The Role of Epsilon Subunit in GABAa Receptor Function

描述(申请人提供)：GABAA受体为哺乳动物中枢神经系统中的神经元提供主要的抑制驱动力。GABAA亚单位基因突变会降低这些受体的功能或表达，从而导致癫痫。另一方面，抗惊厥药苯二氮卓类药物和增加GABAA受体活性的麻醉药是抗惊厥药物。癫痫发作改变了几个GABAA受体亚基的表达，包括e亚基。含有e亚基的重组GABAA受体对麻醉药和苯二氮卓类药物相对不敏感。在功能上，e亚基模拟麻醉剂对GABAA受体的作用，导致自发门控、增强GABA亲和力和延长失活时间。我们的初步数据表明，e亚基参与了海马神经元中自发的(紧张性)GABAA受体活动，这在缺乏外源性GABA的情况下可以看到。定量聚合酶链式反应、E亚基特异性抗体的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亚单位表达的上调可能参与了GABAA受体的重构。该提案的具体目的是：1)研究e亚基对海马锥体神经元突触(相)和突触外(强直)受体的功能贡献；2)检测EEA或癫痫突变体y2(R43Q)亚基引入后GABAA相和强直受体的功能特性；3)使用嵌合e/y2结构来确定&亚基的结构/功能关系。我们将验证这一假设，即e亚基赋予突触外GABAA受体麻醉抗性，并通过诱导麻醉剂结合的受体构象改变受体门控和动力学。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我们更好地理解癫痫过程中神经元抑制的变化。

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.