Antibodies to the Human γ2 Subunit of the γ-Aminobutyric AcidA/Benzodiazepine Receptor

Abstract: A γ-aminobutyric acid_A (GABA_A) receptor (GABA_AR) γ₂ subunit (short form) was cloned from an adult human cerebral cortex cDNA library in bacteriophage λgt11. The 261-bp intracellular loop (IL) located between M3 and M4 was amplified using the polymerase chain reaction and inserted into the expression vectors λgt11 and pGEX-3X. Both γ-galactosidase (LacZ) and glutathione-S-transferase (GST) fusion proteins containing the γ₂IL were purified, and a rabbit antibody to the LacZ–γ₂IL was made. The antibody reacted with the γ₂IL of both LacZ and GST fusion proteins and immunoprecipitated the GABA_AR/ benzodiazepine receptor (GABA_AR/BZDR) from bovine and rat brain. The antibody reacted in affinity-purified GABA_AR/BZDR immunoblots with a wide peptide band of 44,000–49,000 M_r. Immunoprecipitation studies with the anti-γ₂IL antibody suggest that in the cerebral cortex, 87% of the GABA_ARs with high affinity for benzodiazepines and 70% of the GABA_ARs with high affinity for muscimol contain at least a γ subunit, probably a γ₂. These results indicate that there are [³H]muscimol binding GABA_ARs that do not bind [³H]flunitrazepam with high affinity. Immunoprecipitations with this and other anti-GABA_AR/BZDR antibodies indicate that the most abundant combination of GABA_AR subunits in the cerebral cortex involves α₁, γ₂ (or other γ), and β₂ and/or β₃ subunits. These subunits coexist in >60% of the GABA_AR/BZDRs in the cerebral cortex. The results also show that a considerable proportion (20–25%) of the cerebellar GABA_AR/BZDRs is clonazepam insensitive. At least 74% of these cerebellar receptors, which likely contain α₆, also contain γ₂ (or other γ) subunit(s). The α₁ and β₂ or β₃ subunits are also frequently associated with γ₂ (or other γ) and α₆ in these cerebellar receptors.