Abstracts

Rationale: Status epilepticus (STEP) is a progressive and often lethal disorder characterized by continuous or rapidly repeating seizures. During STEP the functions of many neuronal processes are altered but of major significance is decreased synaptic inhibition, mediated by GABA. Fundamental to this loss are reduced potency and efficacy of GABA together with altered transmembrane chloride gradients. In addition, deficits in the function and pharmacology of GABA_ARs are also evident, including reductions in their functional modulation by benzodiazepines. These phenomena are believed to result from decreased functional expression of GABA_A receptors but the underlying mechanisms remain obscure. GABA_ARs are chloride selective ligand gated ion channels that can be assembled from 7 subunit classes; α(1-6), β(1-3), γ(1-3), δ, ε, θ and π, but molecular and cellular studies have illustrated that the majority of benzodiazepine-sensitive GABA_ARs are composed of α, β and γ2 subunits. In contrast, receptors that incorporate α4, α5 and/or δ subunits assemble into a specialized population of high affinity extrasynaptic receptors that mediate tonic inhibtion. Methods: We used hippocampal slices obtained from FVB/N mice induced STEP by pilocarpine for 1 hr. We utilized hippocampal slice biotinylation methods to measure the cell surface GABA_A receptor. Results: In CA1 hippocampal pyramidal cells from treated animals, there was an approximate 40% reduction in the frequency and amplitude of mIPSCs. Using biotinylation it was also evident that, in STEP, the cell surface number of GABA_A receptors, α 1, α2, α4, β3 and γ2 were significantly decreased by 30%, however, α5, δ was increased. In parallel with this decreased cell surface stability in most of the subunits, large deficits in the phosphorylation of serines 408/9 in the β3 subunit were also evident. It has recently been established that phosphorylation of S408/9 in β3 reduces the rate of GABA_A receptor endocytosis by decreasing the binding to the clathrin-adaptor protein 2 (AP-2), a critical regulator of clathrin-mediated endocytosis. In STEP increased association of GABA_A receptors containing subunits with the AP-2 was evident as measured via immunoprecipitation. Moreover, we further illustrate that specifically blocking GABA_A receptor endocytosis or enhancing their levels of phosphorylation, increases the number of cell surface receptors during STEP, together with the amplitude and decay times of miniature inhibitory postsynaptic currents. Conclusions: Our studies have demonstrated that STEP decreases GABA_A receptor phosphorylation, enhancing their interaction with AP-2 promoting their internalization from plasma membrane and thereby increasing the strength of inhibitory synaptic transmission. Therefore inhibiting GABA_A receptor endocytosis or enhancing their phosphorylation may provide novel therapeutic strategies to ameliorate STEP.