Brain Region Differences in α1- and α5-Subunit-Containing GABAA Receptor Proteomes Revealed with Affinity Purification and Blue Native PAGE Proteomics

Abstract

GABA_A receptors are the major inhibitory receptors in the brain. They are hetero-pentamers with a composition of predominantly two α, two β, and one γ or δ subunit. Of the six α subunit genes, the α5 subunit displays a limited spatial expression pattern and is known to mediate both phasic and tonic inhibition. In this study, using immunoaffinity-based proteomics, we identified the α5 subunit containing receptor complexes in the hippocampus and olfactory bulb. The α1-α5 interaction was identified in both brain regions, albeit with significantly different stoichiometries. In line with this, reverse IPs using anti-α1 antibodies showed the α5-α1 co-occurrence and validated the quantitative difference. In addition, we showed that the association of Neuroligin 2 with α1-containing receptors was much higher in the olfactory bulb than in the hippocampus, which was confirmed using blue native gel electrophoresis and quantitative mass spectrometry. Finally, immunocytochemical staining revealed a co-localization of α1 and α5 subunits in the post-synaptic puncta in the hippocampus.

Keywords: GABAA receptor; brain; immunocytochemistry; immunoprecipitation; proteomics; synapse.

Figure 1

Quantitative proteomics of α5, α1, and Nlgn2 IPs performed on extracts of the hippocampus and olfactory bulb. The relative quantity of GABA_AR subunits and known interactors identified from the anti-α5 IP (upper panel), anti-α1 IP (middle panel), and anti-Nlgn2 IP (bottom panel). IP experiments were performed with three biological replicates. The iBAQ abundance values per sample were normalized to the respective bait protein’s abundance, and linear regression was applied to compare relative prey abundances between brain regions. * p value < 0.05 and ** p value < 0.01.

Figure 2

Migration of GABA_AR subunits and known interactors obtained from hippocampus and olfactory bulb extracts with BN-PAGE and identified with mass spectrometry and immunoblotting. Migration profiles of the protein complexes containing (A) GABA_AR α5, (B) Nlgn2, (C) GABA_AR α1, (D) β2, (E) β3, (F) γ2, and (G) δ extracted from the hippocampus and olfactory bulb with BN-PAGE as identified with mass spectrometry. (H) Native GABA_AR run on BN-PAGE from extracts of hippocampi and olfactory bulbs. The migration patterns of the receptors were visualized with immunoblots with antibodies against Gabra1, Gabrb2/3, and Nlgn2. (I) Quantitative analysis of the intensity ratios of bands at 680 kDa over 450 kDa on immunoblots. n = 3 samples from three independent regional brain whole tissue lysates; data are mean ± s.e.m. ** p < 0.01.

Figure 3

The α1-/α5-containing GABA_A receptors demonstrated synaptic localizations in a primary hippocampal neuronal culture. The distributions of α1 and α5 subunits were investigated (A,B) and both subunits were co-stained with the inhibitory presynaptic marker protein vGat (C). (D) The α1 subunit was mostly synaptically localized, opposing vGat, whereas α5 was distributed at both the soma and synapse and often showed co-localization with α1-vGat puncta at the synapse (E,F). (G) The Manders’ coefficient of the α1 subunit (green channel) with an α5 subunit signal (red channel) and colocalized α1 and α5 subunit signals (blue channel) were measured. The latter one is indicated by Manders’ coefficients with vGat; n = 15, fields of view; n = 3, cultures.