Evidence for the Existence of Differential O-Glycosylated α5-Subunits of the γ-Aminobutyric AcidA Receptor in the Rat Brain

: Polyclonal antibodies were raised to synthetic peptides having amino acid sequences corresponding with the N- or C-terminal part of the γ-aminobutyric acidA (GABAA) receptor α5-subunit. These anti-peptide α5(2–10) or anti-peptide α5(427–433) antibodies reacted specifically with GABAA receptors purified from the brains of 5–10-day-old rats in an enzyme-linked immunosorbent assay and were able to dose-dependently immunoprecipitate up to 6.3 or 13.1% of the GABAA receptors present in the incubation, respectively. In immunoblots, each of these antibodies reacted with the same two protein bands with apparent molecular mass of 53 or 57 kDa. After exhaustive treatment of purified GABAA receptors with N-Glycanase, each of these antibodies identified two proteins with apparent molecular masses of 46 and 48 kDa. Additional treatment of GABAA receptors with neuraminidase and O-Glycanase resulted in an apparently single protein with molecular mass of 47 kDa, which again was identified by both the anti-peptide α5(2–10) and the anti-peptide α5(427–433) antibody. These results indicate the existence of at least two different α5-sub-units of the GABAA receptor that differ in their carbohydrate content. In contrast to other α- or β-subunits of GABAA receptors so far investigated, at least one of these two α5-subunits contains O-linked carbohydrates.