Subunit-containing GABA Research Articles

Picrodendrin and related terpenoid antagonists reveal structural differences between ionotropic GABA receptors of mammals and insects

Twenty-eight picrotoxane terpenoids, including picrodendrins isolated from the Euphorbiaceae plant, Picrodendron baccatum (L.) Krug and Urban, have been evaluated for their ability to inhibit the specific binding of [3H]EBOB, the noncompetitive antagonist of ionotropic GABA receptors, to rat-brain and housefly (Musca domestica L.)-head membranes. Picrodendrin Q was the most potent competitive inhibitor of [3H]EBOB binding, with IC50 values of 16nM (rat) and 22nM (Musca). We find that the spiro γ-butyrolactone moiety at the 13-position, which contains a carbonyl group conjugated with an unsaturated bond, and the substituents at the 4-position play important roles in the interaction of picrodendrins with their binding site in rat receptors. In contrast, such structural features are not strictly required in the case of the interaction with Musca receptors; the spiro saturated γ-butyrolactone moiety at the 13-position, which bears the 16-sp3 carbon atom, and the hydroxyl groups at various positions are somewhat tolerated. Quantitative structure-activity studies have clearly shown that the electronegativity of the 16-carbon atom and the presence or absence of the 4- and 8-hydroxyl groups are important determinants of the potency of nor-diterpenes in Musca receptors, while the negative charge on the 17-carbonyl oxygen atom is likely important in the case of rat receptors. These findings indicate that there are significant differences between the structures of the complementary binding sites in rat GABA receptors and Musca GABA receptors. We also infer differences between native Musca GABA receptors and the Drosophila Rdl subunit-containing homo-oligomeric GABA receptors in the structures of their binding sites.

Diazepam protects against rat hippocampal neuronal cell death induced by antisense oligodeoxynucleotide to GABA A receptor γ2 subunit

Antisense oligodeoxynucleotides (ODNs) are used for the selective inhibition of gene expression. Antisense ODNs are promising tools for the investigation of physiological implications of proteins in the central nervous system of rodents in vivo. We have previously demonstrated that a phosphorothioate antisense ODN to the GABA A receptor γ2 subunit, but not sense or mismatch control ODNs, induces a decrease in ex vivo benzodiazepine receptor radioligand binding in rat hippocampus when infused into the hippocampus in vivo [Karle et al., Neurosci. Lett., 202 (1995) 97–100]. This effect is parallelled by a decrease in the number of GABA A receptors and an extensive loss of hippocampal neurones. There is increasing awareness of risks of toxic `non-antisense' effects induced by ODNs, and in particular phosphorothioate ODNs. The present experiments were designed to investigate the specificity of effects induced by the γ2 subunit antisense ODN. The temporal development of changes in [ 3H]flunitrazepam and [ 3H]quinuclidinyl benzilate binding as well as in tissue protein levels supports the notion that the antisense ODN primarily acts by blocking the expression of the targeted receptor subunit protein. Furthermore, it is shown that a threshold for the elicitation of neurodegenerative changes exists. Finally, it is demonstrated that diazepam treatment of rats protects against the development of neuronal cell death induced by the antisense ODN. Collectively, the results support the hypothesis that the neurodegeneration induced by the antisense ODN is a consequence of diminished GABAergic inhibitory tonus following a selective down-regulation of γ2 subunit-containing GABA A receptor complexes.