Abstract

We have recently identified a novel class of allosteric modulators of GABA_A receptors, the ROD compounds that are structurally related to bicuculline. Here, the relationship of their site of action relative to other known modulatory sites of this receptor was investigated. Two types of ROD compounds, R1 (ROD164A, ROD185) and R2 (ROD222 and ROD259) could be differentiated. R1 compounds competitively inhibited binding of benzodiazepines in α1β2γ2 receptors, and their functional effects were partially inhibited by the benzodiazepine antagonist Ro15-1788 in a noncompetitive manner. The enhancement by an R1 compound was not additive with that by diazepam. R2 compounds in contrast failed to inhibit binding of benzodiazepines; the R2 compounds' functional effects were not inhibited by the benzodiazepine antagonist. The enhancement by an R2 compound was additive with that by diazepam. In contrast to benzodiazepines, both R1 and R2 type compounds were still able to enhance α1β2 receptors. ROD164A in α1β2γ2 receptors was found to be partially antagonized by Ro15-1788 in a noncompetitive way. ROD178B did not affect γ-aminobutyric acid induced currents, but was able to inhibit both enhancement by R1 and R2 type compounds as well as enhancement by diazepam. R1 and R2 type compounds as well as diazepam enhanced pentobarbital-induced currents in a Ro15-1788-sensitive way. We conclude that R1 type compounds act at the benzodiazepine binding site and additionally at a different R1 site, and that the R1, but not the R2 site is allosterically coupled to the benzodiazepine binding site. ROD178B is a competitive antagonist at the R1 site in that it shows allosteric interaction with the benzodiazepine binding site and displacement of benzodiazepines, and a negative allosteric modulator at the R2 site.