Localization and functional expression of alternatively spliced forms of the GABA_A receptor γ₂ subunit

Recent studies have identified two alternatively spliced forms of the GABA_A receptor γ₂ subunit that differ by the presence (γ_2L) or absence (γ_2S) of an eight-amino acid segment. This insert in the γ_2L isoform exists in the proposed cytoplasmic loop region, between M3 and M4, and contains a consensus sequence for phosphorylation by protein kinase C. To examine the regional distribution of this novel receptor subunit in the brain, γ_2L subunit mRNA was detected using both in situ hybridization histochemistry and and PCR amplification methods. Hybridization histochemistry with a γ_2L, subunit-specific oligonucleotide probe revealed that the γ_2L, subunit mRNA is widely distributed throughout the mouse brain. The highest levels of expression are found in the cerebral cortex, hippocampus, olfactory lobe, and cerebellum. The presence of the γ_2L, subunit in these regions was confirmed using PCR. Additionally, PCR experiments detected yes subunit mRNA in the cerebral cortex and hippocampus but not in the cerebellum. To examine the functional properties of the γ₂ subunit isoforms, γ_2S and γ_2L, subunit mRNAs were coexpressed with α₁β₁ subunit mRNAs in Xenopus oocytes. These experiments indicate that the γ_2L and γ_2S subunit variants exhibit similar pharmacological properties, including the ability of both isoforms to confer diazepam sensitivity to the receptor complex. In addition, potentiation of GABA responses by pentobarbital in oocytes expressing either subunit isoform is similar. These data indicate that the presence or absence of the additional eight amino acids in the γ₂ subunit isoforms does not appear to alter the response of the GABA_A receptor complex to either benzodiazepines and barbiturates at the level of protein phosphorylation present in the oocyte.