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

Abstract

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 (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 α subunit isoforms: synaptic GABA _A Rs contain the α1-3 subunits whereas extrasynaptic GABA _A Rs contain the α4-6 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 α1- and α4-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 α1 and α4 subunits form separate populations of GABA _A 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 _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 α1α4-containing GABA _A 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 _A Rs, and thus the efficacy of neuronal inhibition.

Keywords: GABAA receptors; phosphorylation; protein purification; subunit composition; trafficking.

FIGURE 1

At steady state, the endogenous α1 and α4 subunits are segregated into distinct GABA_A subtypes. (A) Purified plasma membrane fraction (PM), PM exposed to non-immune IgG, and PM exposed to the α1 subunit antibody for purification of GABA_ARs that contain the α1 subunit (α1 IP) were resolved on Blue Native PAGE (BN-PAGE) gels and probed for the α1 subunit. Major bands at around 250 and 720 kDa, which represent heteropentameric GABA_ARs in isolation and heteropentameric GABA_ARs with binding partners, respectively, were observed. (B) Gel regions containing the 250 kDa band were subjected to liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Analysis of the 250 kDa band with label-free quantification and Welch’s t-test revealed that in α1-containing GABA_ARs, the α1–3, β1–3, γ2–3, and δ subunits are significantly enriched when compared to non-immune IgG control. Detected but non-significant subunits are not shown (**p < 0.01, ***p < 0.001, ****p < 0.0001, n = 5 replicates). (C) Purified PM, PM exposed to non-immune IgG, and PM exposed to the α4 subunit antibody (α4 IP) were resolved on BN-PAGE and probed with the α4 subunit antibody. A band at 250 kDa and a weaker band at 720 kDa were observed with immunoblotting. (D) The contents of the 250 kDa band were analyzed with LC-MS/MS. Only the α4 and β3 subunits are significantly enriched in α4-containing GABA_ARs when compared to non-immune IgG (**p < 0.01, n = 5 replicates). (E) The amount of target subunit recovery in 250 kDa bands is higher for the α1 subunit than the α4 subunit (*p < 0.05, n = 5 replicates).

FIGURE 2

The endogenous α1 subunit interacts with inhibitory scaffolding proteins and cytoskeletal proteins. (A) The 720 kDa band of α1-containing GABA_ARs purified from PM was subjected to LC-MS/MS. Label-free normalized quantification and Welch’s t-test against non-immune control IgG revealed 93 significantly enriched binding proteins of the α1 subunit. The heatmap shows the top 10 binding proteins of α1-containing GABA_ARs by abundance (SI_GI value) across all replicates. The most abundant proteins detected with the α1 subunit are Sptan1, Sptbn1, and Sptbn2 (n = 5 replicates). (B) Pie chart shows the relative abundance of the proteins that are significantly enriched with α1-containing GABA_ARs. Sptan1, Sptbn1, and Sptbn2 together constitute more than 70% of the total amount of proteins. Proteins that make up less than 2% of the total amount of proteins (86 proteins) were grouped together as ‘Other.’ (C) Immunopurified α1-containing GABA_ARs were resolved on BN-PAGE and probed for the α1 subunit, high-abundance proteins (Sptan1, Sptbn1, and Sptbn2), and low-abundance proteins (Erc2, Kcc2, and gephyrin). Sptan1, Sptbn1, and Sptbn2 are observed at around 720 kDa with α1-containing GABA_ARs. Erc2 and Kcc2 are present slightly below 720 kDa and gephyrin is observed higher than 720 kDa. The immunoreactivity of these proteins is specific to purified α1-containing GABA_ARs. (D) The network analysis shows known inhibitory scaffolding proteins such as Actn1, Cyfip2, and Gphn (gephyrin), and a subnetwork of spectrins (Sptan1, Sptbn1, Sptbn2, and Sptb) and ankyrins (Ank2 and Ank3). GO analysis reveals that most proteins are involved in cytoskeletal organization, ion transport, or signal transduction.

FIGURE 3

The endogenous α4 subunit does not interact with inhibitory scaffolding proteins. (A) Analysis of the 720 kDa band of α4-containing GABA_ARs with LC-MS/MS and quantitative analysis identified 19 binding proteins. The heatmap shows the top 10 binding proteins by abundance, which include Ttn (titin), Myo5a, and Dsp (desmoplakin). Myo5a and Vdac2 were detected with both the α1 and α4 subtypes (n = 5 replicates). (B) The relative abundance of binding proteins of the α4 subunit is illustrated as a pie chart. Besides 20% by titin and 13% by Myo5a, most proteins constitute less than 10% of the total amount of proteins detected with α4-containing GABA_ARs. Proteins that make up less than 2% each (6 proteins) were grouped together as ‘Other.’ (C) BN-PAGE blots of purified α4-containing GABA_ARs show the absence of gephyrin in the α4 subtype. (D) The network analysis reveals no known interactions among the proteins detected with the α4 subunit. (E) PCA plot of the significantly enriched binding proteins of the α1 and α4 subunits show high reproducibility of datasets between replicates. It also highlights the difference in the binding proteins of the two receptor subtypes. (F) Target subunit recovery in 720 kDa bands between subtype purifications is comparable (ns ≥ 0.05, n = 5 replicates).

