RDL GABA Receptor Research Articles

Convergent resistance to GABA receptor neurotoxins through plant-insect coevolution.

The molecular mechanisms of coevolution between plants and insects remain elusive. GABA receptors are targets of many neurotoxic terpenoids, which represent the most diverse array of natural products known. Over deep evolutionary time, as plant terpene synthases diversified in plants, so did plant terpenoid defence repertoires. Here we show that herbivorous insects and their predators evolved convergent amino acid changing substitutions in duplicated copies of the Resistance to dieldrin (Rdl) gene that encodes the GABA receptor, and that the evolution of duplicated Rdl and terpenoid-resistant GABA receptors is associated with the diversification of moths and butterflies. These same substitutions also evolved in pests exposed to synthetic insecticides that target the GABA receptor. We used in vivo genome editing in Drosophila melanogaster to evaluate the fitness effects of each putative resistance mutation and found that pleiotropy both facilitates and constrains the evolution of GABA receptor resistance. The same genetic changes that confer resistance to terpenoids across 300 Myr of insect evolution have re-evolved in response to synthetic analogues over one human lifespan.

The mode of action of isocycloseram: A novel isoxazoline insecticide

Isocycloseram is a novel isoxazoline insecticide and acaricide with activity against lepidopteran, hemipteran, coleopteran, thysanopteran and dipteran pest species. Isocycloseram selectively targets the invertebrate Rdl GABA receptor at a site that is distinct to fiproles and organochlorines. The widely distributed cyclodiene resistance mutation, A301S, does not affect sensitivity to isocycloseram, either in vitro or in vivo, demonstrating the suitability of isocylsoseram to control pest infestations with this resistance mechanism. Detailed studies demonstrated that the binding sites relevant to the insecticidal activity of avermectins and isocycloseram are distinct. Isocycloseram was shown to compete for binding with metadiamide insecticides related to broflanilide. In addition, a G335M mutation in the third transmembrane domain of the Rdl GABA receptor, impaired the ability of both isocycloseram and metadiamides to block the GABA mediated response. As such the Insecticides Resistance Action Committee (IRAC) has classified isocycloseram in Group 30 “GABA-Gated Chloride Channel Allosteric Modulators”.

Novel insecticidal 1,6-dihydro-6-iminopyridazine derivatives as competitive antagonists of insect RDL GABA receptors.

The ionotropic γ-aminobutyric acid (GABA) receptor (iGABAR) is an important target for insecticides and parasiticides. Our previous studies showed that competitive antagonists (CAs) of insect iGABARs have the potential to be used for developing novel insecticides and that the structural modification of gabazine (a representative CA of mammalian iGABARs) could lead to the identification of novel CAs of insect iGABARs. In the present study, a novel series of 1,3-di- and 1,3,5-trisubstituted 1,6-dihydro-6-iminopyridazines (DIPs) was designed using a versatile strategy and synthesized using facile methods. Electrophysiological studies showed that several target DIPs (30 μM) exhibited excellent antagonistic activities against common cutworm and housefly iGABARs consisting of RDL subunits. The IC50 values of 3-(4-methoxyphenyl), 3-(4-trifluoromethoxyphenyl), 3-(4-biphenylylphenyl), 3-(2-naphthyl), 3-(3,4-methylenedioxyphenyl), and 3,5-(4-methoxyphenyl) analogs ranged from 2.2 to 24.8μM. Additionally, several 1,3-disubstituted DIPs, especially 3-(4-trifluoromethoxyphenyl) and 3-(3,4-methylenedioxyphenyl) analogs, exhibited moderate insecticidal activity against common cutworm larvae, with >60% mortality at a concentration of 100 mg kg-1 . Molecular docking studies showed that the oxygen atom on the three-substituted aromatic ring could form a hydrogen bond with Arg254, which may enhance the activity of these DIPs against housefly iGABARs. This systematic study indicated that the presence of a carboxyl side chain shorter by one methylene than that of gabazine at the 1-position of the pyridazine ring is effective for maintaining the stable binding of these DIPs in insect iGABARs. Our study provides important information for the design of novel insect iGABAR CAs. © 2022 Society of Chemical Industry.

