Neurosteroid Modulation of Extrasynaptic GABAA Receptors

描述（由申请人提供）：γ -氨基丁酸A型受体（GABAARs）是哺乳动物中枢神经系统中抑制传递的主要介质。GABAARs可以定位于突触后抑制特化位点或突触外位点。突触GABAARs在突触前囊泡释放GABA后被短暂激活，而突触外GABAARs在静息浓度下被持续激活，从而介导张力抑制。这些突触外GABAARs主要由α -4 β 2/3和γ亚基组成，对GABA具有高亲和力，很少或没有脱敏。孕酮和去氧可的松的类固醇代谢物（称为神经类固醇）已被证明是突触外GABAA受体的有效的正变构调节剂。然而，神经类固醇改变突触外GABAARs功能的确切机制尚不清楚。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PKC磷酸化突触外GABAARs以改变受体功能。为了验证这一假设，我们的实验室已经开发出PKC磷酸化的关键丝氨酸突变为丙氨酸（alpha4-S443A和β -3-408/ 409a）的DNA结构。我首先研究了α -4和β -4亚基内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，通过PKC的phobol酯刺激和α -4亚基中的S443A突变，可以防止alpha4beta3介导的gaba能电流的减少。我将评估α -4和β -3亚基内PKC磷酸化位点突变的生理意义，并确定PKC依赖性磷酸化是否在神经类固醇介导的强张抑制增强中发挥作用。这些研究将利用敲入小鼠，其中神经类固醇诱导的GABAARs磷酸化位点已经发生突变，这也将使我们能够检查这种现象的行为意义。神经类固醇水平在不同的生理状态和病理生理条件下波动。这项研究将深入了解神经类固醇介导的神经元兴奋性改变，并可能有助于开发治疗产后抑郁症、癫痫和焦虑症的新药理学靶点。

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.