B3 subunit phosphorylation determines synaptic and extrasynaptic GABAAR assembly

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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.