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

Abstract

GABAA 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

• GABA_A receptor
• immunoprecipitation
• proteomics
• brain
• synapse
• immunocytochemistry