A neolignan from Connarus tuberosus as an allosteric GABAA receptor modulator at the neurosteroid binding site

In a screening of a small library of extracts from plants of the Amazonian and Cerrado biomes, a hexane extract of Connarus tuberosus roots was found to significantly potentiate the GABA induced fluorescence in a fluorescence (FLIPR) assay in CHO cells stably expressing the alpha 1 beta 2 gamma 2 subtype of human GABAA receptors. With the aid of HPLC-based activity profiling the activity was linked to the neolignan connarin. In CHO cells the activity of connarin was not abolished by increasing concentrations of flumazenil, while the effect of diazepam was increased by increasing concentrations of connarin. The effect of connarin was abolished by pregnenolone sulfate (PREGS) in a concentration-dependent manner, and the effect of allopregnanolone was further increased by increasing concentrations of connarin. In a two-microelectrode voltage clamp assay with Xenopus laevis oocytes transiently expressing GABAA receptors composed of human alpha 1 beta 2 gamma 2S and alpha 1 beta 2 subunits connarin potentiated the GABA-induced currents, with EC50 values of 1.2 +/- 0.3 mu M (alpha 1 beta 2 gamma 2S) and 1.3 +/- 0.4 mu M (alpha 1 beta 2), and with a maximum enhancement of currents Emaxof 1959 +/- 70% (alpha 1 beta 2 gamma 2S) and 185 +/- 48% (alpha 1 beta 2). The activation induced by connarin was abolished by increasing concentrations of PREGS.