Study on the mechanism of action of bicuculline on GABA receptor in Musca domestica

Objective In order to explore the inhibitory mechanism of bicuculline on GABA receptor, which was an important target of insecticide.

Method Based on alternately splicing at exon 3 (a or b) and exon 6 (c or d), Musca domestica GABA receptor subunit gene MdRdl yields ac, ad, bc and bd spliceosomes. Two-electrode voltage clamp technique was used to study the inhibitory effect of bicuculline on the ion current of four spliceosomes of MdRdl, characterizing whether its sensitivity was related to alternate splicing of the exons. The molecular docking technique was used to analyze the possible binding sites of bicuculline and GABA receptor of M. domestica. The interaction effect between bicuculline and the allosteric inhibitor of GABA receptors, broflanilide, was verified using the co-stimulation method.

Result The IC₅₀s of bicuculline on the four spliceosomes MdRdl_ac, MdRdl_bc, MdRdl_ad and MdRdl_bd were 1.274, 0.948, 4.702 and 3.698 mmol/L, on their I274F mutants were 15.46, 21.67, 42.83 and 52.33 mmol/L, respectively. The IC₅₀s of four mutants increased by about 11, 22, 8 and 13 times compared to their wildtype spliceosomes, respectively. Bicuculline and broflanilide showed interactions with amino acid residues encoded by the exon 6 of the GABA receptor.

Conclusion The isoleucine residue at position 274 on the GABA receptor was the key target for the interaction between bicuculline and GABA receptor of M. domestica. This isoleucine residue and the amino acid residues encoded by exon 6 together are potentially to be developed as the synergistic allosteric inhibitors of insect GABA receptors.