Polyclonal antibodies directed against an epitope specific for the alpha 4-subunit of GABAA receptors identify a 67-kDa protein in rat brain membranes.

Polyclonal antibodies were raised to the C-terminal part of the gamma-aminobutyric acidA (GABAA) receptor alpha 4-subunit. These anti-peptide alpha 4 (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 alpha 4 (517-523) antibodies and could then be identified by the anti-alpha 4-antibodies as well as by the GABAA receptor subunit-specific monoclonal antibody bd-28. This appears to indicate that the 67-kDa protein is the alpha 4-subunit of GABAA receptors. Intact GABAA receptors appeared to be retained by the immunoaffinity column because other GABAA receptor subunit proteins like the beta 2/beta 3-subunits and the gamma 2-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 alpha 4 (517-523) antibody in the immunoaffinity column eluate. A protein with similar apparent molecular mass was identified by the alpha 1-subunit-specific anti-peptide alpha 1 (1-9) antibody. In contrast to the alpha 1-subunit, the 51-kDa protein identified by the anti-alpha 4 antibody could not be deglycosylated by N-Glycanase. The identity of the 51-kDa protein identified by the anti-alpha 4-antibodies thus must be further investigated.