GABA released from cultured cortical neurons influences the modulation of t-[35S]butylbicyclophosphorothionate binding at the GABAA receptor: Effects of thymol

Abstract

Thymol is a monoterpene that specifically interacts with synaptic neural functions in neuronal GABA-operated Cl- channels. Here we explore the effects of thymol, and propofol as positive control, on t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding in primary cultures of cortical neurons. The study includes a meaningful analysis of the effect of various exposure buffers, and their correlation with GABA released from cells, chloride influx through the GABAA receptor and GABA transporter activity. Cell viability was also determined. Thymol and propofol inhibited the binding of [35S]TBPS to cells exposed to Tris-citrate-NaCl buffer whereas a biphasic effect was observed in HEPES solution. The different effects of the two buffers analysed are due to the higher capacity of Tris-citrate-NaCl buffer to induce the release of endogenous GABA facilitating the binding of [35S]TBPS to its recognition site at the GABAA receptor. Released GABA in the presence of this buffer was inhibited by the neuronal GABA transporter inhibitor SKF 100330-A. Tris-citrate-NaCl buffer also induced a chloride influx, which was reverted by picrotoxinin. TBPS binding in living cells is facilitated by GABA released from the cells, which in turn activates basal GABAA receptor activity. The results deepen on the allosteric mechanism of thymol as positive modulator of the GABAA receptor. Furthermore, we corroborate [35S]TBPS binding as an important test to verify the capacity of drugs to act on and recognize a site at the GABAA receptor.