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.
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.
Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.
Kristin
K
Lees
Department of Biochemistry, University of Oxford, Oxford, United Kingdom.
Maria
M
Musgaard
Department of Biochemistry, University of Oxford, Oxford, United Kingdom.
Siros
S
Suwanmanee
Wolfson Institute for Biomedical Research, Department of Medicine, University College London, London, United Kingdom.
Steven David
SD
Buckingham
Department of Biochemistry, University of Oxford, Oxford, United Kingdom.
Philip
P
Biggin
Wolfson Institute for Biomedical Research, Department of Medicine, University College London, London, United Kingdom.
David
D
Sattelle
D000818:Animals; D017931:DNA Primers / Q000235:genetics; D004330:Drosophila / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins / Q000737:chemistry / Q000378:metabolism*; D018755:GABA Agonists / Q000737:chemistry / Q000494:pharmacology*; D007559:Ivermectin / Q000737:chemistry / Q000494:pharmacology*; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism; D011720:Pyrazoles / Q000737:chemistry / Q000494:pharmacology*; D011963:Receptors, GABA-A / Q000737:chemistry / Q000378:metabolism*; D014982:Xenopus laevis
D017931:DNA Primers; D029721:Drosophila Proteins; D018755:GABA Agonists; D020033:Protein Isoforms; D011720:Pyrazoles; C082030:Rdl protein, Drosophila; D011963:Receptors, GABA-A; D007559:Ivermectin; C082360:fipronil