The Role of Epsilon Subunit in GABA Receptor Function

描述（由申请人提供）：GABAA受体在哺乳动物中枢神经系统中对神经元提供主要的抑制驱动。GABAA亚基基因的突变降低了这些受体的功能或表达，可导致癫痫。另一方面，抗惊厥药物苯二氮卓类药物和增加GABAA受体活性的麻醉剂具有抗惊厥作用。癫痫会改变几种GABAA受体亚基的表达，包括e亚基。含有e亚基的重组GABAA受体对麻醉剂和苯二氮卓类药物相对不敏感。在功能上，e亚基模仿麻醉剂对GABAA受体的作用，引起自发门控，增加GABA亲和力和延长失活时间。我们的初步数据表明，e亚基参与海马神经元自发（强直性）GABAA受体活性，这是在缺乏外源性GABA的情况下看到的。定量PCR、E亚基特异性抗体免疫细胞化学和药理学研究表明，海马锥体神经元表达含有E亚基的天然GABAA受体。此外，癫痫样电活动（EEA）后海马锥体神经元e亚基表达上调可能有助于GABAA受体重塑。本研究的具体目的是：1)研究e亚基对海马锥体神经元突触（相位）和突触外（强直）受体的功能贡献；2)研究EEA或引入癫痫突变体y2（R43Q）亚基后GABAA的相位和强直受体的功能特性；3)使用嵌合e/y2构建体确定&亚基的结构/功能关系。我们将验证e亚基对突触外GABAA受体具有麻醉抗性，并通过诱导麻醉结合受体构象改变受体门控和动力学的假设。这些实验将有助于揭示GABAA受体功能的重要结构决定因素。更好地了解GABAA受体及其适应的潜力将有助于增加我们对癫痫期间神经元抑制变化的理解。

DESCRIPTION (provided by applicant): The GABAA receptor provides the major inhibitory drive to neurons in the mammalian central nervous system. Mutations in GABAA subunit genes that reduce the function or expression of these receptors can cause epilepsy. On the other hand anticonvulsant benzodiazepines and anesthetics that increase GABAA receptor activity are anticonvulsant. Seizures alter the expression of several GABAA receptor subunits including the e subunit. Recombinant GABAA receptors containing the e subunit are relatively insensitive to anesthetics and benzodiazepines. Functionally the e subunit mimics the actions of anesthetics on GABAA receptors causing spontaneous gating, increased GABA affinity and prolonged deactivation. Our preliminary data suggest that e subunits participate in spontaneous (tonic) GABAA receptor activity in hippocampal neurons, which is seen in the absence of exogenous GABA. Quantitative PCR, immunocytochemistry with an E subunit specific antibody, and pharmacological studies suggest that hippocampal pyramidal neurons express native GABAA receptors containing e subunits. Furthermore, up regulation of e subunit expression in hippocampal pyramidal neurons following epileptiform electrical activity (EEA) may contribute to GABAA receptor remodeling. The specific aims of the proposal are to: 1) Examine the functional contribution of the e subunit to synaptic (phasic) and extrasynaptic (tonic) receptors in hippocampal pyramidal neurons, 2) Examine the functional properties of phasic and tonic GABAA receptors following EEA or introduction of the epilepsy mutant y2(R43Q) subunit, 3) Use chimeric e/y2 constructs to determine the & subunit's structure/function relationship. We will test the hypothesis that the e subunit confers anesthetic resistance to extrasynaptic GABAA receptors and alters receptor gating and kinetics by inducing an anesthetic-bound receptor conformation. These experiments will help uncover important structural determinants of GABAA receptor function. A better understanding of GABAA receptors and their potential for adaptation will help increase our understanding of changes in neuronal inhibition during epilepsy.