Molecular genetical analysis on structure and function of GABA_A and GABA_B receptors

对脑内GABA_A和GABA_B受体进行了分子遗传学和分子药理学研究，得到如下结果：1)人脑GABA_A受体α_1亚基由456个氨基酸残基组成，其序列与牛脑相似。2)在人脑中表达GABAA受体α1亚基的单一mRNA(4.6Kb)。3)人脑中GABA_A受体的α_1亚基具有外显子-内含子结构。4)人脑中GABA_A受体的α_1亚基由四个跨膜区组成。5)微量注射人脑内GABA_A受体α_1亚单位的mRNA可诱导功能相关的GABA_A受体的表达。6)小鼠和大鼠脑内GABA_A受体α_1亚基是由两个mRNA(4.1和-4.6k)通过选择性剪接表达的。7)巴氯芬亲和柱层析可使GABA_B受体纯化13000倍。8)GABA_B受体通过GI或GO蛋白与腺苷环化、肌醇磷酸转运体和钙通道负偶联，这些偶联可通过受体的溶解而消除。9)GABA_B受体是一种相对分子质量为80kDa的糖蛋白。10)GABA_A受体可被其激动剂长期作用而下调，这种下调是通过抑制其mRNA的表达而引起的。11)纯化的GABA_A和GABA_B受体均可在磷脂小泡上进行功能性重组。12)将GABA_A和GABA_B受体的单抗(GA-I：IgE，GB-I：IgM)分别注入Balb/C小鼠体内，筛选特异性克隆，获得抗GABAA和GABA_B受体的单抗。13)通过纯化和重组各受体蛋白，可以分析各种药物对GABA_A和GABA_B受体的直接作用。

Molecular genetical and molecular pharmacological studies on cerebral GABA_A and GABA_B receptors have been employed and the following results were obtained.1) The alpha_1-subunits of human brain GABA_A receptor consists of 456 amino acid residues and its sequence is similar to that of bovine brain. 2) The single mRNA (4.6Kb) for GABA_A receptor alpha^<>1-subunit is expressed in human brain. 3) The alpha_1-subunit of GABA_A receptor in the human brain possesses exon-intron organization. 4) The alpha_1-subunit of GABA_A receptor in the human brain consists of four transmembrane domains. 5) The microinjection of mRNA for GABA_A receptor alpha_1-subunit from the human brain induces the expression of functionally relevant GABA_A receptor. 6) The GABA_A receptor alpha_1-subunit in the mouse and rat brains is expressed from two mRNAs (4.1 and-4.6K) by an alternative splicing. 7) The 13, 000 fold purification of GABA_B receptor is achieved by baclofen-affinity column chromatography. 8) The GABA_B receptor is negatively coupled with adenylate cyclas, inositol phosphate turnover and calcium channel via Gi or Go protein and these couplings can be eliminated by solubilization of the receptor. 9) The GABA_B receptor is a glycoprotein having the molecular weight of 80 Kda. 10) The GABA_A receptor can be down-regulated by a prolonged exposure to its agonist and this downregulation is caused by the suppression of the expression of mRNA. 11) Both purified GABA_A and GABA_B receptors can be functionally reconstituted on phospholipid vesicles. 12) Monoclonal antibodies for GABA_A and GABA_B receptors (GA-I : Ige, GB-I : IgM) are obtained by the injection of respective receptor protein to Balb/C mouse and by the subsequent screening of specific clones. 13) The derect actions of various drugs on GABA_A and GABA_B receptors can be analyzed using purified and reconstituted each receptor protein.