Analyzing the mechanisms that facilitate the subtype-specific assembly of γ-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.

Automated summary

Impaired inhibitory signaling is the underlying cause of various neuropsychiatric and neurodevelopmental disorders. Synaptic and extrasynaptic GABA (A) Rs regulate neuronal inhibition through different subunit composition and interacting proteins. Differences in phosphorylation levels result in distinct assembly and expression patterns of GABA (A) Rs in different subtypes.