HOLLIS, D.M.; BOYD, S.K.: Neurosteroid Modulation and Subunit Isolation of a GABA-A-like Receptor in the Amphibian Brain.

The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) has multiple receptor subtypes. The GABA_A receptor is widespread in the mammalian brain, but little is known about GABA receptors in the amphibian brain. Binding of the GABA_A receptor agonist, [³H]muscimol, to membrane preparations isolated from the brain of the adult male bullfrog, Rana catesbeiana, indicate a GABA_A receptor-like protein in the amphibian brain. To further characterize this receptor, we investigated acute neurosteroid modulation of [³H]muscimol binding. In particular, allopregnanolone (3alpha-hydroxy-5alpha-pregnan-20-one) had a positive modulatory effect on the specific binding of [³H]muscimol in concentrations as low as 10 nM and increased specific binding by up to 60% at 10 uM. This modulation was found to be time-dependent, with increased specific binding corresponding to increased radioligand preincubation times. In addition to its modulation, we investigated the putative subunit composition of the bullfrog GABA_A-like receptor. We isolated three cDNA fragments, which corresponded to the mammalian GABA_A receptor alpha-1, beta-1, and gamma-2 subunits. GABA_A receptors in the mammalian brain composed of these subunits are effectively modulated by allopregnanolone. Northern analysis showed these subunits were 9.0 kb or greater. These data further support the hypothesis that the bullfrog brain contains a GABA_A receptor-like protein and that neurosteroid modulation of this receptor may represent a significant mechanism for steroid influence on the amphibian brain. Supported by NSF# IBN99-83020.