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

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 gamma-aminobutyric acid type A receptors (GABA(A)Rs), which mediate phasic and tonic inhibition, respectively. These two GABA(A)R subtypes differ in their function, ligand sensitivity, and physiological properties. Importantly, they contain different alpha subunit isoforms: synaptic GABA(A)Rs contain the alpha 1-3 subunits whereas extrasynaptic GABA(A)Rs contain the alpha 4-6 subunits. While the subunit composition is critical for the distinct roles of synaptic and extrasynaptic GABA(A)R subtypes in inhibition, the molecular mechanism of the subtype-specific assembly has not been elucidated. To address this issue, we purified endogenous alpha 1- and alpha 4-containing GABA(A)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 alpha 1 and alpha 4 subunits form separate populations of GABA(A)Rs and interact with distinct sets of binding proteins. We also discovered that the beta 3 subunit, which co-purifies with both the alpha 1 and alpha 4 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 alpha 1- and alpha 4-containing GABA(A)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 alpha 1 alpha 4-containing GABA(A)Rs. Moreover, in S408/9A mutants, the plasma membrane expression of the alpha 4 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(A)Rs, and thus the efficacy of neuronal inhibition.