Effects of ethanol on recombinant rat GABAA receptors: [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding study

To determine the roles of the alternatively spliced short and long forms of the gamma 2 subunit in the effect of ethanol on the GABAA receptor function, picrotoxin-sensitive [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding was studied in recombinant rat alpha 1 beta 2 gamma 2 and alpha 6 beta 2 gamma 2 receptors expressed in human embryonic kidney 293 cells. Ethanol (10-500 mM) in the absence of added GABA had only minor effects on [35S]TBPS binding irrespective of the gamma 2 splice variant, its effects being greater in alpha 6 beta 2 gamma 2 than in alpha 1 beta 2 gamma 2 receptors. Ethanol (100 mM) decreased the binding in all four subunit combinations at various concentrations of GABA, again an effect independent of the gamma 2 variant. The two gamma 2 variants had different effects on GABA modulation of the binding, with the long gamma 2 variant decreasing the efficiency of GABA inhibition in alpha 6 beta 2 gamma 2 receptors and enhancing the biphasic GABA stimulation and inhibition in alpha 1 beta 2 gamma 2 receptors. The findings confirm the importance of the alpha subunits in the allosteric interactions between the convulsant binding site and other effector sites, which can be modified only to a minor extent by the type of the gamma 2 splice variant.