B3 subunit phosphorylation determines synaptic and extrasynaptic GABAAR assembly

项目总结/摘要 通过γ-氨基丁酸A型受体（GABAARs）的抑制性信号传导受损是病理生理学的基础 包括自闭症谱系障碍在内的几种神经精神疾病的症状。神经元抑制受α 1- 含有突触GABAAR和含有α 4的突触外GABAAR，它们介导相位和紧张性 抑制，分别。这两种亚型都是适当控制抑制性神经传递所必需的，然而， 神经元控制这两种亚型组装的机制尚未阐明。此外，本发明还提供了一种方法， 而β 3亚单位，这是由α 1和α 4-含有GABAAR亚型共享，调节表面 通过丝氨酸408和丝氨酸409残基的磷酸化（S408/9）表达GABAAR， S408/9在介导GABAAR组装中的作用仍有待发现。 该提案结合了生物化学和分子生物学方法来测试新的假设， S408/9的磷酸化调节调节含α 1的突触和含α 4的突触的组装。 突触外GABAAR亚型，突变S408/9损害GABAAR组装， GABA能抑制。检测S408/9的亚基组成、蛋白质组和磷酸化水平 成年C57 Bl/6小鼠脑中含有α 1和α 4的GABAARs的研究将揭示其结构特性， 内源性α 1和α 4 GABAAR组装的潜在机制（目的1）。调查 在S408/9（S408/9A）的丙氨酸取代如何改变亚基组成，蛋白质组，亚细胞 S408/9A动物中含α 1和α 4的GABAAR的定位和半衰期将提供以下信息 S408/9在GABAAR亚型组装中的作用（目的2）。总的来说，这些目标将发现 参与α 1和α 4亚基分选成两种GABAAR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。 为了成功完成这个项目，申请人将接受全面的生物化学，分子生物学， 生物学，成像技术，以及生物信息学。此外，申请人将获得 在科学传播，指导和职业探索培训的众多机会。苔藓 塔夫茨大学的实验室培养了一个环境，在那里申请人将智力参与， 支持所有必要的设施，设备和指导，以确保生产性的研究生培训，并 为申请人作为转化神经科学的独立研究科学家的蓬勃发展做好准备。

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.