Application of computational methods in the analysis of pesticide target-site and resistance mechanisms.

Meta-diamide insecticides including broflanilide have a high insecticidal activity by acting on RDL GABA receptors. Both membrane potential assays and docking studies suggest that the target site of meta-diamides is different from that of conventional noncompetitive inhibitors, such as fipronil. In fact, meta-diamides are effective against cyclodiene- and fipronil-resistant pests that carry target-site mutations. Dinotefuran uniquely possesses a tetrahydrofuran ring, whereas other neonicotinoids possess aromatic rings. Moreover, dinotefuran has been reported to be effective against imidacloprid-resistant strains. A docking study predicted the weak binding of dinotefuran to cytochrome P450s which are associated with imidacloprid resistance. Metabolic assays revealed that dinotefuran was not metabolized by these cytochrome P450s. These findings suggest that the lack of metabolic activity of P450s against dinotefuran causes a low level of cross-resistance.

Important amino acids for function of the insect Rdl GABA receptor.

Insect Rdl GABA receptor is an important insecticide target. To design a novel insecticide, studies on the structures of homologous pentameric ligand-gated ion channels provide information about important amino acids that are necessary for the function of insect Rdl GABA receptors. L9'A, T12'A, T13'A, T13'S, M15'S, and M15'N mutations in the Drosophila Rdl GABA receptor subunit caused the protein to spontaneously adopt the open state conformation. In contrast, the S16'A, S16'T, S17'A, and S17'H mutant homomers showed the same levels of agonist and antagonist sensitivity as the wild-type receptor. The G336M mutation in the Drosophila Rdl GABA receptor abolished the agonist activities of ivermectin and milbemectin, but the F339M mutation did not. Additionally, the F339M mutation caused spontaneous opening of the receptor. In the Drosophila Rdl model, the hydrophobic girdle plays an important role in stabilization of the closed state. Mutations which decrease hydrophobic interactions resulted in spontaneous opening, supporting the importance of the hydrophobic girdle for keeping the channel closed. Through a mutational study of transmembrane 3 (TM3) cytoplasmic domain and Rdl GABA receptor modeling, hydrophobic interactions between TM3 and TM4 and intersubunit interaction were demonstrated to be important for channel gating. Alternatively, the intrasubunit interaction between TM2 and TM3 domains were less important for channel gating in case of Drosophila Rdl GABA receptor. This study demonstrates important amino acids critical to the function of the Drosophila Rdl GABA receptor based on the mutational studies and Drosophila Rdl GABA receptor modeling approach. © 2020 Society of Chemical Industry.

Insights into the synergistic mechanism of target resistance: A case study of N. lugens RDL-GABA receptors and fipronil

It is known that a single mutation exerts moderate resistance to pesticide, while double mutations (DM) cause severe resistance problem through synergistic effect, and even result in failure application of pesticides. However, little is known about how double mutations would synergistically cause much high resistance level. In this work, computational studies were performed on the interaction of fipronil with N. lugens RDL-GABA receptors, to see how single and double mutations changed receptor structure properties and then conferred distinct resistance levels. The A2′S mutation displayed relative weak influence on receptor structure properties. The R0′Q mutation, which has not been detected in the absence of A2′S, however could deeply alter the electrostatic potential around the inner pore region and significantly narrow the bottom region around -2′Pro. For the DM system, the synergistic effect of two mutations lead to similar pore diameters to the WT system, except for the slightly reduced middle part. Docking study and binding free energy calculation revealed that fipronil displayed binding potencies in the order of WT > A2′S > R0′Q > DM systems, coinciding well with the reported fipronil sensitivity trends and resistance levels.

