Study On The Mechanism Of GABA Receptor And Related Drugs In Fish And Its Purification Separation

γ-aminobutyric acid(GABA) is the major inhibitory neurotransmitter in the vertebrates and invertebrates nervous systems. Most of the actions of GABA are mediated via GABAA receptors. GABAA receptors are not only testament to its significance as a CNS drug targets such as epilepsy, insomnia, schizophrenia, alcoholism, Parkinson’s disease, but also are the targets for many insecticides including α-endosulfan, lindane, fipronil and avermectins.Fipronil, the first phenylpyrazole insecticide, is an outstanding new insecticide for crop protection acting at the GABAA receptor as a noncompetitive blocker of the GABA-gated chloride channel. Fipronil at low dosage provides long-term protection against major lepidopterous and orthopterous pests on crops and coleopterous larvae in soil. A distinct advantage of fipronil is that it is one of the most selective of the insecticidal blockers of the GABA-gated chloride channel with a favorable safety factor between insects and mammals. However, fipronil was found highly toxic to aquatic life(such as fish) and non-target organisms, which has attracted extensive attention. The fish is an important part of aquatic ecosystems, thus it is important to study the toxicity mechanism of fipronil to fish.The major innovations in this study are as follow: with respect to the problem of fipronil to fish, ① The interactions between GABAA receptor in fish brain and fipronil and 1,4-benzodiazepine RO7 and were investigated by fluorescence probe technique, respectively. ② The interactions between GABAA receptor in fish brain and fipronil was studied by computational modeling. That is, three-dimensional model of transmembrane domain of zebrafish α1β2γ2 GABAA receptor was established by homology modeling. Moreover, the interactions between the fipronil and zebrafish GABAA receptor were investigated through molecular docking. The molecular mechanisms of fipronil(its metabolites)-receptor interactions were elucidated. ③ The non-magnetic and magnetic monodisperse silica microparticles were fabricated by biotemplate method. Moreover, the agrose-silica as affinity chromatography carrier was successfully prepared by inverse suspension embedding method. ④ The affinity columns make the use of fipronil and 1,4-benzodiazepine compound RO7 as affinity ligands, respectively. The GABAA receptors from the fish brain were purified by two kinds of affinity chromatography columns. The separate conditions of the GABAA receptors from the fish brain by both affinity columns were illustrated.In this paper the main research conclusions are as follows.1) The synthesis of 1,4-benzodiazepine compounds:In the studies of vertebrate GABA receptors, 1, 4-benzodiazepine drugs are of great importance. In this study, 1,4-benzodiazepine drugs RO7 and flurazepam were designed and synthesized, the synthetic conditions were discussed in detail. The structures of the intermediates and final products were elucidated by FTIR, 1H NMR, and smelting point instrument.2) The interaction between the 1,4-benzodiazepine drug and the GABAA receptor in fish brain by Fluorescent probe method: The fluorescent probe RO-FITC(RF) was synthesized by fluorescein isothiocyanate(FITC) labelled 1,4-benzodiazepine drugs RO7. The interactions between the RF and GABAA receptor in fish brain were investigated. Simultaneously, the effects of GABA on the binding of RO7 and GABAA receptor were studied. The results showed that the fluorescent probe RF exhibited the specific binding with the fish GABAA receptor. The equilibrium dissociation constant Kd and total maximum binding capacity [RT] of the ligand RO7 and the receptor were 67±5 nmol/L and 13.8±1.8 pmol/mg protein, respectively. Meanwhile, GABA can improve the binding of RO7 and the receptor. The above results indicated the pharmacological properties of GABAA receptor in fish brain are similar to those in the mammal brain.3) The interaction between the fipronil and the GABAA receptor in fish brain by Fluorescent probe method: The fluorescent probe Fipronil-FITC(FF) was synthesized by fluorescein isothiocyanate(FITC) labelled Fipronil. The interactions between the FF and GABAA receptor in fish brain were investigated. The results showed that the fluorescent probe FF exhibited the excellent specific binding with GABAA receptor the fish brain. The equilibrium dissociation constant