Vertebrate nAChR Research Articles

Potency matters: Impacts of embryonic exposure to nAChR agonists thiamethoxam and nicotine on hatching success, growth, and neurobehavior in larval zebrafish

ABSTRACT Thiamethoxam (TM) is a neonicotinoid insecticide that acts as a nicotinic acetylcholine receptor (nAChR) agonist. While designed to specifically target invertebrate nAChRs, recent studies have reported adverse effects of neonicotinoid exposure in early life-stage fish. This study examined the health and neurobehavioral impacts of chronic exposure to various concentrations of TM or nicotine (NIC) in early life zebrafish (Danio rerio) in conjunction with in-silico molecular docking to compare their ligand–receptor interactions with vertebrate nAChR. Chronic exposure to both reduced survival by approximately 20% (163 µg TM/l) and 25–100% (≥0.49 µg NIC/l). Hatching and growth were impaired following exposure to ≥0.21 µg TM/l or 4.9 µg NIC/l. Both TM and NIC produced morphological and behavioral indicators of neurotoxicity, with more potent effects following NIC exposure. NIC impaired embryonic motor activity by 40% (49 µg NIC/l), while both TM and NIC significantly altered predator escape response in larvae, specifically the latency and the initial burst movement of the response were impacted. Molecular docking predicted variations in the type and strength of interactions that occur between NIC or TM and vertebrate nAChR. These findings demonstrate that chronic exposure to TM might impact general health and neurobehavior of early-stage zebrafish. Our data support hypotheses that TM presents low affinity for vertebrate nAChR but may still pose an adverse risk to larval fish growth and neurobehavior.

Interactions of Nereistoxin and Its Analogs with Vertebrate Nicotinic Acetylcholine Receptors and Molluscan ACh Binding Proteins.

Nereistoxin (NTX) is a marine toxin isolated from an annelid worm that lives along the coasts of Japan. Its insecticidal properties were discovered decades ago and this stimulated the development of a variety of insecticides such as Cartap that are readily transformed into NTX. One unusual feature of NTX is that it is a small cyclic molecule that contains a disulfide bond. In spite of its size, it acts as an antagonist at insect and mammalian nicotinic acetylcholine receptors (nAChRs). The functional importance of the disulfide bond was assessed by determining the effects of inserting a methylene group between the two sulfur atoms, creating dimethylaminodithiane (DMA-DT). We also assessed the effect of methylating the NTX and DMA-DT dimethylamino groups on binding to three vertebrate nAChRs. Radioligand receptor binding experiments were carried out using washed membranes from rat brain and fish (Torpedo) electric organ; [3H]-cytisine displacement was used to assess binding to the predominantly high affinity alpha4beta2 nAChRs and [125I]-alpha-bungarotoxin displacement was used to measure binding of NTX and analogs to the alpha7 and skeletal muscle type nAChRs. While the two quaternary nitrogen analogs, relative to their respective tertiary amines, displayed lower α4β2 nAChR binding affinities, both displayed much higher affinities for the Torpedo muscle nAChR and rat alpha7 brain receptors than their respective tertiary amine forms. The binding affinities of DMA-DT for the three nAChRs were lower than those of NTX and MeNTX. An AChBP mutant lacking the C loop disulfide bond that would potentially react with the NTX disulfide bond displayed an NTX affinity very similar to the parent AChBP. Inhibition of [3H]-epibatidine binding to the AChBPs was not affected by exposure to NTX or MeNTX for up to 24 hr prior to addition of the radioligand. Thus, the disulfide bond of NTX is not required to react with the vicinal disulfide in the AChBP C loop for inhibition of [3H]-epibatidine binding. However, a reversible disulfide interchange reaction of NTX with nAChRs might still occur, especially under reducing conditions. Labeled MeNTX, because it can be readily prepared with high specific radioactivity and possesses relatively high affinity for the nAChR-rich Torpedo nAChR, would be a useful probe to detect and identify any nereistoxin adducts.

Neurotoxic Effects of Neonicotinoids on Mammals: What Is There beyond the Activation of Nicotinic Acetylcholine Receptors?-A Systematic Review.