Mechanisms of resistance to insecticides targeting RDL GABA receptors in planthoppers

Lindane and cyclodienes, such as dieldrin and α-endosulfan, represent the first generation of noncompetitive antagonists (NCAs) against the insect RDL GABA receptor. It has been reported that A2′S and A2′G mutations in the membrane-spanning region M2 of the RDL GABA receptor confer resistance to lindane and cyclodienes. Fipronil is a second-generation NCA, but A2′S and A2′G mutations provide a low level of cross-resistance to fipronil. Moreover, a novel A2′N mutation that confers fipronil resistance was found in M2 of the RDL GABA receptors from fipronil-resistant planthoppers in the heterozygous state. Thus, problems are being caused by a worldwide appearance of cyclodiene-resistant pests. And attention must be paid to development of fipronil-resistant planthoppers, such as Sogatella furcifera and Laodelphax striatellus in Japan.

The neonicotinoid imidacloprid, and the pyrethroid deltamethrin, are antagonists of the insect Rdl GABA receptor.

A mutation in the second transmembrane domain of the GABA receptor subunit, Rdl, is associated with resistance to insecticides such as dieldrin and fipronil. Molecular cloning of Rdl cDNA from a strain of the malaria mosquito, Anopheles gambiae, which is highly resistant to dieldrin revealed this mutation (A296G) as well as another mutation in the third transmembrane domain (T345M). Wild-type, A296G, T345M and A296G + T345M homomultimeric Rdl were expressed in Xenopus laevis oocytes and their sensitivities to fipronil, deltamethrin, 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane (DDT), imidacloprid and spinosad were measured using two-electrode voltage-clamp electrophysiology. Spinosad and DDT had no agonist or antagonist actions on Rdl. However, fipronil, deltamethrin and imidacloprid decreased GABA-evoked currents. These antagonistic actions were either reduced or abolished with the A296G and the A296G + T345M mutations while T345M alone appeared to have no significant effect. In conclusion, this study identifies another mutation in the mosquito Rdl that is associated with insecticide resistance. While T345M itself does not affect insecticide sensitivity, it may serve to offset the structural impact of A296G. The present study also highlights Rdl as a potential secondary target for neonicotinoids and pyrethroids. We show for the first time that deltamethrin (a pyrethroid insecticide) and imidacloprid (a neonicotinoid insecticide) act directly on the insect GABA receptor, Rdl. Our findings highlight Rdl as a potential secondary target of pyrethroids and neonicotinoids mutations in which may contribute to resistance to these widely used insecticides.

昆虫RDL GABA受容体の構造と薬剤感受性に関する研究

昆虫RDL GABA受容体の構造と薬剤感受性に関する研究

昆虫RDL GABA受容体の構造と薬剤感受性に関する研究

昆虫RDL GABA受容体の構造と薬剤感受性に関する研究

Comparison between the modes of action of novel meta-diamide and macrocyclic lactone insecticides on the RDL GABA receptor

Macrocyclic lactones, avermectins, and milbemycins are widely used to control arthropods, nematodes, and endo- and ectoparasites in livestock and pets. Their main targets are glutamate-gated chloride channels. Furthermore, macrocyclic lactones reportedly interact with insect RDL γ-aminobutyric acid (GABA) receptors, but their modes of action on insect RDL GABA receptors remain unknown. In this study, we attempted to better understand the modes of action of macrocyclic lactones on RDL GABA receptors. We observed that ivermectin and milbemectin behaved as allosteric agonists of the Drosophila RDL GABA receptor. G336A, G336S, and G336T mutations had profound effects on the activities of ivermectin and milbemectin, and a G336M mutation abolished the allosteric agonist and antagonist activities of these macrocyclic lactones. These results suggest that G336 in TM3 of the Drosophila RDL GABA receptor is important for the binding of macrocyclic lactones. Recently, it has been suggested that a novel RDL GABA receptor antagonist, 3-benzamido-N-(2-bromo-4-perfluoroisopropyl-6-(trifluoromethyl)phenyl)-2-fluorobenzamide (meta-diamide 7), binds to the transmembrane intersubunit pocket near G336 in the Drosophila RDL GABA receptor. Thus, we compared the effects of mutations around G336 and A302 mutations in TM2 on the activities of macrocyclic lactone and meta-diamide 7. The effects of L281C, V340Q, V340N, A302S, and A302N mutations on the activity of meta-diamide 7 differed from those on ivermectin and milbemectin. Molecular modeling studies showed that macrocyclic lactones docked in the intersubunit pocket near G336 in the Drosophila RDL GABA receptor in the open state. In contrast, meta-diamide 7 docked into the Drosophila RDL GABA receptor in the closed state. This suggests that the modes of action of macrocyclic lactone binding to the wild-type Drosophila RDL GABA receptor differ from those of meta-diamide binding.

