REGULATION OF CLONED GABA RECEPTORS BY PHOSPHORYLATION

通过磷酸化调节克隆的 GABA 受体

• 批准号: 2259684
• 负责人: Lazar John GREENFIELD
• 金额: $ 9.05万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-01 至 1998-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2259684
• 关键词: GABA receptor; barbiturates; benzodiazepine receptor; electrophysiology; membrane channels; molecular cloning; neuropharmacology; neurophysiology; phosphorylation; protein kinase A; protein kinase C; protein structure function; receptor binding; single cell analysis; site directed mutagenesis; transfection; voltage /patch clamp; voltage gated channel

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.

这个项目的长期目标是阐明蛋白质的作用 γ-氨基丁酸（GABA）调节中的磷酸化 受体/通道复合物，或GABAA受体。这种受体/氯离子 通道是GABA的主要靶点，GABA是主要的抑制性神经元， 是哺乳动物大脑的传递器，并且是 用于治疗焦虑症的苯二氮卓类和巴比妥类药物 疾病和癫痫。细胞内受体的磷酸化 蛋白激酶可以改变蛋白质的表达、修饰和组装， 受体/通道复合物、其受体结合和通道门控 特性和/或其脱敏和降解。更好的 了解蛋白质对GABAA通道功能的调节 激酶可能指导新的抗惊厥药和抗焦虑药的开发 药物和影响临床治疗癫痫病。的 拟议的研究将采用和发展神经生理学技术， 神经药理学和分子生物学来表征整个细胞， 表达的克隆GABAA受体产生的单通道电流， 培养细胞 含有编码GABAA受体亚基的cDNA的质粒构建体，和 lacZ（E.大肠杆菌β-半乳糖苷酶基因，用作转染 标记物），将其急性共转染到培养的成纤维细胞（L929）中 细胞在用荧光β- 半乳糖苷酶底物，GABAA电流将使用“全 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）将包含在记录移液器溶液中 并通过电极透析或灌注到细胞中，或到细胞上， 细胞质表面的“外向”补丁。氨基丁酸苯二氮卓类 巴比妥酸盐和神经类固醇将通过压力喷射应用， 附近的移液管，或在细胞或贴片的区域中微灌注， 快速应用和洗脱研究。数据将在线数字化， 随后由计算机程序分析以确定信道 电导状态和门控动力学，激动剂依赖的动力学 脱敏和激动剂非依赖性“衰退。“站点导向 诱变将用于改变GABAA受体的一级结构 通过突变磷酸化位点来确定精确的 结构/功能关系。其他细胞类型将用于 大量表达GABAA受体，用于受体结合 实验来检查响应中受体结合动力学的变化 脱敏或受体磷酸化状态。