y151S/β2), the effects of Nl-lynx1 and Nl-lynx2 on imidacloprid responses, but not acetylcholine response, were different from that in Nlα1/β2. The increased folds in imidacloprid responses by Nl-lynx1 and Nl-lynx2 were much higher in Nlα1^Y151S/β2 (3.25-fold and 2.86-fold) than in Nlα1/β2 (1.72-fold and 1.51-fold), which indicated Nl-lynx1 and Nl-lynx2 might also serve as an influencing factor in target-site insensitivity in N. lugens . These findings indicate that nAChRs chaperone, regulator and modulator may be of importance in assessing the likely impact of the target-site mutations such as Y151S upon neonicotinoid insecticide resistance.     

A hypothesis to account for the selective and diverse actions of neonicotinoid insecticides at their molecular targets,nicotinic acetylcholine receptors: catch and release in hydrogen bond networks

The low mammalian toxicity of neonicotinoid insecticides has been shown to be attributable, at least in part, to their selective actions on insect nicotinic acetylcholine receptors (nAChRs). There are multiple nAChRs in insects and a wealth of neonicotinoid chemicals. Studies to date have discribed a wide range of effects on nAChRs, notably partial agonist, super agonist and antagonist actions. Both the diversity of the neonicotinoid actions and their selectivity for insect over vertebrate nAChRs are the result of physicochemical and steric interactions at their molecular targets (nAChRs). In such interactions, the formation and breakage of hydrogen bond (HB) networks plays a key role. Therefore the loss or gain of even a single HB resulting from either structural changes in neonicotinoids, or the amino acid sequence of a particular nAChR subunit, could result in a drastic modification of neonicotinoid actions. In addition to the amino acid residues, the backbone carbonyl of nAChRs may also be involved in the formation of HB networks with neonicotinoids.     

Neonicotinoids Show Selective and Diverse Actions on Their Nicotinic Receptor Targets: Electrophysiology,Molecular Biology,and Receptor Modeling Studies

Neonicotinoid insecticides, which act selectively on insect nicotinic acetylcholine receptors (nAChRs), are used worldwide for insect pest management. Studies that span chemistry, biochemistry, molecular biology, and electrophysiology have contributed to our current understanding of the important physicochemical and structural properties essential for neonicotinoid actions as well as key receptor residues contributing to the high affinity of neonicotinoids for insect nAChRs. Research to date suggests that electrostatic interactions and possibly hydrogen bond formation between neonicotinoids and nAChRs contribute to the selectivity of these chemicals. A rich diversity of neonicotinoid-nAChR interactions has been demonstrated using voltage-clamp electrophysiology. Computational modeling of nAChR-imidacloprid interaction has assisted in the interpretation of these results.     

Neonicotinoids show selective and diverse actions on their nicotinic receptor targets: electrophysiology, molecular biology, and receptor modeling studies

Neonicotinoid insecticides, which act selectively on insect nicotinic acetylcholine receptors (nAChRs), are used worldwide for insect pest management. Studies that span chemistry, biochemistry, molecular biology, and electrophysiology have contributed to our current understanding of the important physicochemical and structural properties essential for neonicotinoid actions as well as key receptor residues contributing to the high affinity of neonicotinoids for insect nAChRs. Research to date suggests that electrostatic interactions and possibly hydrogen bond formation between neonicotinoids and nAChRs contribute to the selectivity of these chemicals. A rich diversity of neonicotinoid-nAChR interactions has been demonstrated using voltage-clamp electrophysiology. Computational modeling of nAChR-imidacloprid interaction has assisted in the interpretation of these results.     

