B3 subunit phosphorylation determines synaptic and extrasynaptic GABAAR assembly

项目摘要/摘要 γ-氨基丁酸A型受体(GABAARs)抑制信号受损是其病理生理学基础 包括自闭症谱系障碍在内的几种神经精神障碍。神经元抑制受α-1-调节 含有突触GABA受体和含有α4的突触外GABA受体，它们调节时相和强直 分别为抑制作用。这两种亚型都是适当控制抑制性神经传递所必需的，但 神经元控制这两种亚型组装的机制尚未阐明。此外, 而β3亚基由含有α1和α4的GABA亚型所共有，调节表面 通过丝氨酸408和409残基的磷酸化表达GABAARs(S408/9)，其作用 S408/9在介导GABAAR组装中的作用有待发现。 这一建议结合了生物化学和分子生物学的方法来检验新的假设 S408/9的磷酸化调节调节含α1的突触和含α4的突触的组装 突触外GABAAR亚型，突变的S408/9损害GABAAR组装，因此 GABA能抑制。检测磷酸化S408/9的亚基组成、蛋白质组和水平 在成年C57BL/6小鼠脑中对含有α1和α4的GABA受体的研究将揭示其结构特性和 包含α1和α4的内源性GABA受体组装的潜在机制(目标1)。调查 S408/9(S408/9A)的丙氨酸替换如何改变亚基组成、蛋白质组、亚细胞 含有α1和α4的GABA受体在S408/9A动物体内的定位和半衰期将提供关于 S408/9在GABAAR亚型组装中的作用(目标2)。总的来说，这些目标将发现 α1和α4亚基分选为两个调节时相的GABA亚型的机制 和紧张性抑制。该项目的长期目标是确定S408/9作为新的治疗靶点 GABA能抑制受损的疾病状态。 为了成功完成这个项目，申请者将接受彻底的生化、分子方面的培训。 生物学、成像技术以及生物信息学。此外，申请人将获提供 在科学交流、指导和职业探索方面有大量的培训机会。苔藓 塔夫茨大学的实验室培养了申请者参与智力活动的环境，并 支持所有必要的设施、设备和指导，以确保有成效的研究生培训，并 为申请者在翻译神经科学领域的独立研究科学家的职业生涯做好准备。

Project Summary/Abstract Impaired inhibitory signaling via γ-aminobutyric acid type A receptors (GABAARs) underlies the pathophysiology of several neuropsychiatric disorders including autism spectrum disorder. Neuronal inhibition is regulated by α1- containing synaptic GABAARs and α4-containing extrasynaptic GABAARs that mediate phasic and tonic inhibition, respectively. Both subtypes are required for proper control of inhibitory neurotransmission, yet the mechanism by which neurons control the assembly of these two subtypes has not been elucidated. In addition, while the β3 subunits, which are shared by both α1- and α4-containing GABAAR subtypes, regulate the surface expression of GABAARs via phosphorylation of the serine 408 and serine 409 residues (S408/9), the role of S408/9 in mediating GABAAR assembly remains to be discovered. This proposal combines biochemical and molecular biology approaches to test the novel hypothesis that phospho-regulation of S408/9 regulates the assembly of α1-containing synaptic and α4-containing extrasynaptic GABAAR subtypes, and that mutating S408/9 compromises GABAAR assembly and thus GABAergic inhibition. Examining the subunit compositions, proteomes, and levels of phosphorylated S408/9 of α1- and α4-containing GABAARs in adult C57Bl/6 mouse brains will unveil the structural properties and mechanisms underlying the assembly of endogenous α1- and α4-containing GABAARs (Aim 1). Investigating how the alanine substitutions at S408/9 (S408/9A) alter the subunit compositions, proteomes, sub-cellular localizations, and half-lives of α1- and α4-containing GABAARs in S408/9A animals will provide information on the role of S408/9 in the assembly of GABAAR subtypes (Aim 2). Collectively, these aims will discover the mechanisms involved in the sorting of α1 and α4 subunits into the two GABAAR subtypes that mediate phasic and tonic inhibition. The long-term objective of this project is to identify S408/9 as novel therapeutic targets for disease conditions with impaired GABAergic inhibition. For successful completion of this project, the applicant will be thoroughly trained in biochemical, molecular biology, and imaging techniques, as well as in bioinformatics. In addition, the applicant will be provided with numerous opportunities for training in science communication, mentorship, and career exploration. The Moss laboratory at Tufts University fosters an environment where the applicant will be intellectually engaged and supported with all necessary facility, equipment, and guidance to ensure productive graduate training, and to prepare the applicant for a flourishing career as an independent research scientist in translational neuroscience.