Neonicotinoids are a class of insecticides that exert their effect through a specific action on neuronal nicotinic acetylcholine receptors (nAChRs). The success of these insecticides is due to this mechanism of action, since they act as potent agonists of insect nAChRs, presenting low affinity for vertebrate nAChRs, which reduces potential toxic risk and increases safety for non-target species. However, although neonicotinoids are considered safe, their presence in the environment could increase the risk of exposure and toxicity. On the other hand, although neonicotinoids have low affinity for mammalian nAChRs, the large quantity, variety, and ubiquity of these receptors, combined with its diversity of functions, raises the question of what effects these insecticides can produce in non-target species. In the present systematic review, we investigate the available evidence on the biochemical and behavioral effects of neonicotinoids on the mammalian nervous system. In general, exposure to neonicotinoids at an early age alters the correct neuronal development, with decreases in neurogenesis and alterations in migration, and induces neuroinflammation. In adulthood, neonicotinoids induce neurobehavioral toxicity, these effects being associated with their modulating action on nAChRs, with consequent neurochemical alterations. These alterations include decreased expression of nAChRs, modifications in acetylcholinesterase activity, and significant changes in the function of the nigrostriatal dopaminergic system. All these effects can lead to the activation of a series of intracellular signaling pathways that generate oxidative stress, neuroinflammation and, finally, neuronal death. Neonicotinoid-induced changes in nAChR function could be responsible for most of the effects observed in the different studies.

The mechanism of loop C-neonicotinoid interactions at insect nicotinic acetylcholine receptor \u03b11 subunit predicts resistance emergence in pests

Neonicotinoids selectively modulate insect nicotinic acetylcholine receptors (insect nAChRs). Studies have shown that serine with ability to form a hydrogen bond in loop C of some insect nAChR α subunits and glutamate with a negative charge at the corresponding position in vertebrate nAChRs may contribute to enhancing and reducing the neonicotinoid actions, respectively. However, there is no clear evidence what loop C properties underpin the target site actions of neonicotinoids. Thus, we have investigated the effects of S221A and S221Q mutations in loop C of the Drosophila melanogaster Dα1 subunit on the agonist activity of imidacloprid and thiacloprid for Dα1/chicken β2 nAChRs expressed in Xenopus laevis oocytes. The S221A mutation hardly affected either the affinity or efficacy for ACh and imidacloprid, whereas it only slightly reduced the efficacy for thiacloprid on the nAChRs with a higher composition ratio of β2 to Dα1 subunits. The S221Q mutation markedly reduced the efficacy of the neonicotinoids for the nAChRs with a higher composition of the β2 subunit lacking basic residues critical for binding neonicotinoids. Hence, we predict the possibility of enhanced neonicotinoid resistance in pest insect species by a mutation of the serine when it occurs in the R81T resistant populations lacking the basic residue in loop D of the β1 subunit.

Investigation of the Possible Pharmacologically Active Forms of the Nicotinic Acetylcholine Receptor Agonist Anabaseine.

Three major forms of the nicotinic agonist toxin anabaseine (cyclic iminium, cyclic imine and the monocationic open-chain ammonium-ketone) co-exist in almost equal concentrations at physiological pH. We asked the question: Which of these forms is pharmacologically active? First, we investigated the pH dependence of anabaseine inhibition of [3H]-methylcarbamylcholine binding at rat brain α4β2 nicotinic acetylcholine receptors (nAChRs). These experiments indicated that one or both monocationic forms interact with the orthosteric binding site for ACh. However, since they occur at equal concentrations near physiological pH, we employed another approach, preparing a stable analog of each form and examining its agonist activities and binding affinities at several vertebrate brain and neuromuscular nAChRs. Only 2-(3-pyridyl)-1,4,5,6-tetrahydropyrimidine monohydrogen chloride (PTHP), the cyclic iminium analog, displayed nAChR potencies and binding affinities similar to anabaseine. The cyclic imine analog 2,3′-bipyridyl and the open-chain ammonium-ketone analog 5-methylamino-1-(3-pyridyl)-1-pentanone (MAPP), displayed ≤1% of the activity predicted if the one form was solely active. The lower potency of weakly basic 2,3′-bipyridyl can be explained by the presence of a small concentration of its monocationic form. Since the open chain ammonium-ketone monocationic form of anabaseine has some structural similarity to the neurotransmitter GABA, we also tested the ability of anabaseine and its 1,2-dehydropyrrolidinyl analog myosmine to activate a mammalian GABAA receptor, but no activity was detected. We conclude that the monocationic cyclic iminium is the form which avidly binds and activates vertebrate nAChRs.

