Uncoupling of benzodiazepine and GABA binding sites induced by GABA

MARIA LAURA GUTIERREZ; MARIA CLARA GRAVIELLE

Huerta Grande, Cordoba. Argentina

Congreso; First joint meeting of Argentine Society of Neurocience and de Argentine Workshop in Neurocience (SAN-TALLER); 2009

In a previous work we have demonstrated that exposure of rat neocortical cultures to GABA for 5-10 min (t1/2= 3.2 min) initiates a process that results in a reduced interaction between GABA and benzodiazepine binding sites (uncoupling) hours later (t1/2= 12 h), without changes in GABAA receptor number. Uncoupling was accompanied by a decrease in á1, á3, and â1-3 subunit mRNA levels with no change in á2, á4, á5, ã1 and ã2 mRNAs. These alterations in mRNA levels were associated with corresponding changes in subunit protein expression. The decrease in á1 subunit level did not produce, however, a reduction in the proportion of receptors containing á1 as measured by zolpidem binding assays. These results can be explained by the fact that á1 exists in excess and only 20 % of the total subunit becomes part of assembled receptors. The strength of allosteric coupling between GABA and benzodiazepine binding sites depends on the á subunit subtype present in the GABAA receptor (á3 > á1/2). To determine whether uncoupling produces a reduction in the percentage of receptor composed of á3 subunits, inmunoprecipitation experiments followed by western blot assays were performed. Neocortical cultures were incubated with GABA for 10 min, washed and cells were collected 48 h later. Receptor immunoprecipitation assays were carried out using an antibody against ã2 subunits that are present in most of the receptors. Results from western blot experiments using an antibody anti- á3 showed a decrease in the percentage of receptors containing á3. These results suggest that GABA-induced uncoupling involves a change in the subunit composition, resulting in receptors with a reduced allosteric coupling between GABA and benzodiazepine binding sites.