Abstract
Three novel subunit-specific antisera to the beta1, beta2, and beta3 subunits of rat gamma-aminobutyric acid type A (GABAA) receptors have been used to study the native receptor in the rat brain. Affinity-purified anti-beta1, anti-beta2, and anti-beta3 antibodies recognized in immunoblots protein bands of 57, 55, and 57 kDa, respectively. Quantitative immunoprecipitation of solubilized GABAA receptors from various rat brain regions showed that the beta2 subunit was the most abundant isoform in cerebellum (in 96% of the GABAA receptors) and cerebral cortex (64%) but that it was the least abundant isoform in hippocampus (44%). The beta3 subunit was found most abundant in hippocampus (64%) followed by cerebral cortex (48%) and cerebellum (33%). The beta1 subunit was present in a very small proportion of the cerebellar GABAA receptors (3%), but it was present in a high proportion of the GABAA receptors from the hippocampus (49%) and cerebral cortex (32%). Quantitative receptor immunoprecipitation or immunopurification followed by immunoblotting experiments have revealed the existence of colocalization of two different beta subunit isoforms in a significant proportion of the brain GABAA receptors. Thus, in the rat cerebral cortex 33% of the GABAA receptors have both beta2 and beta3 subunits, and 19% of the receptors have both beta1 and beta3 subunits. The extent of colocalization of beta subunit isoforms varied among brain regions, being highest in hippocampus and lowest in cerebellum. These and other results taken together suggest that the number of alpha, beta, and gamma subunits (stoichiometry) in the brain GABAA receptor pentamers might not be unique. It might vary depending on receptor type.
Highlights
The brain GABAA1 receptors are heteropentameric [1] membrane proteins that form ClϪ channels
We report the development of novel  subunit isoform-specific antisera and the use of these antibodies to demonstrate the existence of colocolization of  subunit isoforms in the same brain GABAA receptor
Each antibody-peptide reaction was dose-dependently blocked by the corresponding peptide but not by the others. (iii) Receptor immunoprecipitation was inhibited by the corresponding peptide but not by the others. (iv) Immunoprecipitations of GABAA receptors from various brain regions showed that the relative abundance of the  subunit peptides correlated with the relative abundance of the corresponding mRNAs (29 –32). (v) In immunoblots, each isoform-specific antibody recognized a protein band of the expected size for that particular  subunit [14, 21,22,23, 33,34,35]
Summary
Materials—N-[methyl-3H]Flunitrazepam (FNZ, 87 Ci/mmol) and [methylene-3H]muscimol (12.5 Ci/mmol) were from DuPont NEN. The GABAA receptors (0.3– 0.4 pmol of [3H]FNZ binding activity) in modified radioimmune precipitation buffer (0.137 M NaCl, 1% sodium deoxycholate, 1% Triton X-100, 0.1% sodium dodecyl sulfate, 10 mM Tris-HCl, pH 7.4) containing a mixture of protease. Immunopurification of  Subunit Containing GABAA Receptors— Each anti- subunit antibody was irreversibly coupled through the Fc moiety to Protein A-agarose beads with dimethylpimelimidate, according to the procedure of Schneider et al [25]. The GABAA receptors from rat cerebral cortex membranes were solubilized in modified radioimmune precipitation buffer and were bound onto the immobilized anti- subunit antibody (bound to the agarose beads) by overnight incubation at 4 °C. The beads were extensively washed with modified radioimmune precipitation buffer followed by 10 mM sodium phosphate, pH 6.8, 0.05% Triton X-100. The ELISA was performed as described by De Blas et al [27]
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