The neonicotinoids acetamiprid and imidacloprid impair neurogenesis and alter the microglial profile in the hippocampal dentate gyrus of mouse neonates.

Acetamiprid (ACE) and imidacloprid (IMI) are widely used neonicotinoid pesticides. They bind selectively to insect nicotinic acetylcholine receptors (nAChRs) and are considered non-hazardous to mammals. Few studies have assessed the activation of vertebrate nAChRs and the neurodevelopmental toxicity following in utero or neonatal exposure to neonicotinoids; therefore, we evaluated the effects of ACE or IMI exposure on neurogenesis and microglial profiles in the developing hippocampal dentate gyrus (DG) of mouse neonates. Mice were exposed to ACE, IMI (both 5mg/kg/day) or nicotine (0.5mg/kg/day) from postnatal day (P)12 to P26 by oral gavage. On P27, brains were removed, and neurogenesis and microglial activation in the hippocampal DG were examined via immunohistochemistry. A reduction in neurogenesis in the hippocampal DG of neonates following ACE, IMI and nicotine treatment was found. Additionally, neonicotinoid-exposed newborns showed an increase in the number of amoeboid-type and activated M1-type microglia. These results suggest that exposure to ACE and IMI impairs neurogenesis and alters microglial profiles in the developing hippocampal DG following oral dosing in an early postnatal period. A better understanding of the potential effects of these pesticides on human infant health is an important goal of our research.

Pharmacological profile of Ascaris suum ACR-16, a new homomeric nicotinic acetylcholine receptor widely distributed in Ascaris tissues.

SummaryBackground and PurposeControl of nematode parasite infections relies largely on anthelmintic drugs, several of which act on nicotinic ACh receptors (nAChRs), and there are concerns about the development of resistance. There is an urgent need for development of new compounds to overcome resistance and novel anthelmintic drug targets. We describe the functional expression and pharmacological characterization of a homomeric nAChR, ACR‐16, from a nematode parasite.Experimental ApproachUsing RT‐PCR, molecular cloning and two‐electrode voltage clamp electrophysiology, we localized acr‐16 mRNA in Ascaris suum (Asu) and then cloned and expressed acr‐16 cRNA in Xenopus oocytes. Sensitivity of these receptors to cholinergic anthelmintics and a range of nicotinic agonists was tested.Key ResultsAmino acid sequence comparison with vertebrate nAChR subunits revealed ACR‐16 to be most closely related to α7 receptors, but with some striking distinctions. acr‐16 mRNA was recovered from Asu somatic muscle, pharynx, ovijector, head and intestine. In electrophysiological experiments, the existing cholinergic anthelmintic agonists (morantel, levamisole, methyridine, thenium, bephenium, tribendimidine and pyrantel) did not activate Asu‐ACR‐16 (except for a small response to oxantel). Other nAChR agonists: nicotine, ACh, cytisine, 3‐bromocytisine and epibatidine, produced robust current responses which desensitized at a rate varying with the agonists. Unlike α7, Asu‐ACR‐16 was insensitive to α‐bungarotoxin and did not respond to genistein or other α7 positive allosteric modulators. Asu‐ACR‐16 had lower calcium permeability than α7 receptors.Conclusions and ImplicationsWe suggest that ACR‐16 has diverse tissue‐dependent functions in nematode parasites and is a suitable drug target for development of novel anthelmintic compounds.

Impaired Hippocampal Theta Oscillations in the Mice Null Alpha7 Nicotinic Acetylcholine Receptors

