Polyclonal Antibodies Directed Against an Epitope Specific for the α4‐Subunit of GABAA Receptors Identify a 67‐kDa Protein in Rat Brain Membranes

Abstract

Polyclonal antibodies were raised to the C‐terminal part of the γ‐aminobutyric acid_A (GABA_A) receptor α4‐subunit. These anti‐peptide α4 (517–523) antibodies specifically identified a protein with apparent molecular mass 67 kDa in rat brain membranes. This protein was enriched by immunoaffinity chromatography of brain membrane extracts on Affigel 10 coupled to the anti‐peptide α4 (517–523) antibodies and could then be identified by the anti‐α4‐antibodies as well as by the GABA_A receptor subunit‐specific monoclonal antibody bd‐28. This appears to indicate that the 67‐kDa protein is the α4‐subunit of GABA_A receptors. Intact GABA_A receptors appeared to be retained by the immunoaffinity column because other GABA_A receptor subunit proteins like the β2/β3‐subunits and the γ2‐subunit were detected in the immunoaffinity column eluate. Furthermore, in addition to the 67‐kDa protein, a 51‐kDa protein could be detected by the antibody bd‐28 and the anti‐peptide α4 (517–523) antibody in the immunoaffinity column eluate. A protein with similar apparent molecular mass was identified by the α1‐subunit‐specific anti‐peptide α1 (1–9) antibody. In contrast to the α1‐subunit, the 51‐kDa protein identified by the anti‐α4 antibody could not be deglycosylated by N‐Glycanase. The identity of the 51‐kDa protein identified by the anti‐α4‐antibodies thus must be further investigated.