CLONED GABA RECEPTORS IN INHERITED EPILEPSY

遗传性癫痫中克隆的 GABA 受体

• 批准号: 3410752
• 负责人: DAVID R. BURT
• 金额: $ 13.36万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 1991-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3410752
• 关键词: GABA receptor; Xenopus; brain mapping; clone cells; complementary DNA; electrophysiology; epilepsy; gamma aminobutyrate; gene expression; genetic library; laboratory mouse; messenger RNA; nucleic acid probes; nucleic acid sequence; voltage /patch clamp

The goals of the proposed research are to determine the role of GABAA/benzodiazepine receptors in the development of seizure activity in animal models of inherited epilepsy and to determine whether changes in specific amino acid residues of the receptors or in regulation of expression of unchanged receptors are responsible for any such role. The major approach will involve cloning the receptors from brains of inbred rodent strains chosen on the basis of their susceptibility (SP, seizure prone) or resistance (SR, seizure resistant) to various forms of seizure activity. Gammaaminobutyric acid (GABA) is the major inhibitory neurotransmitter of the brain. Its receptor, which includes a chloride channel as part of its structure, is thought to be the site of action of a number of important anticonvulsant drugs, including benzodiazepines and barbiturates, and certain convulsants. Blockade of GABA receptor function causes seizures and its enhancement prevents them. Recent evidence for changes in GABA-related synaptic markers, including receptor binding, in several forms of inherited epilepsy in rodents suggests that seizure susceptibility may result, at least in part, from changes in GABA receptor structure or expression. To test this hypothesis, we will prepare cDNA libraries in lambda gt10 vector from brains of SP and SR rodents (initially audiogenic seizure prone mice, DBA/2J, vs. 2 controls, one C57BL/6J and one congenic), screen them with probes representing pieces of the cloned bovine GABA receptor, sequence positive clones, look for linkage of receptor differences, as detected with specific oligonucleotide probes, with seizure susceptibility and other markers in recombinant inbred strains, and examine levels of GABA receptor mRNA in different brain regions by in situ hybridization. Crude mRNA from SP and SR brains, prepared as the first step in making cDNA libraries, will also be injected into Xenopus oocytes for electrophysiological studies by patch clamp and intracellular microelectrode techniques. This should reveal any differences in GABA receptor function between SP and SR mice and also give data for later comparison with that obtained using artificial mRNAs prepared from cloned cDNAs, verifying the cloning. These studies may eventually lead to gene therapy of inherited epilepsy.

这项拟议的研究的目标是确定 GABA/苯二氮卓类受体在癫痫发病中的作用 遗传性癫痫动物模型的活动性及测定 受体的特定氨基酸残基是否发生变化 或在调节未改变的受体的表达时 对任何这样的角色负责。主要的方法将包括 近交系啮齿动物大脑受体基因的克隆 根据他们的易感性(SP，癫痫易发)或 对各种形式的癫痫发作的抵抗(SR，抗癫痫) 活动。伽马氨基丁酸(GABA)是主要的 大脑的抑制性神经递质。它的受体，即 包括氯离子通道作为其结构的一部分，被认为 是一些重要的抗惊厥药的作用部位 药物，包括苯二氮卓类和巴比妥类，以及某些 镇静剂。阻断GABA受体功能导致癫痫发作 而它的增强阻止了它们。最近的变化证据 在GABA相关突触标志物中，包括受体结合， 啮齿动物的几种形式的遗传性癫痫表明 癫痫易感性可能至少部分是由以下因素引起的 GABA受体结构或表达。为了检验这一假设， 我们将在脑组织的lambda gt10载体中构建cdna文库。 SP和SR啮齿动物(最初是听源性癫痫易发作小鼠， DBA/2J，对比2个对照，一个C57BL/6J和一个同源基因)，屏幕 它们带有代表克隆的牛GABA片段的探针 受体，序列阳性克隆，寻找受体的连锁 用特定的寡核苷酸探针检测到的差异 重组近交系癫痫易感性及其他标记的研究 并检测不同菌株GABA受体基因的表达水平。 用原位杂交法检测脑区。SP和SP中的粗制mRNA 作为制作cDNA文库的第一步准备的高级大脑， 也将被注射到非洲爪哇卵母细胞中进行电生理 膜片钳和细胞内微电极的研究 技巧。这应该可以揭示GABA受体的任何差异 在SP和SR小鼠之间的作用，也为以后提供数据 与人工制备的mRNA的比较 从克隆的cDNA中，验证克隆。这些研究可能 最终导致遗传性癫痫的基因治疗。