Nicotinic acetylcholine receptor (nAChR) a7 subunits are among the most ancient of vertebrate nAChR subunits and have the capacity to assemble as homomers. Naturally expressed a7-nAChRs in the central nervous system play a critical role in modulating the release of various neurotransmitters, neurite outgrowth, and even neuronal survival or death [1]. a7-nAChRs also have been implicated in development, differentiation, and disease of the nervous system, as well as being targets of nicotine action. a7-nAChRs are highly expressed in the hippocampus, a brain structure associated to learning, memory, and cognitive functions. Accumulating lines of evidence demonstrate that a7-nAChRs are key targets in mediating cholinergic modulations of cognition [2]. Hippocampus plays an important role in declarative memory and learning, and possesses a large amplitude rhythm at theta frequency (4–12 Hz), which is postulated to support mnemonic processes in humans and rodents [3,4]. It has been reported that nicotine modulates hippocampal theta oscillations likely through a4b2-nAChRs [5,6], but the role of a7-nAChRs in hippocampal theta oscillations is still uncertain. Whether or not a7-nAChR knockout (KO) mice exhibit hippocampal theta oscillation is unknown. Here, we present, for the first time, that a7-nAChR KO mice selectively lack hippocampal theta oscillations. All experimental procedures were approved by the Institutional Animal Care and Use Committee at the Barrow Neurological Institute. The data were collected from 27 hippocampal slices from 8 wild-type (WT) mice (postnatal 28 3 days) and 16 hippocampal slices from 6 a7-nAChR KOmice (postnatal 26 3 days, P > 0.05 between WT and a7-nAChR KO mice, t-test). Transverse hippocampal slices (450 lm) were prepared as previously reported [7]. External solution contained (mM) NaCl 124, NaHCO3 26, KCl 3, KH2PO4 1.2, CaCl2 2.4, MgSO4 1.3, D-glucose 10, which was continuously bubbled with 95% O2–5% CO2 during the entire experimental period. Field potential recordings were performed in the CA1 cell layer using a glass electrode filled with 2 M NaCl. During recordings, the recording chamber was set to 31°C. Hippocampal theta oscillations were induced by bath application of 50 lM carbachol (CCh) for 50–60 min, as previously described [5,8]. Under this experimental protocol, only one slice showed no response to CCh in 27 tested slices from WT mice, while six slices showed no response to CCh in 16 tested slices from a7-nAChR KO mice. As shown in Figure 1, in the continuous presence of 50 lM CCh, there were typically three types of oscillations, slow (delta: 0.5 0.06 Hz, n = 18), theta (6.3 0.46 Hz, n = 23), and super-fast (146.1 7.0 Hz, n = 19) oscillations in WT hippocampal slices (Figure 1A). However, in a7-nAChR KO hippocampal

A Fungal Metabolite Asperparaline A Strongly and Selectively Blocks Insect Nicotinic Acetylcholine Receptors: The First Report on the Mode of Action

Asperparalines produced by Aspergillus japonicus JV-23 induce paralysis in silkworm (Bombyx mori) larvae, but the target underlying insect toxicity remains unknown. In the present study, we have investigated the actions of asperparaline A on ligand-gated ion channels expressed in cultured larval brain neurons of the silkworm using patch-clamp electrophysiology. Bath-application of asperparaline A (10 µM) had no effect on the membrane current, but when delivered for 1 min prior to co-application with 10 µM acetylcholine (ACh), it blocked completely the ACh-induced current that was sensitive to mecamylamine, a nicotinic acetylcholine receptor (nAChR)-selective antaogonist. In contrast, 10 µM asperparaline A was ineffective on the γ-aminobutyric acid- and L-glutamate-induced responses of the Bombyx larval neurons. The fungal alkaloid showed no-use dependency in blocking the ACh-induced response with distinct affinity for the peak and slowly-desensitizing current amplitudes of the response to 10 µM ACh in terms of IC50 values of 20.2 and 39.6 nM, respectively. Asperparaline A (100 nM) reduced the maximum neuron response to ACh with a minimal shift in EC50, suggesting that the alkaloid is non-competitive with ACh. In contrast to showing marked blocking action on the insect nAChRs, it exhibited only a weak blocking action on chicken α3β4, α4β2 and α7 nAChRs expressed in Xenopus laevis oocytes, suggesting a high selectivity for insect over certain vertebrate nAChRs.

