Immunochemical Identification of the α1- And α3–Subuntis of the Gaba_A-Receptor in Rat Brain

Abstract

To identify subunit variants of the GABAA-receptor antisera were developed against specific cDNA-derived peptide sequences of the alpha 1- and alpha 3-subunits of rat brain. The alpha 1-subunit antiserum selectively recognized a protein of 50 +/- 1 kDa in rat and bovine GABAA-receptor preparations, while the alpha 3-subunit antiserum interacted with a protein doublet of 59 +/- 2 kDa and 61 +/- 3 kDa. The alpha 1-subunit immunoreactivity resides in a large population of GABAA-receptors as shown by immunoprecipitation of 63 +/- 6% of [3H]flumazenil binding sites with the alpha 1-subunit antiserum. In contrast, only 24 +/- 3% of receptor binding sites were precipitated with the alpha 3-subunit antiserum. Co-precipitation studies suggest that the alpha 1- and alpha 3-subunit immunoreactivities do not share the same receptor population while the gamma 2-subunit immunoreactivity is associated with the alpha 1-subunit immunoreactivity.

Five subtypes of type A gamma-aminobutyric acid receptors identified in neurons by double and triple immunofluorescence staining with subunit-specific antibodies.

“…The subunit specificity of the corresponding antisera is evidenced by (i) the selective recognition of subunits in purified GABAA receptors by Western blotting (13)(14)(15); (ii) the correlation between the regional distribution of the subunit IR and the respective mRNA visualized by in situ hybridization (8)(9)(10)(11)21), except for the a3 subunit in cerebellum (12,28); (iii) the correlation between the absence of subunit IR and lack of subunit mRNA signal, most clearly seen for the al subunit in striatum and olfactory bulb granule cells (9) (Table 1). An association of these subunits in receptor complexes is made likely by several lines of evidence.…”

“…The pattern of subunit immunoreactivity (IR) was analyzed at the cellular and subcellular levels by confocal laser microscopy. To visualize the al, a3, and 'Y2 subunits, polyclonal antisera were used whose specificity had been assessed by Western blotting, immunoprecipitation, and immunohistochemistry (13)(14)(15). Since antibodies for single P subunits are not available, the monoclonal antibody bd-17 (16,17), which recognizes both the P2 and the f3 subunit (18), was used to represent the /-subunit family.…”

“…Sections were cut from frozen blocks with a sliding microtome and processed free-floating for double and triple immunofluorescence staining (25). Polyclonal antisera directed against subunit-specific peptides (a,, residues 1-16; a3, 1-15; Y2, 1-15) raised in rabbits (al, a3, Y2) (13)(14)(15) and guinea pigs (a3, V2), as well as the mouse monoclonal antibody bd-17 (16,18) (#2 and 183 subunits), were used as primary antibodies. After overnight incubation of sections with antibodies diluted in phosphatebuffered saline containing 2% normal serum and 0.2% Triton X-100, secondary antibodies coupled to dichlorotriazinylaminofluorescein, Texas Red, or Cascade blue were applied.…”

GABAAReceptor Subunit Composition and Functional Properties of Cl−Channels with Differential Sensitivity to Zolpidem in Embryonic Rat Hippocampal Cells

“…and rabbit polyclonal antibodies specific for ␣1 (1-9 residues), ␣4 (379 -421 residues), ␣5 (2-10 residues), ␤1 (350 -404 residues), ␤2 (351-405 residues), ␤3 (345-408 residues), ␥1 (324 -366 residues), ␥2 (319 -366 residues), and ␥3 (322-372 residues) subunits (generated by W.S.). The characterization and specificity of these antibodies have been described in detail elsewhere (Benke et al, 1991(Benke et al, , 1997Buchstaller et al, 1991;Marksitzer et al, 1993;Mertens et al, 1993;Mossier et al, 1994;Todd et al, 1996;Sperk et al, 1997). The cells were immunoreacted with guinea pig antibodies diluted 1:1000 or rabbit antibodies diluted 1:100 -300 overnight at room temperature.…”

GABA Expression Dominates Neuronal Lineage Progression in the Embryonic Rat Neocortex and Facilitates Neurite Outgrowth via GABAAAutoreceptor/Cl−Channels

“…GABA A receptor antibodies included guinea pig polyclonals specific for ␣2 (1-9) and ␣3 (1-15) subunits (obtained from Dr. Jean-Marc Fritschy, University of Zurich, Zurich, Switzerland) and rabbit polyclonals specific for ␣1 (1-9), ␣4 (379 -421), ␣5 (2-10), ␣6 (429 -434), ␤1 (350 -404), ␤2 (351-405), ␤3 (345-408), ␥1 (324 -366), ␥2 (319 -366), ␥3 (322-372), and ␦ (1-44) subunits (obtained from Dr. Werner Sieghart, University C linic for Psychiatry, Vienna, Austria); GABA B receptor antibodies included guinea pig polyclonals specific for R1 (both R1a and R1b isoforms) and R2 subunits (Chemicon, Temecula, CA). The characterization and specificity of the aforementioned antibodies have been described elsewhere (Benke et al, 1991(Benke et al, , 1997Buchstaller et al, 1991;Marksitzer et al, 1993;Martin and Rimvall, 1993;Mertens et al, 1993;Rimvall et al, 1993;Mossier et al, 1994;Todd et al, 1996;Sperk et al, 1997;Jones et al, 1998;Kaupmann et al, 1998;White et al, 1998;Durkin et al, 1999;Martin et al, 1999;Yung et al, 1999;C alver et al, 2000;Princivalle et al, 2000).…”