Studies on picrotoxin binding sites of GABA A receptors in rat cortical synaptoneurosomes

Abstract

Experiments were performed to characterize [ 35S]TBPS binding in rat cortical Synaptoneurosomes, which have vesicular structures containing both pre- and postsynaptic elements. Scatchard analysis revealed a single component of [ 35S]TBPS binding sites with K D and B max values of 76.1 nM and 1.97 pmoles/mg protein, respectively, under physiological conditions. GABA and muscimol inhibited [ 35S]TBPS binding in a concentration-dependent manner. IC 50 values of these GABA A agonists in displacing synaptoneurosomal [ 35S]TBPS binding are comparable to previously reported EC 50 values of the agonist-stimulated 36C1 − uptake in Synaptoneurosomes by these agents. Furthermore, the IC 50 values of these GABA A agonists were better correspondence to those determined by [ 3H]muscimol binding in synaptoneurosomal preparations as reported by Delorey and Brown (3) than those determined in membrane preparations. Although bicuculline increased [ 35S]TBPS binding in a concentration dependent manner in cortical membranes, it did not affect synaptoneurosomal [ 35S]TBPS binding. Benzodiazepine agonists and inverse agonists (0.1 to 10 μM) did not show any effects on the binding in the absence of muscimol. However, benzodiazepine agonists potentiated and inverse agonists antagonized muscimol-induced inhibition of synaptoneurosomal [ 35S]TBPS binding. In addition, an anesthetic steroid, THDOC, and pentobarbital inhibited synaptoneurosomal [ 35S]TBPS binding in a concentration dependent manner. These results suggest that allosteric modulation of [ 35S]TBPS binding by various ligands which interact with GABA A supramolecular complexes remain intact in Synaptoneurosomes. It appears that this preparation is useful for investigating correlation between functional 36Cl − uptake and individual binding studies of each of the GABA A receptor complex.