REGULATION OF CLONED GABA RECEPTORS BY PHOSPHORYLATION

通过磷酸化调节克隆的 GABA 受体

• 批准号: 3084804
• 负责人: Lazar J Greenfield
• 金额: $ 7.99万
• 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 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3084804
• 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.

该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/通道复合体，或 GABAA 受体。该受体/氯离子 通道是 GABA 的主要靶标，GABA 是主要的抑制性神经元 哺乳动物大脑的递质，是作用部位 用于治疗焦虑症的苯二氮卓类药物和巴比妥类药物 疾病和癫痫。细胞内受体的磷酸化 蛋白激酶可能会改变表达、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 激酶可能指导新型抗惊厥药和抗焦虑药的开发 剂并影响癫痫病的临床治疗。这 拟议的研究将采用和开发神经生理学技术， 神经药理学和分子生物学来表征全细胞和 由克隆的 GABAA 受体产生的单通道电流 培养的细胞。 含有编码GABAA受体亚基的cDNA的质粒构建体，和 lacZ（大肠杆菌β-半乳糖苷酶基因，用作转染 标记），将被急性共转染到培养的成纤维细胞（L929）中 细胞。用荧光 β- 鉴定转染细胞后 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来研究 GABAA 受体 药理。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 “从外到外”斑块的细胞质表面。 GABA、苯二氮卓类药物、 巴比妥类和神经类固醇将通过压力喷射来应用 附近的移液器，或在细胞或补片区域进行微灌注 快速应用和冲洗研究。数据将在线数字化并 随后通过计算机程序进行分析以确定频道 电导态和门控动力学，激动剂依赖性动力学 脱敏和不依赖激动剂的“纲要”。现场定向 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亚基 结构/功能关系。其他细胞类型将用于 表达大量 GABAA 受体以进行受体结合 检查受体结合动力学响应变化的实验 脱敏或受体磷酸化状态。