Unique Neonicotinoid Binding Conformations Conferring Selective Receptor Interactions

Neonicotinoid agonists selectively act on the insect nicotinic acetylcholine receptor (nAChR). The molecular basis for this specificity is deciphered by comparisons of two acetylcholine binding proteins (AChBPs) with distinct pharmacological profiles that serve as structural homologues for the nAChR subtypes. Aplysia AChBP has high neonicotinoid sensitivity, whereas Lymnaea AChBP has low neonicotinoid sensitivity, pharmacologies reminiscent of insect and vertebrate nAChR subtypes, respectively. The ligand-receptor interactions for these AChBPs were established by chemical and structural neurobiology approaches. Neonicotinoids and nicotinoids bind in a single conformation with Aplysia AChBP, wherein the electronegative nitro or cyano pharmacophore of the neonicotinoid faces in a reversed orientation relative to the cationic nicotinoid functionality. For Lymnaea AChBP, the neonicotinoids have two binding conformations in this vertebrate receptor model, which are completely inverted relative to each other, whereas nicotinoids are nestled in only one conserved conformation. Therefore, the unique binding conformations of nicotinic agonists determine the selective receptor interactions.

An Ion Selectivity Filter in the Extracellular Domain of Cys-loop Receptors Reveals Determinants for Ion Conductance

Neurotransmitter binding to Cys-loop receptors promotes a prodigious transmembrane flux of several million ions/s, but to date, structural determinants of ion flux have been identified flanking the membrane-spanning region. Using x-ray crystallography, sequence analysis, and single-channel recording, we identified a novel determinant of ion conductance near the point of entry of permeant ions. Co-crystallization of acetylcholine-binding protein with sulfate anions revealed coordination of SO4(2-) with a ring of lysines at a position equivalent to 24 A above the lipid membrane in homologous Cys-loop receptors. Analysis of multiple sequence alignments revealed that residues equivalent to the ring of lysines are negatively charged in cation-selective receptors but are positively charged in anion-selective receptors. Charge reversal of side chains at homologous positions in the nicotinic receptor from the motor end plate decreases unitary conductance up to 80%. Selectivity filters stemming from transmembrane alpha-helices have similar pore diameters and compositions of amino acids. These findings establish that when the channel opens under a physiological electrochemical gradient, permeant ions are initially stabilized within the extracellular vestibule of Cys-loop receptors, and this stabilization is a major determinant of ion conductance.

Molecular Recognition of Neonicotinoid Insecticides: The Determinants of Life or Death

Until the mid-20th century, pest insect control in agriculture relied on largely inorganic and botanical insecticides, which were inadequate. Then, the remarkable insecticidal properties of several organochlorines, organophosphates, methylcarbamates, and pyrethroids were discovered, leading to an arsenal of synthetic organics. The effectiveness of these insecticides, however, diminished over time due to the emergence of resistant insect strains with less sensitive molecular targets in their nervous systems. This created a critical need for a new type of neuroactive insecticide with a different yet highly sensitive target. Nicotine in tobacco extract was for centuries the best available agent to prevent sucking insects from damaging crops, although this alkaloid was hazardous to people and not very effective. The search for unusual structures and optimization revealed a new class of potent insecticides, known as neonicotinoids, which are similar to nicotine in their structure and action as agonists of the nicotinic acetylcholine receptor (nAChR). Fortunately, neonicotinoids are much more toxic to insects than mammals due in large part to differences in their binding site interactions at the corresponding nAChRs. This Account discusses the progress that has been made in defining the structural basis of neonicotinoid and nicotinoid potency and selectivity. The findings are based on comparisons of two acetylcholine binding proteins (AChBPs) with distinct pharmacological profiles that serve as structural surrogates for the extracellular ligand-binding domain of the nAChRs. Saltwater mollusk (Aplysia californica) AChBP has high neonicotinoid sensitivity, whereas freshwater snail (Lymnaea stagnalis) AChBP has low neonicotinoid and high nicotinoid sensitivities, pharmacologies reminiscent of insect and vertebrate nAChR subtypes, respectively. The ligand-receptor interactions for these AChBPs were established by photoaffinity labeling and X-ray crystallography. Both azidopyridinyl neonicotinoid and nicotinoid photoprobes bind in a single conformation with Aplysia AChBP; this is consistent with high-resolution crystal structures. Surprisingly, though, the electronegative nitro or cyano moiety of the neonicotinoid faced in a reversed orientation relative to the cationic nicotinoid functionality. For the Lymnaea AChBP, the azidoneonicotinoid probes modified two distinct and distant sites, while the azidonicotinoid probes, surprisingly, derivatized only one point. This meant that the neonicotinoids have two bound conformations in the vertebrate receptor model, which are completely inverted relative to each other, whereas nicotinoids appear buried in only one conserved conformation. Therefore, the unique binding conformations of nicotinic agonists in these insect and vertebrate receptor homologues define the basis for molecular recognition of neonicotinoid insecticides as the determinants of life or death.