Kd and total maximum binding capacity [RT] of the ligand RO7 and the receptor were 346±6 nmol/L and 40.6±3.5 pmol/mg protein, respectively. Compared with the mammals, fipronil shows a higher affinity to fish GABAA receptor. This may be reason that the fipronil is more highly toxic to fish than mammal.4) The interactions between GABAA receptor and fipronil by computational modeling: To elucidate the toxicity of fipronil to fish, three-dimensional model of transmembrane domain of zebrafish α1β2γ2 GABAA receptor was established by homology modeling, using nicotinic acetylcholine receptor as a template. Through molecular docking and molecular dynamics simulations, the interactions between the fipronil and zebrafish GABAA receptor were investigated. The results showed that the strong interaction between fipronil and 2′,6′, 9′ amino acid of inside receptor ion channel. The benzene ring and CF3 of fipronil are located in the intracellular region of receptor, and the pyrazole ring and SOCF3 of fipronil are located in extracellular region of receptor. It’s worth noting that fipronil produce three hydrogen bonding with 6′ hydrophilic amino acid residues threonine. It’s a very key issue for the binding between fipronil and fish GABAA receptor. This may be reason that the fipronil is more highly toxic to fish.Meanwhile, the interactions between the four metabolites of fipronil and zebrafish GABAA receptor were investigated. The results showed that CDOCKER interaction energy of four metabolites have higher toxicity to fish GABAA receptor than fipronil, especially MB46136 and Fipronil-desulfinyl.The three-dimensional model of transmembrane domain of rat α1β2γ2 GABAA receptor was established by homology modeling, using nicotinic acetylcholine receptor as a template. Through molecular docking and molecular dynamics simulations, the results showed that fipronil produce only one hydrogen bonding with rat α1β2γ2 GABAA receptor. It’s a very key issue for the binding between fipronil and fish GABAA receptor. This may be reason that the fipronil is more highly toxic to fish. The above results showed that the fipronil is more highly toxic to fish than mammal.5) The preparation of affinity chromatography carrier: Biotemplating is an effective strategy to obtain morphology-controllable materials with structural specificity, complexity, and corresponding unique functions. Herein, solid silica microspheres were prepared using biotemplate technique. Meanwhile, we take advantage of the unique heavy-metal-ion biosorption behavior of cyanobacteria cells to fabricate Fe3O4/cell composites. Simultaneously, cyanobacteria cells served as a biotemplate for fabricating magnetic silica microspheres. The obtained produces retained the original morphology of the cells, and exhibited excellent monodispersity and uniform spherical shape with narrow size distribution.Finally, the agarose- silica composite particles were prepared after surface modification with Sepharose CL-6B. The composite particles were activated by 1,4-butanediol diglycidyl ether and The density of epoxy groups was 65.27μmol/g gel. The good chromatographic property of the resultant composite particles was demonstrated in the fish GABAA receptor protein separation chromatography.6) The solubilization of GABAA receptors from the fish brain with the six kinds of detergents: The GABAA receptors from the fish brain were solubilized with the six kinds of detergents from the fish brain. The results showed that the maximum dissolution quantity(729μg/m L) of receptor protein was obtained with 1% Nonidet P-40.7)In this study, the best purifying method of the GABAA receptors from the fish brain were evaluated. The GABAA receptors from the fish brain were purified by two kinds of affinity chromatography columns. The affinity column were prepared with fipronil and 1,4-benzodiazepine compound RO7 as affinity ligands, and the agarose-silica composite particles as affinity matrix. The purified receptor from RO7 affinity column showed four bands, and the molecular weight were 56, 54, 43 and 37 k D, respectively. However, the purified receptor from fipronil affinity column showed only two bands, and the molecular weight were 54 and 44 k D, respectively. The above results indicated that fipronil and 1, 4-benzodiazepine compound RO7 interacted with GABAA receptors formed by different subunits. This may be reason that the fipronil is more highly toxic to fish than mammal.