Functional Characteristics of the Lepidopteran Ionotropic GABA Receptor 8916 Subunit Interacting with the LCCH3 or the RDL Subunit

Abstract

The ionotropic γ-aminobutyric acid (iGABA) receptor is commonly considered as a fast inhibitory channel and is an important insecticide target. Since 1990, RDL, LCCH3, and GRD have been successively isolated and found to be potential subunits of the insect iGABA receptor. More recently, one orphan gene named 8916 was found and considered to be another potential iGABA receptor subunit according to its amino acid sequence. However, little information about 8916 has been reported. Here, the 8916 subunit from Chilo suppressalis was studied to determine whether it can form part of a functional iGABA receptor by co-expressing this subunit with CsRDL1 or CsLCCH3 in the Xenopus oocyte system. Cs8916 or CsLCCH3 did not form functional ion channels when expressed alone. However, Cs8916 was able to form heteromeric ion channels when expressed with either CsLCCH3 or CsRDL1. The recombinant heteromeric Cs8916/LCCH3 channel was a cation-selective channel, which was sensitive to GABA or β-alanine. The current of the Cs8916/LCCH3 channel was inhibited by dieldrin, endosulfan, fipronil, or ethiprole. In contrast, fluralaner, broflanilide, and avermectin showed little effect on the Cs8916/LCCH3 channel (IC50s > 10 000 nM). The Cs8916/RDL1 channel was sensitive to GABA, but was significantly different in EC50 and I max for GABA to those of homomeric CsRDL1. Fluralaner, fipronil, or dieldrin showed antagonistic actions on Cs8916/RDL1. In conclusion, Cs8916 is a potential iGABA receptor subunit, which can interact with CsLCCH3 to generate a cation-selective channel that is sensitive to several insecticides. Also, as Cs8916/RDL1 has a higher EC50 than homomeric CsRDL1, Cs8916 may affect the physiological functions of CsRDL1 and therefore play a role in fine-tuning GABAergic signaling.

Molecular Targets of Neurotoxic Insecticides in Apis mellifera

“…Similarly to mammals, GABA is the principal neurotransmitter mediating fast inhibitory neurotransmission in insects. Genomic and phylogenic analysis have revealed the existence of at least 4 different genes encoding GABA A subunits, namely the RDL (for Resistance to Dieldrin), GRD (GABA and glycine‐like receptor of Drosophila), LCCH3 (ligand‐gated chloride channel homologue 3) and CG8916 (also known as LCCH‐14 A) subunits [46] . All these subunits belong to the insect LGIC family and share similar structural features with mammalian Cys‐loop receptors.…”

“…Genomic and phylogenic analysis have revealed the existence of at least 4 different genes encoding GABA A subunits, namely the RDL (for Resistance to Dieldrin), GRD (GABA and glycine-like receptor of Drosophila), LCCH3 (ligand-gated chloride channel homologue 3) and CG8916 (also known as LCCH-14 A) subunits. [46] All these subunits belong to the insect LGIC family and share similar structural features with mammalian Cys-loop receptors. Genomes of insect species comprises a specific number of genes encoding GABA A subunits.…”

“…Recent structures obtained for mammalian LIGCs [84,85] will undoubtedly help to understand molecular events at work for selecting anion or cation in the various LGICs. The GRD amino‐acid sequence at M2 helice resembles that of CG8916 and it has been suggested recently that rice striped stem borer Chilo suppressalis CG8916 can reconstitute functional cationic receptors when co‐expressed with LCCH3 [46] . Therefore, it will be interesting to compare the pharmacology of these two potential GABA‐mediated cationic receptors in honeybee.…”

“…The GRD amino-acid sequence at M2 helice resembles that of CG8916 and it has been suggested recently that rice striped stem borer Chilo suppressalis CG8916 can reconstitute functional cationic receptors when co-expressed with LCCH3. [46] Therefore, it will be interesting to compare the pharmacology of these two potential GABA-mediated cationic receptors in honeybee.…”

5-(4-Pyridinyl)-3-isothiazolols as Competitive Antagonists of Insect GABA Receptors: Design, Synthesis, and a New Mechanism Leading to Insecticidal Effects

“…The first insect iGABAR subunit was identified from Drosophila melanogaster and named “resistant to dieldrin (RDL)” . Although the subunit composition of native insect iGABARs has yet to be determined, , RDL subunits have been reported to form GABA-gated chloride channels that resemble several aspects of native GABARs when expressed in Xenopus laevis oocytes or other cells . Insect iGABARs composed of RDL subunits (RDLRs) are relevant to vertebrate iGABARs, as they open intrinsic chloride channels upon GABA binding.…”