Atypical nicotinic agonist bound conformations conferring subtype selectivity

The nicotinic acetylcholine (ACh) receptor (nAChR) plays a crucial role in excitatory neurotransmission and is an important target for drugs and insecticides. Diverse nAChR subtypes with various subunit combinations confer differential selectivity for nicotinic drugs. We investigated the subtype selectivity of nAChR agonists by comparing two ACh-binding proteins (AChBPs) as structural surrogates with distinct pharmacological profiles [i.e., Lymnaea stagnalis (Ls) AChBP of low neonicotinoid and high nicotinoid sensitivities and Aplysia californica (Ac) AChBP of high neonicotinoid sensitivity] mimicking vertebrate and insect nAChR subtypes, respectively. The structural basis of subtype selectivity was examined here by photoaffinity labeling. Two azidoneonicotinoid probes in the Ls-AChBP surprisingly modified two distinct and distant subunit interface sites: loop F Y164 of the complementary or (-)-face subunit and loop C Y192 of the principal or (+)-face subunit, whereas three azidonicotinoid probes derivatized only Y192. Both the neonicotinoid and nicotinoid probes labeled Ac-AChBP at only one position at the interface between loop C Y195 and loop E M116. These findings were used to establish structural models of the two AChBP subtypes. In the Ac-AChBP, the neonicotinoids and nicotinoids are nestled in similar bound conformations. Intriguingly, for the Ls-AChBP, the neonicotinoids have two bound conformations that are inverted relative to each other, whereas nicotinoids appear buried in only one conserved conformation as seen for the Ac-AChBP subtype. Accordingly, the subtype selectivity is based on two disparate bound conformations of nicotinic agonists, thereby establishing an atypical concept for neonicotinoid versus nicotinoid selectivity between insect and vertebrate nAChRs.

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.

ShAR2β, a divergent nicotinic acetylcholine receptor subunit from the blood fluke Schistosoma

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that mediate the fast actions of the neurotransmitter, acetylcholine. Invertebrate nAChRs are of interest as they are targets of widely-selling insecticides and drugs that control nematode parasites. Here, we report the cloning of ShAR2beta, a candidate nAChR subunit from the blood fluke, Schistosoma haematobium, which is the third trematode nAChR subunit to be characterized. While ShAR2beta possesses key structural features common to all nAChRs, its amino acid sequence shares considerably low identity with those of insect, nematode and vertebrate nAChR subunits. In particular, the second transmembrane domain of ShAR2beta, which lines the ion channel, bears unusual amino acid residues which will likely give rise to a receptor with distinct functional properties. Phylogenetic analysis shows that ShAR2beta is a divergent nAChR subunit that may define a clade of trematode-specific subunits. We discuss our findings in the context of potentially exploiting this receptor as a target for controlling schistosome parasites.

Mechanisms of action of insecticides on ligand-gated ion channels

Ligand-gated ion channels (LGICs) mediate fast synaptic neurotransmission and are important targets for insecticides. Thus, the actions of several insecticides have been explored in electrophysiological studies on recombinant and native insect neuronal LGICs. I have shown that non-competitive antagonists of γ-aminobutyric acid gated Cl− channels also act on glutamate-gated Cl− channels, albeit at higher concentrations. Neonicotinoids are more potent agonists on recombinant hybrid nicotinic acetylcholine receptors (nAChRs) consisting of Drosophila Dα2 and vertebrate β2 subunits than those consisting of only vertebrate nAChR subunits (α4β2). Using this hybrid nAChR, clothianidin and related compounds containing a acyclic guanidine moiety were found to be super-agonists. Similar super-agonist actions of neonicotinoids were also observed on cultured Drosophila cholinergic neurons. Single channel nAChR recordings show that a clothianidin analogue induces a high conductance state in channel opening more frequently than acetylcholine, thereby offering a possible explanation for its super-agonist action. Unlike the case for clothianidin, imidacloprid attenuates the acetylcholine-induced re-sponse of native neuronal nAChRs when co-applied with ACh. These new discoveries add to our understanding of both the selectivity and the diverse actions of insecticides targeting LGICs.

