Neurosteroid Modulation of Extrasynaptic GABAA Receptors

描述(申请人提供)：γ-氨基丁酸A型受体(GABAARs)是哺乳动物中枢神经系统抑制性传递的主要媒介。GABA受体可定位于突触后抑制性专化或突触外部位。突触GABA从突触前小泡释放后，突触GABAARs被短暂激活，而突触外GABAARs被静息浓度持续激活，从而介导紧张性抑制。这些突触外GABA受体主要由α-4β2/3和伽马亚基组成，对GABA有很高的亲和力，几乎没有脱敏作用。孕酮和脱氧皮质醇的类固醇代谢物(称为神经类固醇)已被证明是突触外GABAA受体的强有力的正变构调节剂。然而，神经类固醇改变突触外GABA受体功能的确切机制还不是很清楚。先前的实验表明，蛋白激酶C(PKC)活性是神经类固醇介导的GABAARs调节所必需的。我们假设神经类固醇介导突触外GABAARs的PKC磷酸化，从而改变受体功能。为了验证这一假说，我们的实验室已经开发出对PKC磷酸化至关重要的丝氨酸的DNA结构已经突变为丙氨酸(Alpha4-S443a和beta-3-408/409a)。我首先研究了α-4和β-4亚基中PKC磷酸化位点的这些点突变如何改变神经类固醇介导的GABA能电流大小的变化。我已经证明，佛波酯刺激PKC和α-4亚单位中的S443A突变可以阻止α4beta3介导的GABA能电流的下降。我将评估α-4和β-3亚基中PKC磷酸化位点突变的生理学意义，并确定PKC依赖的磷酸化是否在神经类固醇介导的紧张性抑制增强中发挥作用。这些研究将利用神经类固醇诱导的GABAARs磷酸化位点发生突变的敲入小鼠，这也将使我们能够检查这一现象的行为意义。众所周知，神经类固醇水平会在不同的生理状态和病理生理条件下波动。这项资助中提出的工作将深入了解神经类固醇对神经元兴奋性的影响，并可能有助于开发治疗产后抑郁症、癫痫和焦虑症的新的药理靶点。 公共卫生相关性：介导紧张性抑制的GABAA受体亚型对神经类固醇的作用特别敏感。类固醇诱导的GABAA受体亚单位表达的波动导致神经元兴奋性的改变，并与产后抑郁症和经前综合征等综合征有关。这项应用中的研究将深入了解神经类固醇介导的神经元兴奋性的变化，并可能有助于开发新的产后抑郁、癫痫和焦虑症的治疗方法。

DESCRIPTION (provided by applicant): Gamma-aminobutyric acid type A receptors (GABAARs) are the principal mediators of inhibitory transmission in the mammalian central nervous system. GABAARs can be localized at postsynaptic inhibitory specializations or at extrasynaptic sites. While synaptic GABAARs are activated transiently following the release of GABA from presynaptic vesicles, extrasynaptic GABAARs are activated continuously by resting concentrations and thus mediate tonic inhibition. These extrasynaptic GABAARs are predominantly composed of alpha-4 beta2/3 and gamma subunits, exhibit high affinity for GABA and have little or no desensitization. Steroid metabolites of progesterone and deoxycortisone (known as neurosteroids) have been shown to be potent positive allosteric modulators of extrasynaptic GABAA receptors. However the exact mechanisms by which neurosteroids alter extrasynaptic GABAARs function are not well understood. Previous experiments have suggested that Protein Kinase C (PKC) activity is required for neurosteroid-mediated modulation of GABAARs. We hypothesize that neurosteroids mediate PKC phosphorylation of extrasynaptic GABAARs to alter receptor function. In order to test this hypothesis, our laboratory has developed DNA constructs that were critical serines for PKC phosphorylation have been mutated to alanines (alpha4-S443A and beta-3-408/409A). I have first examined how these point mutations in PKC phosphorylation sites within alpha-4 and beta-4 subunits alter the neurosteroid-mediated changes in the magnitude of GABAergic currents. I have shown that the rundown of alpha4beta3-mediated GABAergic currents is prevented by phorbol ester stimulation of PKC and by the S443A mutation in the alpha-4 subunit. I will assess the physiological significance of mutations in PKC phosphorylation sites within the alpha-4 and beta-3 subunits and determine whether PKC-dependant phosphorylation plays a role in neurosteroid-mediated enhancement of tonic inhibition. These studies will utilize knock-in mice in which sites of neurosteroid-induced phosphorylation of GABAARs have been mutated, which will also allow us to examine the behavioral significance of this phenomenon. Neurosteroid levels are known to fluctuate during various physiological states and pathophysiological conditions. The work proposed in this grant will provide insight into neurosteroid-mediated alterations in neuronal excitability and might contribute to the development of novel pharmacological targets for the treatment of postpartum depression, epilepsy and anxiety disorders. PUBLIC HEALTH RELEVANCE: GABAA receptor subtypes that mediate tonic inhibition are uniquely sensitive to the actions of neurosteroids. Steroid-induced fluctuations in GABAA receptor subunit expression result in alterations in neuronal excitability and are implicated in syndromes such as postpartum depression and premenstrual syndrome. The studies in this application will provide insight into neurosteroid-mediated alterations in neuronal excitability an might contribute to the development of novel treatments for postpartum depression, epilepsy and anxiety disorders.