FIGURE 4

The β3 subunit is more highly phosphorylated at S408 and S409 in α1-containing GABA_ARs than in the α4 subtype. (A) Immunoblots of purified α1- and α4-containing GABA_ARs were resolved on SDS-PAGE and probed for the target subunit (α1 or α4 subunit), β3 subunit, and phosphorylated S408/9 (p-S408/9). (B) The percentage of p-S408/9 in respect to total β3 subunit is significantly higher in the α1 subtype than in the α4 subtype (*p < 0.05, n = 4 replicates). (C) Spectra searches were performed on LC-MS/MS data obtained from purified α1- and α4-containing GABA_ARs to identify phosphorylated serine (S), threonine (T), or tyrosine (Y) residues within the β3 subunit. Three known (S383, S408, and S409) and seven novel (T322, S330, S332, Y391, S396, T402, and T419) phosphorylation sites were detected. S332 was detected in the α1 subtype only (n = 5 replicates). (D) Illustration of a GABA_AR β3 subunit with phosphorylation sites detected by LC-MS/MS is shown. Known and novel sites are in pink and green, respectively.

FIGURE 5

Mutation of S408/9 does not change the total expression level of the α1 or α4 subunit. (A) Immunoblots of forebrain tissues from WT and S408/9A mice were probed for the α1 subunit, α4 subunit, and GAPDH. (B) Signal intensity of the α1 subunit was normalized to that of GAPDH. There is no significant change in the total level of the α1 subunit in the forebrain between WT and S408/9A mice (ns ≥ 0.05, n = 3). (C) The S408/9A mutation does not affect the total level of the α4 subunit in the forebrain (ns ≥ 0.05, n = 3).

FIGURE 6

Mutation of S408/9 leads to the formation of α1α4-containing GABA_ARs. (A) Purified PM from S408/9A mice were exposed to non-immune control IgG or the α1 subunit antibody and probed for the α1 subunit. Major bands at 250 and 720 kDa were observed. (B) Quantitative analysis of the contents of the 250 kDa band revealed that the α4 and α5 subunits are significantly enriched in α1-containing GABA_ARs in S408/9A mice, along with the α1, α3, β1–3, γ2, and δ subunits (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 5 replicates). (C) Contribution of each α subunit variant to the total amount of α subunits is compared between α1-containing GABA_ARs from WT and S408/9A mice. With the S408/9A mutation, the percentages of the α1 and α2 subunits decrease whereas those of the α3, α4, and α5 subunits increase. (D) The α4 subunit antibody recognized a major band at 250 kDa in purified α4-containing GABA_ARs from S408/9A mice. (E) In α4-containing GABA_ARs from S408/9A mice, only the α4 and β3 subunits are significantly enriched (**p < 0.01, n = 5 replicates). (F) The S408/9A mutation does not affect the efficacy of the purification of either α1- or α4-containing GABA_ARs (ns ≥ 0.05, n = 5 replicates).

FIGURE 7

The S408/9A mutation does not lead to gross changes in global β3 subunit phosphorylation. (A) Phosphorylated residues within the β3 subunit of α1-containing GABA_ARs are compared between WT and S408/9A mice. The mutation does not induce aberrant phosphorylation on the β3 subunit, as indicated by the detection of the same phosphorylation sites in WT and S408/9A mice. Two sites – T322 and T419 – are more highly phosphorylated in S408/9A than in WT mice (*p < 0.05, n = 5 replicates). (B) Phosphorylated residues within the β3 subunit of α4-containing GABA_ARs from WT and S408/9A mice are shown. Phosphorylated S332 was only detected in the α4 subtype from S408/9A mice (n = 5 replicates).

FIGURE 8

The S408/9A mutation selectively affects the distribution of the α4 subunit across subcellular compartments. (A) Immunoblots of total forebrain lysate (TL), PM, and ER fractions from WT animals were probed for N-cadh, α1, α4, and β3 subunits. (B) The distribution of each GABA_AR subunit between PM and ER was calculated as a percentage of total expression in TL, PM, and ER. Higher percentage of the α1 subunit is expressed in the PM than in the ER (**p < 0.01, n = 3). (C) Similar levels of the α4 subunit are observed in the PM and ER (ns ≥ 0.05, n = 3). (D) The β3 subunit is more significantly enriched in the PM than in the ER (**p < 0.01, n = 3). (E) Levels of N-Cadh, α1, α4, and β3 subunits were assessed in immunoblots of TL, PM, and ER fractions from S408/9A animals. (F–H) In S408/9A animals, higher levels of the α1, α4, and β3 subunits are expressed in the PM than in the ER (*p < 0.05, ***p < 0.001, ****p < 0.0001, n = 3).

FIGURE 9

Colocalization of the α4 subunit with calreticulin is reduced in cultured neurons from S408/9A mice. (A) Representative images of the soma of DIV21 neurons cultured from WT and S408/9A mice stained with the α4 subunit and calreticulin antibodies are shown. Individual channels and areas of colocalization for each cell are shown on the right. (B) Total area of the α4 subunit staining in the soma is not affected by the S408/9A mutation (ns ≥ 0.05, n = 18 cells from three individually prepared cultures). (C) There is no significant difference in calreticulin staining in the soma between WT and S408/9A cultures (ns ≥ 0.05, n = 18 cells from three individually prepared cultures). (D) Colocalized area of the α4 subunit with calreticulin is reduced in S408/9A cultures when compared to WT cultures (*p ≤ 0.05, n = 18 cells from three individually prepared cultures).