EFFECTS OF ETHANOL ON RECOMBINANT RAT GABA(A) RECEPTORS - [S-35] T-BUTYLBICYCLOPHOSPHOROTHIONATE ([S-35]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 GABA(A) receptor function, picrotoxin-sensitive [S-35]t-butylbicyclophosphorothionate ([S-35]TBPS) binding was studied in recombinant rat alpha 1 beta 2y2 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 [S-35]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 a 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.