The differential antagonism by bicuculline and SR95531 of pentobarbitone-induced currents in cultured hippocampal neurons

Abstract

In voltage clamped cultured hippocampal neurons, application of γ-aminobutyric acid (GABA) or pentobarbitone induced chloride current in a dose-dependent manner. The dose dependence of these agonists were well described by ED 50 and Hill coefficients of 14.7 ± 7 μM and 1.2 ± 0.5, and 299 ± 17.3 μM and 1.6 ± 0.1, for GABA and pentobarbitone, respectively. The effects of two GABA A receptor antagonists, bicuculline and 2-(3-carboxypropyl)-3-amino-6-methoxyphenyl-pyridazinium bromide (SR95531) were evaluated by coapplication of increasing concentrations of the antagonists with a fixed equipotent (approximately ED 30) dose of GABA or pentobarbitone. Both bicuculline and SR95531 blocked the GABA induced current with ID 50 and Hill coefficients of 0.74 ± 0.07 μM and 0.96 ± 0.07, and 0.44 ± 0.02 μM and 1.22 ± 0.06, respectively. Bicuculline similarly blocked the pentobarbitone induced current with a ID 50 and Hill coefficient of 0.69 ± 0.04 μM and 1.2 ± 0.1. However, pentobarbitone induced current was poorly blocked by SR95531 retaining 86.5% of current amplitude at a concentration of SR95531, 200 times the IC 50 for GABA induced current. Current induced by etomidate, another intravenous general anesthetic with GABA A receptor agonistic property, is likewise resistant to SR95531 blockade. Three-dimensional modeling of bicuculline and SR95531 with alignment of the molecules along the suggested GABA-receptor binding moiety indicates that these two antagonist molecules have distinct steric properties. We suggest that GABA and pentobarbitone act at nearby but non-identical sites on the hippocampal GABA A receptor to open the chloride ionophore, and that these sites can be distinguished by bicuculline and SR95531. This is the first demonstration that the prototypic GABA A site antagonists bicuculline and SR95531 have different effects on currents induced by GABA and pentobarbitone.