The mode of action of a nitroconjugated neonicotinoid and the effects of target site mutation Y151S on its potency

Neonicotinoid insecticides, such as imidacloprid, are selective agonists of the insect nicotinic acetylcholine receptors (nAChRs) with -NO₂ or -CN group in trans-configuration. Previously we reported the excellent insecticidal activity of a series of nitroconjugated neonicotinoids with -NO₂ or -CN group in cis-configuration by replacing nitromethylene pharmacophore with a nitroconjugated system. To understand the action mode of these nitroconjugated neonicotinoids, a representative member IPPA152201 was chosen to perform toxicity and pharmacology studies. IPPA152201 showed a comparable toxicity with imidacloprid against Nilaparvata lugens in a susceptible strain and had no significant cross-resistance in an imidacloprid resistant strain. IPPA152201 showed good efficacies on the isolated cockroach neurons (pEC₅₀ = 5.91 ± 0.14) and the evoked responses by IPPA152201 could be blocked by the typical nAChRs antagonists methyllycaconitine citrate (MLA) and dihydro-??-erythroidine (DH??E), with pIC₅₀ of 6.56 ± 0.07 and 6.89 ± 0.12. The efficacy of IPPA152201 on hybrid receptors Nl??1/??2 in Xenopus oocytes and response inhibition by MLA and DH??E were also observed. These data demonstrate that IPPA152201 acts on insect nAChRs as an agonist. In addition, the influence of a Nl??1 mutation (Y151S), which has been linked to the lab-generated neonicotinoid resistance in N. lugens, has been examined. Compared to the wildtype Nl??1/??2, this mutation reduced I_max for IPPA152201 to 63.2% and caused a 1.5-fold increase in EC₅₀, which is much smaller than the effects on imidacloprid. The high insecticidal activity and little influence by Y151S mutation make IPPA152201 to be a potential insecticide to manage N. lugens.     

Amino acids outside of the loops that define the agonist binding site are important for ligand binding to insect nicotinic acetylcholine receptors

Nicotinic acetylcholine (ACh) receptors (nAChRs) are the targets of several kinds of insecticides. Based on the mutagenesis studies of Torpedo californica nAChRs and solved structure of a molluscan, glial-derived soluble ACh-binding protein, a model of the agonist site was constructed with contributing amino acids from three distinct loops (A, B, and C) of the α subunits and another three loops (D, E, and F) of the non-α subunits. According to this model, most insect nAChR subunits can form the functional heteromeric or homomeric receptors. Actually, insect subunits themselves did not form any functional receptor at various combinations as yet, and only part of them can form the functional receptors with vertebrate non-α subunits. These findings suggested that the agonist binding for insect nAChRs was not only contributed by those key amino acids in six loops, but also some unidentified amino acids from other regions. In our previous studies on nAChRs for Nilaparvata lugens , a target-site mutation (Y151S) was found within two α subunits (Nlα1 and Nlα3). In Drosophila S2 cells and Xenopus oocytes, Nlα1 can form functional receptors with rat β2 subunit. However, the same thing was not observed in Nlα3. In the present paper, by exchanging the corresponding regions between Nlα1 and Nlα3 to generate different chimeras, amino acid residues or residue clusters in the regions outside the six loops were found to play essential roles in agonist binding, especially for the amino acid clusters between loop B and C. This result indicated that the residues in the six loops could be necessary, but not enough for the activity of agonist binding.     

The insecticidal activity and action mode of an imidacloprid analogue, 1‐(3‐pyridylmethyl)‐2‐nitroimino‐imidazolidine

Neonicotinoids, such as imidacloprid, are key insecticides extensively used for control of Nilaparvata lugens. However, imidacloprid resistance has been reported in many Asian countries in recent years. To understand the roles of the chlorine atom of pyridyl group on insecticidal activity and resistance, the atom was removed to generate an imidacloprid analogue DC‐Imi (DesChlorine Imidacloprid). DC‐Imi showed significantly higher toxicity than imidacloprid in the susceptible strain of N. lugens, but had medium level cross‐resistance in an imidacloprid‐resistant strain. In Xenopus oocyte expressed nicotinic acetylcholine receptors (nAChRs) Nlα1/rβ2, the inward currents evoked by DC‐Imi were detected and could be blocked by typical nAChRs antagonist dihydro‐β‐erythroidine (DHβE), which demonstrated that DC‐Imi acted as an agonist on insect nAChRs. The efficacy of DC‐Imi on Nlα1/rβ2 was 1.8‐fold higher than that of imidacloprid. In addition, the influence of an imidacloprid resistance associated mutation (Y151S) on agonist potencies was evaluated. Compared with the wild‐type receptor, the mutation reduced maximal inward current of DC‐Imi to 55.6% and increased half maximal effective concentration (EC₅₀) to 3.53‐fold. Compared with imidacloprid (increasing EC₅₀ to 2.38‐fold of wild‐type receptor), Y151S mutation decreased DC‐Imi potency more significantly. The results indicated that the selective and possibly high toxicities could be achieved through the modification of 6‐chloro‐3‐pyridyl group in imidacloprid and other neonicotinoids.     

