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Interaction proteomics of cortical GABAA receptor subunits identifies novel interactors

ABSTRACT: GABAA receptors (GABAAR) are pentameric ligand-gated ion channels responsible for most of the inhibitory synaptic transmission in the brain. The distinct physiological and pharmacological properties of these receptors are underlain by unique subunit composition and receptor associated proteins, where the most abundant form of synaptic GABAAR comprises of 2 copies of α1, 2 copies of β, and one copy of γ2 subunits. To reveal novel receptor protein complexes, we performed affinity-based proteomics analysis from mouse cortex extract using antibodies against GABAAR α1, β2/3, and γ2 subunits, as well as antibodies against the known GABAAR interacting proteins Nlgn2. From this analysis various interactions between GABAAR α subunits were identified. Focusing on the intersection of the three subunit proteomes yielded a more precise proteome analysis of a specific GABAAR subtype, and revealed Camkv and Gpc as novel GABAAR interactors. Their direct interaction was validated by co-expression and co-immunoprecipitation experiments.

ORGANISM(S): Mus Musculus

SUBMITTER: Kawan Li

PROVIDER: PXD048233 | iProX | Thu Jan 04 00:00:00 GMT 2024

REPOSITORIES: iProX

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Project description: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 γ-aminobutyric acid type A receptors (GABAARs), which mediate phasic and tonic inhibition, respectively. These two GABAAR subtypes differ in their function, ligand sensitivity, and physiological properties. Importantly, they contain different α subunit isoforms: synaptic GABAARs contain the α1-3 subunits whereas extrasynaptic GABAARs contain the α4-6 subunits. While the subunit composition is critical for the distinct roles of synaptic and extrasynaptic GABAAR 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 GABAARs 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 GABAARs 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 GABAARs, 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 GABAARs. 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 GABAARs, and thus the efficacy of neuronal inhibition.

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Interaction proteomics of cortical GABAA receptor subunits identifies novel interactors

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