Altered Function of a GABA-A receptor epilepsy mutation

遗传性癫痫综合征称为GEFS(全身性癫痫伴发热性癫痫发作加)，现在已知是遗传性癫痫的一种常见形式。在已确认的GEF突变中，至少有三个发生在编码GABA A受体的Gamma2亚单位的基因中，该基因负责大脑中大多数突触抑制。一个这样的突变，Gamma2(R43Q)，用谷氨酰胺取代了高度保守的精氨酸残基。分子模拟预测，这种替换可能会破坏Gamma2和Beta2亚基之间的盐桥相互作用，这可能是正常受体功能和药理学的关键。 我们对含有人Gamma2(R43Q)的GABA A受体的初步研究揭示了戏剧性的动力学变化，包括增强的受体脱敏，这可能通过在高强度神经活动期间导致突触抑制的“衰弱”而导致GEFS。这项拟议研究的总体目标是了解GABAA受体靠近Gamma2(R43Q)区域的变化如何改变受体功能并促进癫痫的发生。其具体目的是：1)通过研究切除的膜片中的通道动力学，确定突变引起受体功能改变的生物物理机制；2)通过检查旨在挽救预测的点突变，确定改变的结构基础 为了进一步控制盐桥的形成，以及b)以受控的方式进一步净化它，3)确定Gamma2(R43Q)突变对表达突变亚单位而不是野生型等位基因的转基因小鼠突触传递的影响，以及4)确定突触是否因突变而经历增强的脱敏，以及是否导致皮层、丘脑和海马脑片的网络超兴奋性增加。从这些研究中获得的知识将1)提供关于受癫痫突变Gamma2(R43Q)影响的受体区域的结构/功能作用的有价值的信息，2)使我们更好地了解与癫痫有关的抑制的关键方面，3)提供 为研究GABAA受体脱敏在功能性突触和网络兴奋性中的生理作用提供了前所未有的机会；4)进一步发展了一种新的动物模型，用于未来对GEFS癫痫的研究。

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.