Neonicotinoid insecticides display partial and super agonist actions on native insect nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) are present in high density in insect nervous tissue and are targeted by neonicotinoid insecticides. Improved understanding of the actions of these insecticides will assist in the development of new compounds. Here, we have used whole-cell patch-clamp recording of cholinergic neurons cultured from the central nervous system of 3rd instar Drosophila larvae to examine the actions of acetylcholine (ACh) and nicotine, as well as the neonicotinoids imidacloprid, clothianidin and P-CH-clothianidin on native nAChRs of these neurons. Dose-response data yield an EC(50) value for ACh of 19 microm. Both nicotine and imidacloprid act as low efficacy agonists at native nAChRs, evoking maximal current amplitudes 10-14% of those observed for ACh. Conversely, clothianidin and P-CH-clothianidin evoke maximal current amplitudes up to 56% greater than those evoked by 100 microm ACh in the same neurons. This is the first demonstration of 'super' agonist actions of an insecticide on native insect nAChRs. Cell-attached recordings indicate that super agonism results from more frequent openings at the largest (63.5 pS) conductance state observed.     

A novel halogen bond and a better-known hydrogen bond cooperation of neonicotinoid and insect nicotinic acetylcholine receptor recognition

Neonicotinoid insecticides target the insect nicotinic acetylcholine receptor (nAChR) and are highly effective against the piercing-sucking pests. To explore the molecular interaction mechanism between the neonicotinoids and the insect nAChR, some key neonicotinoid compounds were docked into Aplysia californica acetylcholine binding protein (Ac-AChBP), which serves as a suitable structural surrogate of the insect nAChR. The binding mode study showed that the hydrogen bond force between the electronegative pharmacophore of the neonicotinoids and Cys190NH of the target binding pocket is crucial to the high efficiency of the neonicotinoids. Increasing the coplanarity between the guanidine or amidine and the electronegative pharmacophore of the neonicotinoids could increase the Π-Π stacking effect with Tyr188 of the Ac-AChBP and thus enhance the insecticidal potency. The introduction of an azide group to the chloropyridine ring of the neonicotinoids would reduce its binding ability due to the disappearance of a novel halogen bonding interaction. A series of novel neonicotinoid molecules were designed based on the halogen bonding interaction and two compounds with 6-bromopyridine-3-yl and 6-(trifluoromethyl)-3-pyridinyl were found to be with potential insecticidal activities.     

Insect nicotinic acetylcholine receptor gene families: from genetic model organism to vector,pest and beneficial species

Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic transmission in the insect nervous system and are targets of a major group of insecticides, the neonicotinoids. Analyses of genome sequences have shown that nAChR gene families remain compact in diverse insect species, when compared to their mammalian counterparts. Thus, Drosophila melanogaster and Anopheles gambiae each possess 10 nAChR genes while Apis mellifera has 11. Although these are among the smallest nAChR gene families known, receptor diversity can be considerably increased by alternative splicing and mRNA A-to-I editing, thereby generating species-specific subunit isoforms. In addition, each insect possesses at least one highly divergent nAChR subunit. Species-specific subunit diversification may offer promising targets for future rational design of insecticides that act on particular pests while sparing beneficial insects. Electrophysiological studies on cultured Drosophila cholinergic neurons show partial agonist actions of the neonicotinoid imidacloprid and super-agonist actions of another neonicotinoid, clothianidin, on native nAChRs. Recombinant hybrid heteromeric nAChRs comprising Drosophila Dα2 and a vertebrate β2 subunit have been instructive in mimicking such actions of imidacloprid and clothianidin. Unitary conductance measurements on native nAChRs indicate that more frequent openings of the largest conductance state may offer an explanation for the superagonist actions of clothianidin.     

