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/苯二氮卓受体在发作的发育中在遗传癫痫的动物模型中的活性并确定受体的特定氨基酸残基的变化是否变化或调节未改变受体的表达是负责任何此类角色。主要方法将涉及从选择的近交啮齿动物菌株的大脑中克隆受体根据它们的敏感性（SP，癫痫发作）或电阻（SR，癫痫发作）对各种形式的癫痫发作活动。伽马米丁酸（GABA）是主要的大脑的抑制性神经递质。它的受体，它包括氯化物通道作为其结构的一部分，被认为是成为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，某些药物抽搐者。 GABA受体功能的封锁导致癫痫发作它的增强阻止了它们。最新的变化证据在与GABA相关的突触标记中，包括受体结合，在啮齿动物中几种形式的遗传癫痫表明癫痫发作的易感性可能至少部分是由于变化 GABA受体结构或表达。为了检验这一假设，我们将从大脑中准备Lambda GT10载体的cDNA库 SP和SR啮齿动物（最初是听力癫痫发作的小鼠， DBA/2J，vs. 2个控件，一个C57BL/6J和1个同类），屏幕它们用代表克隆牛的碎片的探针受体，序列阳性克隆，寻找受体的联系用特定的寡核苷酸探针检测到的差异，重组的癫痫发作易感性和其他标记菌株并检查不同的GABA受体mRNA水平通过原位杂交进行大脑区域。来自SP的原油mRNA和 Sr Brains，作为制作cDNA库的第一步准备的，还将注入电生理学的武士卵母细胞通过斑块夹和细胞内微电极进行研究技术。这应该揭示GABA受体的任何差异 SP和SR小鼠之间的功能，还提供了以后的数据与制备的人工mRNA获得的比较从克隆的cDNA中验证克隆。这些研究可能最终导致遗传癫痫的基因疗法。