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

Authors


Address correspondence and reprint requests to Dr. W. Sieghart at Department of Biochemical Psychiatry, University Clinic for Psychiatry, Währinger Gürtel 18-20, A-1090 Vienna, Austria.

Abstract

Abstract: Polyclonal antibodies were raised to the C-terminal part of the γ-aminobutyric acidA (GABAA) 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 GABAA receptor subunit-specific monoclonal antibody bd-28. This appears to indicate that the 67-kDa protein is the α4-subunit of GABAA receptors. Intact GABAA receptors appeared to be retained by the immunoaffinity column because other GABAA 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.

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