The neonicotinoid electronegative pharmacophore plays the crucial role in the high affinity and selectivity for the Drosophila nicotinic receptor: an anomaly for the nicotinoid cation--pi interaction model

Cation-pi interaction, a prominent feature in agonist recognition by neurotransmitter-gated ion channels, does not apply to the anomalous action of neonicotinoids at the insect nicotinic acetylcholine receptor (nAChR). Insect-selective neonicotinoids have an electronegative pharmacophore (tip) in place of the ammonium or iminium cation of the vertebrate-selective nicotinoids, suggesting topological divergence of the agonist-binding sites in insect and vertebrate nAChRs. This study defines the molecular and electronic basis for the potent and selective interaction of the neonicotinoid electronegative pharmacophore with a unique subsite of the Drosophila but not of the vertebrate alpha4beta2 nAChR. Target site potency and selectivity are retained when the usual neonicotinoid N-nitroimine (=NNO(2)) electronegative tip is replaced with N-nitrosoimine (=NNO) or N-(trifluoroacetyl)imine (=NCOCF(3)) in combination with an imidazolidine, imidazoline, thiazolidine, or thiazoline heterocycle. X-ray crystallography establishes coplanarity between the heterocyclic and imine planes, including the electronegative substituent in the trans configuration. The functional tip is the coplanar oxygen atom of the N-nitrosoimine or the equivalent oxygen of the N-nitroimine. Quantum mechanics in the gas and aqueous phases fully support the conserved coplanarity and projection of the strongly electronegative tip. Further, a bicyclic analogue with a nitro tip in the cis configuration but retaining coplanarity has a high potency, whereas the N-trifluoromethanesulfonylimine (=NSO(2)CF(3)) moiety lacking coplanarity confers very low activity. The coplanar system between the electronegative tip and guanidine-amidine moiety extends the conjugation and facilitates negative charge (delta(-)) flow toward the tip, thereby enhancing interaction with the proposed cationic subsite such as lysine or arginine in the Drosophila nAChR.     

Pretreatment of the cockroach cercal afferent/giant interneuron synapses with nicotinoids and neonicotinoids differently affects acetylcholine and nicotine-induced ganglionic depolarizations

We have recently demonstrated that neonicotinoid insecticides were able to act as agonists of postsynaptic nicotinic acetylcholine receptors (nAChRs) expressed at the synapse between the cercal nerve XI and the giant interneurons, in the sixth abdominal ganglion. In this work, we demonstrated that nicotinoids such as nornicotine acted as an agonist of nicotinic acetylcholine receptors expressed at cercal afferent/giant interneurons while cotinine was a poor agonist. Indeed, nornicotine induced a ganglionic depolarization which was blocked by the nicotinic antagonist mecamylamine. In addition, we found that pretreatment of the sixth abdominal ganglion with 1 and 10 μM nornicotine and cotinine had no significant effect on acetylcholine and nicotine-induced depolarization. But pretreatment with 1 and 10 μM acetamiprid and imidacloprid had a strong effect. 1 and 10 μM acetamiprid completely blocked acetylcholine-induced depolarization, whereas imidacloprid had a partial effect. The present work therefore suggests, in agreement with previous studies, that nornicotine and cotinine bind to distinct cockroach postsynaptic nAChRs, whereas acetamiprid and imidacloprid have competitive effects with acetylcholine and nicotine on ganglionic depolarization.     

