Abstracts

Rationale: The activity-dependent, subunit-dependent trafficking of GABA_A receptors (GABARs) is one potential mechanism to explain both the reduction in GABA-mediated inhibition and the development of benzodiazepine pharmacoresistance that occur during the prolonged seizures of status epilepticus (SE). The internalization of these receptors occurs via a clathrin-mediated process following the binding of the clathrin adaptor AP2. The intracellular domains of the GABAR β and γ subunits contain putative patch-binding motifs for AP2 that are accessible upon the dephosphorylation of these subunits. Prior studies have demonstrated that a decrease in the phosphorylation of the β3 subunit occurs during SE. Whether the decrease in phosphorylation of this subunit occurs as the result of a site-selective process or whether a decrease in the phosphorylation of both sites is required for GABAR endocytosis during SE is not known. The objective of this study was to evaluate phosphorylation state of these AP2 binding sites during SE. Methods: SE was induced in postnatal (P) 23-25 SD rats (SE-treated) either by a combination of lithium and pilocarpine (LiPilo) or kainic acid (KA). Surface expression of the γ2 subunit of the GABAR was determined in whole hippocampal slices from SE-treated and naïve control animals using a biotinylation pull-down assay. Phosphorylation of the γ2 and β3 subunits of the GABAR was determined by standard Western blotting techniques. Results: GABAR surface expression: We confirmed our prior observation of SE model-dependent changes in the surface expression of GABARs during SE: the surface expression of the γ2 subunit was 59 ± 7% compared to controls (n=5, p<0.05) after 1 hr of LiPilo-induced SE and unchanged (83 ± 18%) compared to controls after 3 hours of KA-induced SE. The surface expression of the γ2 subunit was also unchanged (116 ± 33%) compared to controls in animals in which LiPilo-induced SE was prevented by the administration of 10 mg/kg diazepam prior to the injection of pilocarpine demonstrating that the reduced surface expression of the γ2 subunit in this model is not a direct pharmacological effect of these agents.