Eguchi Y, Ihara M, Ochi E, Shibata Y, Matsuda K, Fushiki S, Sugama H, Hamasaki Y, Niwa H, Wada M, Ozoe F, Ozoe Y
Department of Applied Biological Chemistry, Faculty of Agriculture, Kinki University, Nara, Japan.
Insect Mol Biol. 2006 Dec;15(6):773-83. doi: 10.1111/j.1365-2583.2006.00680.x.
Ligand-gated chloride channels (LGICs) are important targets for insecticides and parasiticides. Genes encoding subunits of two LGICs, a glutamate-gated chloride channel (MdGluCl-alpha) and a gamma-aminobutyric acid (GABA)-gated chloride channel (MdRdl), were cloned from house-flies (Musca domestica L.). These genes were first expressed independently in Xenopus laevis oocytes by cRNA injection in order to investigate the pharmacology of these ligand-gated channels using two-electrode voltage-clamp electrophysiology. It was found that L-glutamate and GABA activated the MdGluCl-alpha homo-oligomers with an EC(50) value of 30 microM and the MdRdl homo-oligomers with an EC(50) value of 101 microM, respectively. Both channels were chloride ion-permeable, and the MdRdl channel was more sensitive to chloride channel blockers, such as gamma-hexachlorocyclohexane (gamma-HCH), fipronil and picrotoxinin, than the MdGluCl-alpha channel. MdGluCl-alpha required only 1-2 days of incubation after cRNA injection to be expressed in oocytes, whereas 4-7 days of incubation was necessary to achieve MdRdl expression. However, when the cRNA of MdGluCl-alpha was injected at a dose of 1% (w/w) 1 day after the injection of the cRNA of MdRdl, a significant increase in the current amplitude of responses to GABA was observed, and the incubation period necessary for MdRdl expression became shorter. These results suggest that MdGluCl-alpha assists in the expression of MdRdl when the two are coexpressed.
配体门控氯离子通道(LGICs)是杀虫剂和杀寄生虫剂的重要作用靶点。从家蝇(Musca domestica L.)中克隆出了编码两种LGICs亚基的基因,即一种谷氨酸门控氯离子通道(MdGluCl-α)和一种γ-氨基丁酸(GABA)门控氯离子通道(MdRdl)。为了利用双电极电压钳电生理学研究这些配体门控通道的药理学特性,首先通过注射cRNA将这些基因分别在非洲爪蟾卵母细胞中独立表达。结果发现,L-谷氨酸和GABA分别以30μM的EC50值激活MdGluCl-α同型寡聚体和以101μM的EC50值激活MdRdl同型寡聚体。两种通道均对氯离子通透,并且MdRdl通道比MdGluCl-α通道对氯离子通道阻滞剂(如γ-六六六(γ-HCH)、氟虫腈和印防己毒素)更敏感。注射cRNA后,MdGluCl-α在卵母细胞中表达仅需1-2天的孵育时间,而MdRdl表达则需要4-7天的孵育时间。然而,当在注射MdRdl的cRNA 1天后以1%(w/w)的剂量注射MdGluCl-α的cRNA时,观察到对GABA反应的电流幅度显著增加,并且MdRdl表达所需的孵育期变短。这些结果表明,当两者共表达时,MdGluCl-α有助于MdRdl的表达。