Meta-diamide insecticides acting on distinct sites of RDL GABA receptor from those for conventional noncompetitive antagonists

The RDL GABA receptor is an attractive target of insecticides. Here we demonstrate that meta-diamides [3-benzamido-N-(4-(perfluoropropan-2-yl)phenyl)benzamides] are a distinct class of RDL GABA receptor antagonists showing high insecticidal activity against Spodoptera litura. We also suggest that the mode of action of the meta-diamides is distinct from that of conventional noncompetitive antagonists (NCAs), such as fipronil, picrotoxin, lindane, dieldrin, and α-endosulfan. Using a membrane potential assay, we examined the effects of the meta-diamide 3-benzamido-N-(2-bromo-4-(perfluoropropan-2-yl)-6-(trifluoromethyl)phenyl)-2-fluorobenzamide (meta-diamide 7) and NCAs on mutant Drosophila RDL GABA receptors expressed in Drosophila Mel-2 cells. NCAs had little or no inhibitory activity against at least one of the three mutant receptors (A2′S, A2′G, and A2′N), which were reported to confer resistance to NCAs. In contrast, meta-diamide 7 inhibited all three A2′ mutant receptors, at levels comparable to its activity with the wild-type receptor. Furthermore, the A2′S·T6′V mutation almost abolished the inhibitory effects of all NCAs. However, meta-diamide 7 inhibited the A2′S・T6′S mutant receptor at the same level as its activity with the wild-type receptor. In contrast, a G336M mutation in the third transmembrane domain of the RDL GABA receptor abolished the inhibitory activities of meta-diamide 7, although the G336M mutation had little effect on the inhibitory activities of conventional NCAs. Molecular modeling studies also suggested that the binding site of meta-diamides was different from those of NCAs. Meta-diamide insecticides are expected to be prominent insecticides effective against A2′ mutant RDL GABA receptors with a different mode of action.

A comparison of the modes of action of novel meta-diamide insecticides and conventional noncompetitive antagonists on the <i>Spodoptera litura</i> RDL GABA receptor

A comparison of the modes of action of novel meta-diamide insecticides and conventional noncompetitive antagonists on the <i>Spodoptera litura</i> RDL GABA receptor

Fipronil resistance in Sogatella furcifera: Molecular cloning and functional expression of wild-type and mutant RDL GABA receptor subunits

The whitebacked planthopper Sogatella furcifera is a serious pest of rice that has developed fipronil resistance in Asia. We found a novel R340Q mutation in the cytoplasmic loop between M3 and M4 of the S. furcifera RDL γ-aminobutyric acid (GABA) receptor subunit (SF-RDL). The R340Q mutation was found in clones carrying the A2′N mutation, which was previously implicated in fipronil resistance and suggested to be a heterozygous mutation. To investigate the influence of mutations, Drosophila Mel-2 cells were transfected with wild-type or mutant SF-RDL genes, either individually or together. A membrane potential assay showed that the influence of the A2′N·R340Q double mutation on fipronil resistance was more profound than that of the A2′N single mutation in the heterozygous expression of wild-type and mutant SF-RDL genes.

