Immunoaffinity purification of gamma-aminobutyric acidA (GABAA) receptors containing gamma 1-subunits. Evidence for the presence of a single type of gamma-subunit in GABAA receptors.
Two polyclonal antibodies were raised against the gamma 1-subunit of gamma-aminobutyric acidA (GABAA) receptors. One of these antibodies could be used for the immunopurification of GABAA receptors containing gamma 1-subunits. These receptors exhibited [3H]muscimol and [3H]flunitrazepam binding, and their benzodiazepine binding properties were dramatically different from those of receptors precipitated by anti-gamma 2- or anti-gamma 3-antibodies. Western blot analysis of the immunopurified GABAA receptors indicated that the gamma 1-subunit exhibits an apparent molecular mass of 45-51 kDa. Furthermore, in addition to gamma 1-subunits, beta 2/3-, alpha 1-, alpha 2-, alpha 3-, and alpha 5-subunits could be detected in immunoaffinity column eluates from total brain. In contrast, gamma 2- or gamma 3-subunits could not be identified in GABAA receptors immunopurified by anti-gamma 1-antibodies. Similarly, gamma 1- and gamma 3-subunits or gamma 1- and gamma 2-subunits could not be identified in GABAA receptors purified by anti-gamma 2- or anti-gamma 3-antibodies, respectively. These data seem to indicate that GABAA receptors contain only a single type of gamma-subunit.
Mossier
B
B
Department of Biochemical Psychiatry, University Clinic for Psychiatry, Vienna, Austria.
Tögel
M
M
Fuchs
K
K
Sieghart
W
W
D000818:Animals; D000918:Antibody Specificity; D001483:Base Sequence; D015153:Blotting, Western; D001923:Brain Chemistry; D002846:Chromatography, Affinity; D002998:Clonazepam / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005442:Flumazenil / Q000378:metabolism; D005445:Flunitrazepam / Q000378:metabolism; D007158:Immunologic Techniques; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009118:Muscimol / Q000378:metabolism; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D011487:Protein Conformation; D051381:Rats; D011963:Receptors, GABA-A / Q000145:classification / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology
D010446:Peptide Fragments; D011963:Receptors, GABA-A; D011993:Recombinant Fusion Proteins; D009118:Muscimol; D005442:Flumazenil; D002998:Clonazepam; D005445:Flunitrazepam