A Family of Acetylcholine-gated Chloride Channel Subunits in Caenorhabditis elegans

The genome of the nematode Caenorhabditis elegans encodes a surprisingly large and diverse superfamily of genes encoding Cys loop ligand-gated ion channels. Here we report the first cloning, expression, and pharmacological characterization of members of a family of anion-selective acetylcholine receptor subunits. Two subunits, ACC-1 and ACC-2, form homomeric channels for which acetylcholine and arecoline, but not nicotine, are efficient agonists. These channels are blocked by d-tubocurarine but not by alpha-bungarotoxin. We provide evidence that two additional subunits, ACC-3 and ACC-4, interact with ACC-1 and ACC-2. The acetylcholine-binding domain of these channels appears to have diverged substantially from the acetylcholine-binding domain of nicotinic receptors.

Molecular characterization of Dalpha6 and Dalpha7 nicotinic acetylcholine receptor subunits from Drosophila: formation of a high-affinity alpha-bungarotoxin binding site revealed by expression of subunit chimeras.

Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic transmission in the insect brain and are target sites for neonicotinoid insecticides. Seven nAChR subunits (four alpha-type and three beta-type) have been cloned previously from Drosophila melanogaster, the model insect system and characterized by heterologous expression. Recently, three further putative nAChR alpha subunits (Dalpha5, Dalpha6 and Dalpha7) with sequence similarity to the vertebrate alpha7 subunit have been identified from Drosophila genome sequence data but there have been no reports, as yet, of their characterization by heterologous expression. In the present study, we report the first isolation of a full-length Dalpha7 cDNA and the independent molecular cloning of Dalpha6. Binding of nicotinic radioligands was not detected to full-length Dalpha6 or Dalpha7 subunits when expressed alone or when or co-expressed with other nAChR subunits in Drosophila or mammalian cell lines, but specific cell-surface binding of [(125)I]alpha-bungarotoxin (K(d) = 0.68 +/- 0.22 nm) and [(3)H]methyllycaconitine (K(d) = 0.27 +/- 0.06 nm) was detected after expression of a subunit chimera containing the ligand-binding domains of Dalpha6 fused to the C-terminal domain of the 5-hydroxytryptamine receptor 5HT(3A). Although cell-surface binding was not detected with a Dalpha7/5HT(3Alpha) chimera expressed alone, co-expression of the two subunit chimeras resulted in significantly enhanced levels of nicotinic radioligand binding (with no change in affinity). This is the first evidence for the formation of a nAChR binding site by heterologously expressed Drosophila nAChR subunits in the absence of a co-expressed vertebrate nAChR subunit. In addition to the formation of homomeric nAChR complexes, evidence has been obtained from both radioligand binding and co-immunoprecipitation studies for the co-assembly of Dalpha6 and Dalpha7 into heteromeric cell surface complexes.

The nicotinic acetylcholine receptor gene family of the pufferfish, Fugu rubripes☆

Nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission at nerve–muscle junctions and in the brain. However, the complete gene family of nAChRs has not so far been reported for any vertebrate organism. We have identified the complete nAChR gene family from the reference genome of the pufferfish, Fugu rubripes. It consists of 16 α and 12 non-α candidate subunits, making it the largest vertebrate nAChR gene family known to date. The gene family includes an unusual set of muscle-like nAChR subunits comprising two α1s, two β1s, one δ, one ε, and one γ. One of the β1 subunits possesses an aspartate residue and N-glycosylation sites hitherto shown to be necessary for δ-subunit function. Potential Fugu orthologs of neuronal nAChR subunits α2–4, α6, and β2–4 have been identified. Interestingly, the Fugu α5 counterpart appears to be a non-α subunit. Fugu possesses an expanded set of α7–9-like subunits and no α10 ortholog has been found. Two new candidate β subtypes, designated β5 and β6, may represent subunits yet to be found in the human genome. The Fugu nAChR gene structures are considerably more diverse than those of higher vertebrates, with evidence of “intron gain” in many cases. We show, using RT-PCR, that the Fugu nAChR subunits are expressed in a variety of tissues.

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.