Regorafenib Single cell reverse transcriptase PCR on A melli
Single-cell reverse transcriptase-PCR on A. mellifera antennal lobe (AL) cells showed the expression of RDL as well as another GABA receptor like subunit, LCCH3 (Dupuis et al., 2010). Based on whole cell patch-clamp electrophysiology it was concluded that the predominant GABA receptors in AL cells are homomeric RDL complexes (Dupuis et al., 2010). In line with this, the GABA EC50 observed in AL cells (12.3±0.33μM) is similar to that of homomeric Amel_RDL expressed in Xenopus oocytes (Table 1). The GABA receptors in AL cells, however, appear to be slightly less sensitive to fipronil (IC50 0.823±0.19μM) when compared to Amel_RDL in this study (Table 1). This difference could be due to the presence of RDL+LCCH3 heteromeric receptors in AL cells or perhaps the association of GABA receptors with accessory proteins found in native cells that may affect ion channel properties (Dupuis et al., 2010).
Conclusions It is concluded that the diversity of the insect GABA receptor, RDL, is broadened by differential splicing in the TM3-TM4 intracellular loop. These variants do not appear to modulate GABA potency but can affect the actions of fipronil in a species-specific manner. When studying RDL from a certain species, several clones should be analysed to ascertain whether there are intracellular loop variants. A major challenge is to determine whether the intracellular loop variants are expressed in different tissues, at different development stages or co-assemble to form a heterogeneous population of ion channels. The heterologous expression of RDL variants found in A. mellifera may provide a useful counter-screen tool to assess the effects of agrochemical compounds on the GABA receptor of an important pollinator.
Acknowledgements The financial support of the Leverhulme Trust (JT-W) and Oxford Brookes University (JH and CC) are gratefully acknowledged.
Introduction γ-Aminobutyric Regorafenib (GABA) is a major inhibitory neurotransmitter that acts on metabotropic and ionotropic GABA receptors (GABARs) in the nervous systems of animals. The metabotropic GABAR belongs to the family of G protein-coupled receptors, whereas the ionotropic GABAR is a member of the Cys-loop family of ligand-gated ion channels. Both ionotropic and metabotropic GABARs are found in the insect nervous system, and the former is an important target of insecticides, such as fipronil.1, 2 A gene encoding an insect GABAR subunit was first cloned from dieldrin-resistant Drosophila melanogaster and was hence named rdl (resistant to dieldrin).3, 4 Subsequently, orthologous genes have been cloned from various insect species.5, 6 Heterologous expression of the insect GABAR subunit RDL in Xenopus laevis oocytes demonstrated a relationship with mammalian ionotropic GABARs due to their ability to open chloride channels upon activation by GABA. A number of studies have demonstrated that insect RDL GABARs contain multiple binding sites for agonists and different antagonists.6, 7 Traditional GABAR-targeting insecticides such as dieldrin and fipronil, which block channels by interacting with channel-lining amino-acid residues, are noncompetitive antagonists (NCAs) of insect GABARs. Novel classes of insecticidal GABAR NCAs that act at a unique allosteric site(s), including isoxazolines and benzamides, have overcome the emerging resistance to conventional NCA insecticides.8, 9, 10, 11, 12 Moreover, competitive antagonists (CAs) exhibit insecticidal activities, as they stabilize the closed conformation of GABAR channels by binding to the orthosteric binding site. Given that the mechanism of action and the binding sites of GABAR CAs are different from those of NCAs, CAs have significant potential for development as novel insecticides to overcome the increasing insect resistance to GABAR-targeting NCA insecticides. In our previous studies, several series of insect GABAR CAs, including gabazine analogues, 5-(4-piperidyl)-3-isothiazolol (thio-4-PIOL) analogues, and 5-carbamoyl-3-isoxazolol (5-CIOL) analogues, reportedly exhibited moderate inhibitory potencies, with micromolar IC50 values (Fig. 1).13, 14, 15, 16, 17 We observed enhanced antagonism of insect GABARs by introducing bicyclic aromatic rings into the 3-position of the pyridazine ring of gabazine, the 4-position of the 3-isothiazolol ring of thio-4-PIOL, or the 4-position of the 3-isoxazolol ring of 5-CIOL (Fig. 1).13, 14, 15, 16, 17 These data indicated that the 3-isoxazolol and 3-isothiazolol are effective scaffolds for insect GABAR CAs without a significant loss of affinity. However, the activity of 4-monocyclic aryl-5-CIOL analogues in insect GABARs has not been studied to date. To characterize more powerful CAs for insect GABARs, in the present study, we synthesized a series of 4-aryl-5-carbamoyl-3-isoxazolols and examined their antagonism in insect GABARs expressed in Xenopus oocytes using a two-electrode voltage clamp (TEVC) method. We also examined the insecticidal activities of these 4-aryl-5-carbamoyl-3-isoxazolols and performed ligand-docking studies to predict their interaction mechanism in insect GABARs.