posted on 2005-03-31, 00:00authored byMatthew Jamin Asay
Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the vertebrate brain. GABA activates both ionotropic (GABAA and GABAC) and metabotropic (GABAB) receptors. Very little information about the GABAergic properties of the amphibian brain is available and even less is known about amphibian GABAB receptors. The aim of this project was to systematically characterize the GABAB receptor in the brain of an anuran amphibian, the adult male bullfrog (Rana catesbeiana). Receptor binding assays with the mammalian GABAB receptor antagonist, [3H]CGP54626A, revealed a single, high affinity binding site. Binding was time and temperature dependent, saturable and specific. The affinity (KD) of [3H]CGP54626A was 2.97 nM with a Bmax of 2.62 pmol/mg protein. Specific binding of [3H]CGP54626A was inhibited by several mammalian GABAB receptor specific analogs. The GABAA receptor ligands muscimol and SR95531 had very low affinity for the receptor and the GABAA receptor antagonists bicuculline and bicuclline methiodide did not inhibit binding of [3H]CGP54626A. Binding of GABA was modulated by the mammalian allosteric modulator CGP7930. Taurine did not modulate GABA binding or compete with [3H]CGP54626A. Quantitative in vitro autoradiography with [3H]CGP54626A showed GABAB-like receptors distributed throughout the brain with highest expression levels in the pallium, striatum, septum and habenula. Cloning of GABAB receptor subunits with PCR and RACE yielded cDNA fragments for the GABAB1 and GABAB2 receptor subunits. The subunits are similar to the mammalian forms. The putative GABAB1 subunit represents approximately 93% of the rat gene and is 80% identical at the amino acid level. The putative GABAB2 subunit represents approximately 41% of the rat gene and is 89% identical at the amino acid level. Residues important for ligand binding, G-protein coupling and heterodimerization are conserved in the bullfrog GABAB-like receptor subunits. The pharmacological and molecular data suggest the amphibian brain expresses a functional GABAB-like heterodimeric receptor. The distribution throughout the bullfrog brain suggests the GABAB-like receptor plays a role in sensory and motor integration processes.