REGULATION OF CLONED GABA RECEPTORS BY PHOSPHORYLATION

这个项目的长期目标是阐明蛋白质(S)的作用 γ-氨基丁酸(GABA)调节中的磷酸化 受体/通道复合体，或GABAA受体。该受体/氯离子 通道是GABA的主要靶点，GABA是GABA的主要抑制神经元。 哺乳动物大脑的传递者，也是 苯二氮卓类药物和巴比妥类药物在焦虑治疗中的应用 精神障碍和癫痫。细胞内对受体的磷酸化作用 蛋白激酶可能改变蛋白的表达、修饰和组装 受体/通道复合体及其受体结合和通道门控 特性，和/或其脱敏和降解。更好的 蛋白质对GABAA通道功能调节的认识 激酶可能指导新的抗惊厥和抗焦虑药物的开发 药物和影响临床治疗癫痫的障碍。这个 拟议的研究将使用和开发神经生理学的技术， 神经药理学和分子生物学来表征整个细胞和 克隆的GABAA受体产生的单通道电流在 培养的细胞。 含有编码GABAA受体亚单位的cDNA的质粒构建体，以及 LacZ(大肠杆菌的β-半乳糖苷酶基因，用作转染剂 标记)，将其与成纤维细胞(L929)急性共转染 细胞。在用荧光贝塔鉴定转基因细胞后- 半乳糖苷酶底物中，GABAA电流将被记录下来 细胞“和单通道膜片钳记录研究GABAA受体 药理学。蛋白激酶A(催化亚基)和蛋白激酶C (催化活性)将包括在记录吸管溶液中 并通过电极透析或灌流进入细胞，或进入细胞 “外向外”贴片的细胞质表面。GABA，苯二氮卓类， 巴比妥酸盐和神经类固醇将通过压力喷射从 附近的吸管，或在细胞或贴片的区域内微灌流 快速应用和洗涤研究。数据将在线数字化，并 随后由计算机程序分析以确定通道 电导态和门控动力学，激动剂依赖的动力学 脱敏和非激动剂非依赖性“减退”。站点定向 突变将被用来改变GABAA受体的一级结构 通过突变磷酸化位点来确定亚基的精确 结构/功能关系。将使用其他单元格类型来 大量表达GABAA受体与受体结合 检测受体结合动力学响应变化的实验 至脱敏或受体磷酸化状态。

The long-term goal of this project is to elucidate the role(s) of protein phosphorylation in the regulation of the gamma-aminobutyric acid (GABA) receptor/channel complex, or GABAA receptor. This receptor/chloride ion channel is the primary target of GABA, the major inhibitory neuro- transmitter of the mammalian brain, and is the site of action of benzodiazepines and barbiturates used in the treatment of anxiety disorders and epilepsy. Phosphorylation of the receptor by intracellular protein kinases may alter the expression, modification and assembly of the receptor/channel complex, its receptor binding and channel gating characteristics, and/or its desensitization and degradation. A better understanding of the regulation of GABAA channel function by protein kinases may guide the development of new anticonvulsant and anxiolytic agents and influence the clinical treatment of seizure disorders. The proposed studies will employ and develop techniques in neurophysiology, neuropharmacology, and molecular biology to characterize whole cell and single channel currents generated by cloned GABAA receptors expressed in cultured cells. Plasmid constructs containing cDNAs encoding GABAA receptor subunits, and lacZ (the E. coli beta-galactosidase gene, used as a transfection marker), will be acutely co-transfected into cultured fibroblast (L929) cells. After identifying transfected cells with a fluorescent beta- galactosidase substrate, GABAA currents will be recorded using "whole cell" and single channel patch clamp recordings to study GABAA receptor pharmacology. Protein kinase A (catalytic subunit) and protein kinase C (catalytically active) will be included in the recording pipette solution and dialyzed or perfused through the electrode into the cell, or onto the cytoplasmic surface of an "outside-out" patch. GABA, benzodiazepines, barbiturates and neurosteroids will be applied by pressure ejection from a nearby pipette, or microperfused in the area of the cell or patch for rapid application and washout studies. Data will be digitized on-line and subsequently analyzed by computer programs to determine channel conductance states and gating kinetics, the kinetics of agonist-dependent desensitization and agonist-independent "rundown." Site-directed mutagenesis will be used to alter the primary structure of GABAA receptor subunits by mutating phosphorylation sites to determine precise structure/function relationships. Other cell types will be used to express GABAA receptors in larger quantities for receptor binding experiments to examine changes in receptor binding kinetics in response to desensitization or receptor phosphorylation states.