Actions of Agonists, Fipronil and Ivermectin on the Predominant In Vivo Splice and Edit Variant (RDLbd, I/V) of the Drosophila GABA Receptor Expressed in Xenopus laevis Oocytes

Abstract

Ionotropic GABA receptors are the targets for several classes of insecticides. One of the most widely-studied insect GABA receptors is RDL (resistance to dieldrin), originally isolated from Drosophila melanogaster. RDL undergoes alternative splicing and RNA editing, which influence the potency of GABA. Most work has focussed on minority isoforms. Here, we report the first characterisation of the predominant native splice variant and RNA edit, combining functional characterisation with molecular modelling of the agonist-binding region. The relative order of agonist potency is GABA> muscimol> TACA> β-alanine. The I/V edit does not alter the potency of GABA compared to RDLbd. Docking calculations suggest that these agonists bind and activate RDLbdI/V through a similar binding mode. TACA and β-alanine are predicted to bind with lower affinity than GABA, potentially explaining their lower potency, whereas the lower potency of muscimol and isoguvacine cannot be explained structurally from the docking calculations. The A301S (resistance to dieldrin) mutation reduced the potency of antagonists picrotoxin, fipronil and pyrafluprole but the I/V edit had no measurable effect. Ivermectin suppressed responses to GABA of RDLbdI/V, RDLbd and RDLbdI/VA301S. The dieldrin resistant variant also showed reduced sensitivity to Ivermectin. This study of a highly abundant insect GABA receptor isoform will help the design of new insecticides.

Species specific RNA A-to-I editing of mosquito RDL modulates GABA potency and influences agonistic, potentiating and antagonistic actions of ivermectin

“…Lees et al (Lees et al, 2014) reported that the predominant edit in D. melanogaster RDL, I283V, which 288…”

Novel GABA receptor pesticide targets

“…Ivermectin was the essential agent in greatly reducing the incidence of river blindness in millions of people by controlling the schistisome vector [69]. Ave is a positive allosteric modulator of several ligand-gated channels including GABA-and glutamategated chloride channels and the α7-nicotinic receptor [70][71][72][73]. The GABA-R target for ave is designated here as AVE. [ 3 H]Ave is very effective as a radioligand for both insects and mammals in defining AVE action [55,73].…”

“…[ 3 H]Ave binding in Musca is not inhibited by GABA, fipronil or cyclodienes but is by flu. The C. elegans glutamategated chloride channel was important in structural definition of the RDL AVE site although a muscle glutamate receptor may be involved in contributing to or the cause of the toxicity [69][70][71][72][73][74][75]. Decreased binding is conferred by in silico mutations to A/Q6 and B1/S58 [75].…”

Exon 3 Splicing and Mutagenesis Identify Residues Influencing Cell Surface Density of Heterologously Expressed Silkworm (Bombyx mori) Glutamate-Gated Chloride Channels

“…Notably, the RNA editing occurs more frequently in adults than in embryonic and larval stages (Jones et al, 2009). Recently, a detailed characterization of the pharmacology of the most abundantly expressed splice/edit isoform of Drosophila RDL has been described (Lees et al, 2014).…”