AUTHOR=Choi Catherine , Smalley Joshua L. , Lemons Abigail H. S. , Ren Qiu , Bope Christopher E. , Dengler Jake S. , Davies Paul A. , Moss Stephen J. 

TITLE=Analyzing the mechanisms that facilitate the subtype-specific assembly of γ-aminobutyric acid type A receptors

JOURNAL=Frontiers in Molecular Neuroscience

VOLUME=15

YEAR=2022

URL=https://www.frontiersin.org/journals/molecular-neuroscience/articles/10.3389/fnmol.2022.1017404

DOI=10.3389/fnmol.2022.1017404

ISSN=1662-5099

ABSTRACT=<p>Impaired inhibitory signaling underlies the pathophysiology of many neuropsychiatric and neurodevelopmental disorders including autism spectrum disorders and epilepsy. Neuronal inhibition is regulated by synaptic and extrasynaptic <italic>γ</italic>-aminobutyric acid type A receptors (GABA<sub><italic>A</italic></sub>Rs), which mediate phasic and tonic inhibition, respectively. These two GABA<sub><italic>A</italic></sub>R subtypes differ in their function, ligand sensitivity, and physiological properties. Importantly, they contain different α subunit isoforms: synaptic GABA<sub><italic>A</italic></sub>Rs contain the α1–3 subunits whereas extrasynaptic GABA<sub><italic>A</italic></sub>Rs contain the α4–6 subunits. While the subunit composition is critical for the distinct roles of synaptic and extrasynaptic GABA<sub><italic>A</italic></sub>R subtypes in inhibition, the molecular mechanism of the subtype-specific assembly has not been elucidated. To address this issue, we purified endogenous α1- and α4-containing GABA<sub><italic>A</italic></sub>Rs from adult murine forebrains and examined their subunit composition and interacting proteins using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and quantitative analysis. We found that the α1 and α4 subunits form separate populations of GABA<sub><italic>A</italic></sub>Rs and interact with distinct sets of binding proteins. We also discovered that the β3 subunit, which co-purifies with both the α1 and α4 subunits, has different levels of phosphorylation on serines 408 and 409 (S408/9) between the two receptor subtypes. To understand the role S408/9 plays in the assembly of α1- and α4-containing GABA<sub><italic>A</italic></sub>Rs, we examined the effects of S408/9A (alanine) knock-in mutation on the subunit composition of the two receptor subtypes using LC-MS/MS and quantitative analysis. We discovered that the S408/9A mutation results in the formation of novel α1α4-containing GABA<sub><italic>A</italic></sub>Rs. Moreover, in S408/9A mutants, the plasma membrane expression of the α4 subunit is increased whereas its retention in the endoplasmic reticulum is reduced. These findings suggest that S408/9 play a critical role in determining the subtype-specific assembly of GABA<sub><italic>A</italic></sub>Rs, and thus the efficacy of neuronal inhibition.</p>