荷包牡丹碱对家蝇GABA受体作用机理探究

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

[目的]探究荷包牡丹碱对杀虫剂重要靶标GABA受体抑制机理。[方法]基于家蝇Musca domestica GABA受体亚基基因MdRdl在第3外显子(a或b)和第6外显子(c或d)交替拼接产生ac、ad、bc和bd剪接体,采用双电极电压钳技术研究荷包牡丹碱对MdRdl 4个剪接体离子电流的抑制效果,鉴定其敏感性是否与外显子交替拼接有关。并采用分子对接技术分析荷包牡丹碱与家蝇GABA受体的可能结合位点。同时,采用共刺激法验证荷包牡丹碱和GABA受体变构抑制剂溴虫氟苯双酰胺是否存在互作效应。[结果]荷包牡丹碱对MdRdlac、MdRdlbc、MdRdlad和MdRdlbd4个剪接体的IC50分别为1.274、0.948、4.702和3.698 mmol/L;对I274F突变体的IC50分别为15.46、21.67、42.83和52.33 mmol/L;与野生型剪接体相比,4个突变体IC50分别增大约11、22、8和13倍。荷包牡丹碱和溴虫氟苯双酰胺与GABA受体外显子6编码的氨基酸残基存在互作效应。[结论]第274位异亮氨酸残基为荷包牡丹碱与家蝇GABA受体互作的关键靶点,且与外显子6编码的氨基酸残基一起,具有开发协同增效作用的昆虫GABA受体别构抑制剂的潜力。

[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 MdRdlac,MdRdlbc,MdRdlad and MdRdlbd 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.