Amplification of a Cytochrome P450 Gene Is Associated with Resistance to Neonicotinoid Insecticides in the Aphid Myzus persicae

The aphid Myzus persicae is a globally significant crop pest that has evolved high levels of resistance to almost all classes of insecticide. To date, the neonicotinoids, an economically important class of insecticides that target nicotinic acetylcholine receptors (nAChRs), have remained an effective control measure; however, recent reports of resistance in M. persicae represent a threat to the long-term efficacy of this chemical class. In this study, the mechanisms underlying resistance to the neonicotinoid insecticides were investigated using biological, biochemical, and genomic approaches. Bioassays on a resistant M. persicae clone (5191A) suggested that P450-mediated detoxification plays a primary role in resistance, although additional mechanism(s) may also contribute. Microarray analysis, using an array populated with probes corresponding to all known detoxification genes in M. persicae, revealed constitutive over-expression (22-fold) of a single P450 gene (CYP6CY3); and quantitative PCR showed that the over-expression is due, at least in part, to gene amplification. This is the first report of a P450 gene amplification event associated with insecticide resistance in an agriculturally important insect pest. The microarray analysis also showed over-expression of several gene sequences that encode cuticular proteins (2–16-fold), and artificial feeding assays and in vivo penetration assays using radiolabeled insecticide provided direct evidence of a role for reduced cuticular penetration in neonicotinoid resistance. Conversely, receptor radioligand binding studies and nucleotide sequencing of nAChR subunit genes suggest that target-site changes are unlikely to contribute to resistance to neonicotinoid insecticides in M. persicae.     

Crystal structures of Lymnaea stagnalis AChBP in complex with neonicotinoid insecticides imidacloprid and clothianidin

Neonicotinoid insecticides, which act on nicotinic acetylcholine receptors (nAChRs) in a variety of ways, have extremely low mammalian toxicity, yet the molecular basis of such actions is poorly understood. To elucidate the molecular basis for nAChR-neonicotinoid interactions, a surrogate protein, acetylcholine binding protein from Lymnaea stagnalis (Ls-AChBP) was crystallized in complex with neonicotinoid insecticides imidacloprid (IMI) or clothianidin (CTD). The crystal structures suggested that the guanidine moiety of IMI and CTD stacks with Tyr185, while the nitro group of IMI but not of CTD makes a hydrogen bond with Gln55. IMI showed higher binding affinity for Ls-AChBP than that of CTD, consistent with weaker CH-pi interactions in the Ls-AChBP-CTD complex than in the Ls-AChBP-IMI complex and the lack of the nitro group-Gln55 hydrogen bond in CTD. Yet, the NH at position 1 of CTD makes a hydrogen bond with the backbone carbonyl of Trp143, offering an explanation for the diverse actions of neonicotinoids on nAChRs.     

Insecticidal and neural activities of candidate photoaffinity probes for neonicotinoid binding sites

Photoreactive derivatives of imidacloprid and its nitromethylene analogue were synthesized as candidate photoaffinity probes for identifying the amino acid residues of nicotinic acetylcholine receptors (nAChRs) that interact with the neonicotinoid insecticides. When the candidate probes were injected into American cockroaches, the nerve cord neural activity initially increased, then ceased and death of the insect followed. Both the nerve cord and toxicity were enhanced by changing the photoreactive substituent from the para position to the meta position on the spacer benzyl moiety. When tested on a Drosophila SAD/chicken beta2 hybrid, recombinant nAChR expressed in Xenopus oocytes, the nitromethylene candidate probes showed agonist activity similar to that previously observed for imidacloprid.     

Nicotinic acetylcholine receptors: targets for commercially important insecticides

Nicotinic acetylcholine receptors (nAChRs) are major excitatory neurotransmitter receptors in both vertebrates and invertebrates. In insects, nAChRs are the target site for several naturally occurring and synthetic compounds that exhibit potent insecticidal activity. Several compounds isolated from plants are potent agonists or antagonists of nAChRs, suggesting that these may have evolved as a defence mechanism against insects and other herbivores. Nicotine, isolated from the tobacco plant, has insecticidal activity and has been used extensively as a commercial insecticide. Spinosad, a naturally occurring mixture of two macrocyclic lactones isolated from the microorganism Saccharopolyspora spinosa, acts upon nAChRs and has been developed as a commercial insecticide. Since the early 1990s, one of the most widely used and rapidly growing classes of insecticides has been the neonicotinoids. Neonicotinoid insecticides are potent selective agonists of insect nAChRs and are used extensively in both crop protection and animal health applications. As with other classes of insecticides, there is growing evidence for the evolution of resistance to insecticides that act on nAChRs.