The role of GABA and glutamate receptors in susceptibility and resistance to chloride channel blocker insecticides

Despite a point mutation in the pore-forming segment of the Rdl GABA receptor subunit that is widespread and persistent in insect populations and confers high levels of resistance to dieldrin and other polychlorocycloalkane (PCCA) insecticides, the phenylpyrazole insecticide fipronil, which binds at same site, has proven to be effective in controlling many insects, including dieldrin-resistant populations. Fipronil and its major sulfone metabolite are unique among chloride channel blocking insecticides in that they also potently block GluCls. We present here a patch clamp study of the action of fipronil sulfone on native GABA receptors and GluCl receptors from susceptible and dieldrin-resistant German cockroaches, to provide a better understanding of the effect of the Rdl mutation on the function and insecticide sensitivity of these two targets, and its role in resistance. Dieldrin blocked GABA currents with an IC 50 of 3 nM in wild-type cockroaches, and 383 nM in resistant insects, yielding a resistance ratio of 128. Fipronil sulfone blocked GABA currents with an IC 50 of 0.8 nM in susceptible insects and 12.1 nM, or 15-fold higher, in resistant insects. While both GluClD (desensitizing) and GluClN (non-desensitizing) receptors were found in German cockroach neurons, GluClN receptors were rare and could not be included in this study. GluClD receptors from resistant insects had reduced sensitivity to glutamate and a lower rate of desensitization than those from susceptible insects, but their sensitivity to block by fipronil sulfone was not significantly changed, with an IC 50 of 38.5 ± 2.4 nM ( n = 8) in the susceptible strain and 40.3 ± 1.0 nM ( n = 7) in the resistant strain. Fipronil sulfone also slowed the decay time course of GluClD currents. These results suggest that GluClD receptors contain the Rdl subunit, but their sensitivity to fipronil sulfone is not altered in resistant insects.

Development of a Drosophila seizure model for in vivo high‐throughput drug screening

An important application of model organisms in neurological research has been to identify and characterise therapeutic approaches for epilepsy, a recurrent seizure disorder that affects > 1% of the human population. Proconvulsant-treated rodent models have been widely used for antiepileptic drug discovery and development, but are not suitable for high-throughput screening. To generate a genetically tractable model that would be suitable for large-scale, high-throughput screening for antiepileptic drug candidates, we characterized a Drosophila chemical treatment model using the GABA(A) receptor antagonist picrotoxin. This proconvulsant, delivered to Drosophila larvae via simple feeding methods suitable for automated screening, generated robust generalised seizures with lethality occurring at doses between 0.3 and 0.5 mg/mL. Electrophysiological analysis of CNS motor neuron output in picrotoxin-treated larvae revealed generalised seizures within minutes of drug exposure. At subthreshold doses for seizure induction, picrotoxin produced an increased frequency of motor neuron action potential bursting, indicating that CNS GABAergic transmission regulates patterned activity. Mutants in the Drosophila Rdl GABA(A) receptor are resistant to picrotoxin, confirming that seizure induction occurs via a conserved GABA(A) receptor pathway. To validate the usefulness of this model for in vivo drug screening, we identified several classes of neuroactive antiepileptic compounds in a pilot screen, including phenytoin and nifedipine, which can rescue the seizures and lethal neurotoxicity induced by picrotoxin. The well-defined actions of picrotoxin in Drosophila and the ease with which compounds can be assayed for antiseizure activity makes this genetically tractable model attractive for high-throughput in vivo screens to identify novel anticonvulsants and seizure susceptibility loci.

Preliminary analysis of the GABA-induced current in cultured CNS neurons of the cutworm moth, Spodoptera litura

Properties of GABA-induced current in cultured CNS (ganglion) neurons of cutworm moths ( Spodoptera litura) were studied using a whole-cell patch-clamp technique. CNS neurons ranging from 10 to 20 μm in diameter were cultured for 4–7 days in MGM-464 medium. GABA-induced a current response in these neurons in a sigmoidally dose dependent manner where the Hill coefficient and EC 50 were 2.2 and 33.0 μM, respectively. The reversal potential of GABA-induced current was −2.5 mV, which is close to the Cl − equilibrium potential that was calculated from chloride ion concentrations in the present experimental environment. Furthermore, the GABA-induced current response depended on the extracellular chloride ion concentration, indicating that the receptor regulates the Cl − permeability of cells. The GABA-induced current was completely inhibited by the GABA A antagonist, SR95531, and activated by the GABA A agonist, muscimol but not by the GABA B agonist, baclofen. On the other hand, the GABA C agonist, CACA, also induced a little smaller current than the GABA-induced response. The pharmacological behaviors of the GABA-induced currents suggest that these cells contain GABA receptors that belong to the GABA receptor family including the Rdl GABA receptors identified in Drosophila neurons.

