CLONED GABA RECEPTORS IN INHERITED EPILEPSY

遗传性癫痫中克隆的 GABA 受体

基本信息

批准号：
3410755
负责人：
DAVID R. BURT
金额：
$ 13.75万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE
依托单位国家：
美国
项目类别：
财政年份：
1988
资助国家：
美国
起止时间：
1988-07-01 至 1992-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3410755
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

项目摘要

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.

拟议研究的目标是确定 GABAA/苯二氮卓受体在癫痫发作中的作用遗传性癫痫动物模型中的活性并确定受体的特定氨基酸残基是否发生变化或在未改变受体的表达调节中负责任何此类角色。主要方法将涉及从所选近交啮齿动物品系的大脑中克隆受体根据他们的易感性（SP，癫痫发作倾向）或对各种形式的癫痫发作的抵抗力（SR，癫痫抵抗）活动。 γ氨基丁酸（GABA）是主要的大脑的抑制性神经递质。它的受体，包括氯离子通道作为其结构的一部分，被认为是许多重要抗惊厥药的作用部位药物，包括苯二氮卓类药物和巴比妥类药物，以及某些惊厥。 GABA 受体功能阻断导致癫痫发作而它的增强则阻止了它们。最近的变化证据 GABA 相关突触标记，包括受体结合，啮齿类动物遗传性癫痫的几种形式表明癫痫易感性可能至少部分由以下因素的变化引起： GABA 受体结构或表达。为了检验这个假设，我们将从大脑中制备 lambda gt10 载体的 cDNA 文库 SP 和 SR 啮齿动物（最初有听源性癫痫发作倾向的小鼠， DBA/2J，对比 2 个对照，一个 C57BL/6J 和一个同类），筛选它们带有代表克隆牛 GABA 片段的探针受体，测序阳性克隆，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中的癫痫易感性和其他标记菌株，并检查不同菌株中 GABA 受体 mRNA 的水平通过原位杂交分析大脑区域。来自 SP 的粗 mRNA 和 SR大脑，作为制作cDNA文库的第一步而准备，还将被注射到非洲爪蟾卵母细胞中进行电生理学检查膜片钳和细胞内微电极研究技术。这应该揭示 GABA 受体的任何差异 SP 和 SR 小鼠之间的功能，并为以后提供数据与使用人工制备的 mRNA 获得的结果进行比较从克隆的 cDNA 中提取，验证克隆。这些研究可能最终导致遗传性癫痫的基因治疗。