Altered Function of a GABA-A receptor epilepsy mutation

GABA-A 受体癫痫突变的功能改变

• 批准号: 6670300
• 负责人: MATHEW V JONES
• 金额: $ 32.88万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-15 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6670300
• 关键词: GABA receptor; benzodiazepines; binding sites; biological signal transduction; biophysics; chemical models; epilepsy; febrile seizure; gene mutation; genetically modified animals; hippocampus; laboratory mouse; protein structure function; receptor expression; synapses; thalamus; tissue /cell culture

A genetic epilepsy syndrome called GEFS+ (Generalized Epilepsy with Febrile Seizures plus) is now known to be a common form of genetic epilepsy. At least three of the identified GEFS+ mutations occur in the gene encoding the gamma2 subunit of the GABA A receptor, responsible for most synaptic inhibition in the brain. One such mutation, gamma2(R43Q), replaces a highly conserved arginine residue with a glutamine. Molecular modeling predicts that this substitution may disrupt salt bridge interactions between the gamma2 and beta2 subunits, which may be critical for normal receptor function and pharmacology. Our initial studies of human gamma2(R43Q)-containing GABA A receptors reveal dramatic kinetic changes, including enhanced receptor desensitization, that could contribute to GEFS+ by causing a "rundown" in synaptic inhibition during high intensity neural activity. The overall Objective of the proposed research is to understand how alterations in the region of the GABAA receptor near gamma2(R43Q) change receptor function and contribute to epilepsy. The Specific Aims are to 1) determine the biophysical mechanism of the changes in receptor function caused by the mutation, by studying channel kinetics in excised membrane patches, 2) determine the structural basis of the changes by examining point mutations designed to a) rescue the predicted salt bridge formation and b) to further purturb it in controlled manner, 3) determine the impact of the gamma2(R43Q) mutation on synaptic transmission in transgenic mice that express the mutant subunit in place of the wild type allele, and 4) determine whether synapses undergo enhanced desensitization as a result of the mutation, and whether this causes increased network hyperexcitability in cortical, thalamic and hippocampal brain slices. The knowledge gained from these studies will 1) provide valuable information about the structural/functional role of the receptor region affected by the epilepsy mutation gamma2(R43Q), 2) allow us to better understand critical aspects of inhibition that are causally related to epilepsy, 3) provide an unprecedented opportunity to study the physiological role of GABA A receptor desensitization at functional synapses and in network excitability; and 4) further develop a novel animal model for future studies of GEFS+ epilepsy.

遗传性癫痫综合征称为GEFS+（全身性癫痫伴热性惊厥+），现在已知是遗传性癫痫的一种常见形式。至少有三个已鉴定的GEFS+突变发生在编码GABA A受体γ 2亚基的基因中，该基因负责大脑中的大多数突触抑制。一个这样的突变，γ 2（R43 Q），用谷氨酰胺替换高度保守的精氨酸残基。分子模型预测，这种取代可能会破坏γ 2和β 2亚基之间的盐桥相互作用，这可能对正常的受体功能和药理学至关重要。 我们对含人γ 2（R43 Q）的GABA A受体的初步研究揭示了显著的动力学变化，包括增强的受体脱敏，这可能通过在高强度神经活动期间引起突触抑制的“减弱”而导致GEFS+。该研究的总体目标是了解γ 2（R43 Q）附近GABAA受体区域的改变如何改变受体功能并导致癫痫。具体目的是：1）通过研究切除的膜斑中的通道动力学，确定突变引起的受体功能变化的生物物理机制，2）通过检查点突变确定变化的结构基础， 盐桥形成和B）以受控方式进一步纯化B，3）确定γ 2（R43 Q）突变对表达突变亚基代替野生型等位基因的转基因小鼠中突触传递的影响，和4）确定突触是否由于突变而经历增强的脱敏，以及这是否引起皮质中增加的网络过度兴奋性，丘脑和海马脑切片。从这些研究中获得的知识将1）提供关于受癫痫突变γ 2（R43 Q）影响的受体区域的结构/功能作用的有价值的信息，2）使我们能够更好地理解与癫痫有因果关系的抑制的关键方面，3）提供一种新的抗癫痫治疗方法。 研究GABA A受体脱敏在功能性突触和网络兴奋性中的生理作用的前所未有的机会;和4）进一步开发用于GEFS+癫痫的未来研究的新型动物模型。