Resistance to fipronil in Drosophila simulans: influence of two point mutations in the RDL GABA receptor subunit

The Eyguieres 42 strain of Drosophila simulans, obtained by laboratory selection, displayed approximately 20,000-fold resistance to the insecticide fipronil. Molecular cloning of the cDNA encoding the RDL GABA receptor subunit of this strain revealed the presence of two mutations: the Rdl mutation (A301G) and an additional mutation in the third transmembrane domain (T350M). In order to assess the individual and combined roles of the two mutations in fipronil resistance, the functional properties of wild-type, A301G, T350M and A301G/T350M homomultimeric RDL receptors were compared by expression in Xenopus oocytes. In wild-type receptors, the inhibition of GABA (EC(30))-induced currents by fipronil and picrotoxin was enhanced by repeated GABA applications. The A301G mutation nearly abolished this effect, decreased the sensitivity to fipronil and picrotoxin and increased the reversibility of inhibition. The T350M mutation also reduced the sensitivity to both antagonists. Of the four receptor variants tested, the double mutant showed the highest resistance to fipronil, following repeated GABA applications. In conclusion, the present study emphasizes new aspects of the pharmacological alterations induced by the Rdl mutation and shows that resistance to GABA receptor-directed insecticides may implicate a mutation distinct from Rdl.

Single channel analysis of the blocking actions of BIDN and fipronil on a Drosophila melanogaster GABA receptor (RDL) stably expressed in a Drosophila cell line.

Single channel recordings were obtained from a Drosophila S2 cell line stably expressing the wild-type RDL(ac) Drosophila melanogaster homomer-forming ionotropic GABA receptor subunit, a product of the resistance to dieldrin gene, RDL: GABA (50 microM) was applied by pressure ejection to outside-out patches from S2-RDL cells at a holding potential of -60 mV. The resulting inward current was completely blocked by 100 microM picrotoxin (PTX). The unitary current-voltage relationship was linear at negative potentials but showed slight inward rectification at potentials more positive than 0 mV. The reversal potential of the current (E(GABA)=-1.4 mV) was close to the calculated chloride equilibrium potential. The single channel conductance elicited by GABA was 36 pS. A 71 pS conductance channel was also observed when the duration of the pulse, used to eject GABA, was longer than 80 ms. The mean open time distribution of the unitary events was fitted best by two exponential functions suggesting two open channel states. When either 1 microM fipronil or 1 microM BIDN was present in the external saline, the GABA-gated channels were completely blocked. When BIDN or fipronil was applied at a concentration close to the IC(50) value for suppression of open probability (281 nM, BIDN; 240 nM, fipronil), the duration of channel openings was shortened. In addition, the blocking action of BIDN resulted in the appearance of a novel channel conductance (17 pS). The effects of co-application of BIDN and fipronil were examined. Co-application of BIDN (300 nM) with various concentrations (100-1000 nM) of fipronil resulted in an additional BIDN-induced dose-dependent reduction of the maximum P(o) value. Thus both BIDN and fipronil shorten the duration of wild-type RDL(ac) GABA receptor channel openings but appear to act at distinct sites.

Target site mediated insecticide resistance: what questions remain?

Recent advances in the molecular genetics of insecticide resistance have identified the point mutations associated with target site insensitivity in the genes encoding the three major insecticide targets: the Rdl GABA receptor, the para voltage gated sodium channel and insect acetylcholinesterase. However central questions relating to the origin, selection and fitness of these mutations in natural populations remain. This review examines the extent to which we understand how a specific subset of potential resistance associated mutations are selected, how often they may arise and/or recombine, and whether we can explain any potential fitness disadvantages based on our knowledge of the molecular mechanisms of resistance involved.