Blue Native PAGE–Antibody Shift in Conjunction with Mass Spectrometry to Reveal Protein Subcomplexes: Detection of a Cerebellar α1/α6-Subunits Containing γ-Aminobutyric Acid Type A Receptor Subtype

Abstract

The pentameric γ-Aminobutyric acid type A receptors (GABAARs) are ligand-gated ion channels that mediate the majority of inhibitory neurotransmission in the brain. In the cerebellum, the two main receptor subtypes are the 2α1/2β/γ and 2α6/2β/δ subunits. In the present study, an interaction proteomics workflow was used to reveal additional subtypes that contain both α1 and α6 subunits. Immunoprecipitation of the α6 subunit from mouse brain cerebellar extract co-purified the α1 subunit. In line with this, pre-incubation of the cerebellar extract with anti-α6 antibodies and analysis by blue native gel electrophoresis mass-shifted part of the α1 complexes, indicative of the existence of an α1α6-containing receptor. Subsequent mass spectrometry of the blue native gel showed the α1α6-containing receptor subtype to exist in two main forms, i.e., with or without Neuroligin-2. Immunocytochemistry on a cerebellar granule cell culture revealed co-localization of α6 and α1 in post-synaptic puncta that apposed the presynaptic marker protein Vesicular GABA transporter, indicative of the presence of this synaptic GABAAR subtype.

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

“…Mutations in Nlgn2 genes, leading to impaired folding and trafficking of Nlgn2, have been found in autism subjects [ 52 ]. As previously reported, the Nlgn2-interacting and naked forms of the GABA A receptor can be clearly differentiated by their molecular weight [ 33 ]. Surprisingly, we found that the Nlgn2-interacting GABA A receptor was relatively more abundant in the olfactory bulb compared with the hippocampus, suggesting that the processing and regulation of sensory odor inputs requires a larger quantity of synaptic GABA A receptors.…”

“…Nlgn2 co-migrated with the receptors only at ~680 kDa ( Figure 2 B). The 450 kDa α5-containing protein complex detected here could be assigned to the naked form of the GABA A R, as the GABA A R alone was found to migrate ~450 kDa in the BN gel [ 33 ].…”

“…Immunoprecipitation and SDS-PAGE were similarly prepared as previously described [ 33 , 34 ]. In short, mouse hippocampi and olfactory bulbs were dissected and homogenized in 1% maltose-neopentyl glycol (MNG) buffer (25 mm HEPES, 150 mm NaCl (pH 7.4), and proteinase inhibitor (Roche)) using a homogenizer (Sartorius, Göttingen, Germany; 12 strokes, 900 rpm).…”

“…For each step, a brief wash with water and centrifugation of the plate were performed to remove the supernatant. The sample preparation for mass spectrometry was performed as described previously [ 33 ].…”

“…In the present study, we applied a previously reported interaction–proteomics research platform on hippocampus and olfactory bulb extraction [ 33 ] to reveal the α5 GABA A receptor composition and found substantial heterogeneity of α5-containing receptor subtypes. In particular, the receptors with α1α5 subunits in the hippocampus are more abundant than in the olfactory bulb.…”