Novel actions of neurosteroids on GABA (A) receptor trafficking

描述(申请人提供)：紧张性抑制通过特定人群的持续活动决定神经元的兴奋性和神经元回路的活动。 高亲和力突触外？-氨基丁酸A受体(GABAARs)，是神经类固醇的主要靶点。神经类固醇被广泛认为是GABA能抑制的内源性调节剂。神经类固醇水平的波动被认为在癫痫中起着关键作用，也与自闭症、焦虑、抑郁、经前综合征和精神分裂症有关。然而，到目前为止，在了解神经类固醇水平的波动如何改变突触外GABAAR亚单位的表达水平方面存在着根本的差距，这是在大量神经精神疾病中发生的GABA能抑制改变的一个重要因素。我们的长期目标是充分了解神经类固醇影响突触外GABA受体表达水平的机制。在这项提案中，我们将讨论神经类固醇在蛋白激酶C(PKC)依赖的含4亚单位的GABAARs的磷酸化中所起的作用。我们将确定这如何影响介导紧张性抑制的GABAAR亚型的细胞表面积累。我们的中心假设是，神经类固醇调节由4和3组装的GABAARs的磷酸化。亚基，它介导了齿状回和丘脑的大部分紧张性抑制。神经类固醇特异性地增强依赖于PKC的丝氨酸443(S443)亚基的磷酸化。这种增强的磷酸化导致由4和3组成的受体的插入增强。亚基进入质膜，起源于分泌途径。这些受体细胞表面稳定性的增强是紧张性抑制效果持续增加的原因。在强大的初步数据的指导下，这一假设将通过追求三个具体目标来检验：(1)确定PKC依赖的磷酸化在调节介导紧张性抑制的GABAARs的特定细胞表面聚集中的作用；(2)可视化神经类固醇介导的细胞表面稳定性和GABAARs膜转运的变化；(3)确定神经类固醇依赖的磷酸化对GABAARs的活性和紧张性抑制的有效性的影响。综上所述，这项提议将展示一种新的和意想不到的机制，通过这种机制，神经类固醇在紧张性抑制的疗效中发挥持久和持续的变化。了解神经类固醇介导的GABAARs磷酸化和细胞表面表达的变化有可能转化为更好地理解神经精神障碍。此外，这些信息可能会导致开发出更有效的治疗焦虑、抑郁、经前综合症、精神分裂症和药物滥用的方法。

DESCRIPTION (provided by applicant): Tonic inhibition determines the excitability of neurons and the activity of neuronal circuits through the persistent activity of specialized populations of high affinity extrasynaptic ?-aminobutyric acid A receptors (GABAARs), that are the principle targets of neurosteroids. Neurosteroids are widely accepted as endogenous regulators of GABAergic inhibition. Fluctuations in the levels of neurosteroids are accepted to play a critical role in epilepsy, and are also relevant to autism, anxiety, depression, premenstrual syndrome and schizophrenia. However, to date, there is a fundamental gap in understanding how fluctuations in the levels of neurosteroids change extrasynaptic GABAAR subunit expression levels, which are a significant factor in the changes in GABAergic inhibition that occur in a plethora of neuropsychiatric disorders. Our long-term goal is to fully understand the mechanism by which neurosteroids influence the expression levels of extrasynaptic GABAARs. In this proposal we will address the role that neurosteroids play in protein kinase C (PKC)-dependent phosphorylation of ¿4 subunit-containing GABAARs. We will determine how this influences the cell surface accumulation of the GABAAR subtypes that mediate tonic inhibition. Our central hypothesis is that neurosteroids modulate the phosphorylation of GABAARs assembled from ¿4¿3 and ? subunits, which mediate the majority of tonic inhibition in the dentate gyrus and thalamus. Neurosteroids specifically potentiate PKC-dependent phosphorylation of serine 443 (S443) in the ¿4 subunit. This increased phosphorylation leads to enhanced insertion of receptors composed of ¿4¿3 and ? subunits into the plasma membrane that originate within the secretory pathway. These enhancements of receptor cell surface stability are responsible for sustained increases in the efficacy of tonic inhibition. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: (1) Determining the role of PKC-dependent phosphorylation in modulating the specific cell surface accumulation of GABAARs that mediate tonic inhibition (2) Visualizing neurosteroid- mediated changes in cell surface stability and membrane trafficking of GABAARs and (3) Ascertaining the effects of neurosteroid-dependent phosphorylation on the activity of GABAARs and the efficacy of tonic inhibition. In summary this proposal will demonstrate a new and unexpected mechanism by which neurosteroids exert persistent and sustained changes in the efficacy of tonic inhibition. Understanding neurosteroid-mediated changes in phosphorylation and cell surface expression of GABAARs has the potential to translate into better understanding of neuropsychiatric disorders. Moreover such information is likely to lead to the development of more efficacious treatments for anxiety, depression, premenstrual syndrome, schizophrenia and substance abuse.