Novel actions of neurosteroids on GABA (A) receptor trafficking

神经活性类固醇(NASS)，如别孕酮(ALLO)，通过以下途径在调节行为中发挥核心作用 其有效的抗焦虑、抗惊厥、镇静和催眠作用。因此，对 NASS会导致焦虑、自闭症谱系障碍、抑郁症、癫痫和经前综合症。 经典地说，NASS被认为是通过正变构调节来迅速增强神经元抑制作用的 γ-氨基丁酸A型受体(GABAARs)活性。除了它们的变构作用外，我们还有 最近发现，NASS通过一种依赖蛋白激酶C的机制来增强蛋白激酶C的磷酸化。 包括β3亚基中的丝氨酸408和409的残基(S408/9)，这是一个增加伽巴尔数的过程 在质膜上导致GABA能抑制效果持续增强。虽然我们 已经证明，NASS并不直接激活PKC，而PKC是NASS导致 GABAAR亚基的磷酸化尚不清楚。有迹象表明，除了它们的正变构外 GABAARs的调节，NASS可直接激活膜孕酮受体；G蛋白 调节PKC信号的偶联受体。然而，目前还没有关于MPR所起作用的信息 在调节GABAAR活性方面。同样，持续的MPR介导的行为意义 NASS的代谢性作用仍未被探索。为了解决这些问题，我们创造了老鼠，在其中 β3亚基中的S408/9已经突变为丙氨酸，预计这种突变会降低 NASS对GABAAR函数的代谢作用使用这些工具的初步研究使我们能够 制定一个将在这里进行测试的中心假设；NASS激活MPR以增强 GABAARs在β3亚单位中包括S408/9残基上的磷酸化，这是一种 是它们抗惊厥功效的基础。相反，它们的抗焦虑作用是通过变构来实现的。 GABA活性的增强，这是一个依赖于α2亚基中Q241的过程。我们的实验将 重点关注以下目标。 目的1.验证NAS诱导GABA能持续效应的假设 抑制作用依赖于β3亚单位中的S408/9A。 目的2.验证NASS抗焦虑和抗惊厥疗效依赖的假设 在β3亚基的S408/9上。 目的3.检验NASS通过对GABA的代谢调节作用的假设 激活MPR。 总而言之，我们的提案将确定NAS发挥治疗作用的分子机制。 这些信息可能有助于开发新的治疗策略来减轻焦虑的负担， 自闭症谱系障碍、抑郁、癫痫和经前综合征。

Neuroactive steroids (NASs) such as allopregnanolone (ALLO) play a central role in regulating behavior via their potent anxiolytic, anticonvulsant, sedative, and hypnotic actions. Accordingly, modifications in the levels of NASs contribute to anxiety, autism spectrum disorders, depression, epilepsy, and premenstrual syndrome. Classically, NASs are thought to act by rapidly boosting neuronal inhibition by positive allosteric modulation of the activity of γ-aminobutyric acid type A receptors (GABAARs). In addition to their allosteric actions, we have recently shown that NASs act via a protein kinase C-dependent mechanism to enhance the phosphorylation of residues including Serine’s 408 and 409 in the β3 subunit (S408/9), a process that increases GABAAR number on the plasma membrane leading to a sustained increase in the efficacy of GABAergic inhibition. Although we have shown that NASs do not directly activate PKC, the mechanism by which NASs lead to changes in phosphorylation of GABAAR subunits are unknown. It is emerging that in addition to their positive allosteric modulation of GABAARs, NASs can directly activate membrane progesterone receptors (mPRs); G-protein coupled receptors that regulate PKC signaling. However, no information is available on the role that mPRs play in regulating GABAAR activity. Likewise, the behavioral significance of the sustained mPR-mediated metabotropic actions of NASs remains unexplored. To address these issues we have created mice in which S408/9 in the β3 subunit have been mutated to alanines, mutations that are predicted to reduce the metabotropic actions of NASs on GABAAR function. Preliminary studies using these tools have allowed us to formulate a central hypothesis that will be tested here; NASs activate mPRs to enhance the phosphorylation of GABAARs on residues including S408/9 in the β3 subunit, a mechanism that underlies their anticonvulsant efficacy. In contrast, their anxiolytic efficacy is mediated via allosteric potentiation of GABAAR activity, a process dependent upon Q241 in the α2 subunit. Our experiments will focus on the following aims. Aim 1. To test the hypothesis that the ability of NAS to induce sustained effects on GABAergic inhibition is dependent upon S408/9A in the β3 subunit. Aim 2. To test the hypothesis that the anxiolytic, and anticonvulsant efficacy of NASs is dependent upon S408/9 in the β3 subunit. Aim 3. To test the hypothesis that NASs mediate their metabotropic effects on GABAARs via the activation of mPRs. Collectively, our proposal will identify the molecular mechanisms by which NAS exert their therapeutic actions. This information may aid the development of new therapeutic strategies to alleviate the burdens of anxiety, autism spectrum disorders, depression, epilepsy, and premenstrual syndrome.