Blue Native PAGE-Antibody Shift in Conjunction with Mass Spectrometry to Reveal Protein Subcomplexes: Detection of a Cerebellar alpha 1/alpha 6-Subunits Containing gamma-Aminobutyric Acid Type A Receptor Subtype

The pentameric gamma-Aminobutyric acid type A receptors (GABA(A)Rs) 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 2 alpha 1/2 beta/gamma and 2 alpha 6/2 beta/delta subunits. In the present study, an interaction proteomics workflow was used to reveal additional subtypes that contain both alpha 1 and alpha 6 subunits. Immunoprecipitation of the alpha 6 subunit from mouse brain cerebellar extract co-purified the alpha 1 subunit. In line with this, pre-incubation of the cerebellar extract with anti-alpha 6 antibodies and analysis by blue native gel electrophoresis mass-shifted part of the alpha 1 complexes, indicative of the existence of an alpha 1 alpha 6-containing receptor. Subsequent mass spectrometry of the blue native gel showed the alpha 1 alpha 6-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 alpha 6 and alpha 1 in post-synaptic puncta that apposed the presynaptic marker protein Vesicular GABA transporter, indicative of the presence of this synaptic GABA(A)R subtype.