Epilepticus (SE) is a major neurological emergency affecting over 150,000 people/year in the US. Despite advances in treatment strategies, SE is still associated with a significant level of mortality. It has been well established that a significant number of SE patients respond poorly to treatment. These resistant patients have a much higher mortality rate than patients that respond rapidly to treatment. Therefore, understanding the cellular and molecular mechanisms that result in decreased responsiveness to therapy is essential to develop intervention strategies to better treat this life-threatening condition. SE has been shown to alter GABAA receptor (GABAAR) function in a duration-dependent manner. This acute and progressive alteration of GABAAR function has been attributed to a post-translational modification of the receptor. A significant inhibition of Calcium/calmodulin-dependent protein kinase II (CaM kinase II) activity has been observed in multiple animal and in vitro models of SE. In addition, CaM kinase II has been shown to augment GABAAR function and SE-induced inhibition of CaM kinase II results in decreased GABAAR subunit phosphorylation. This observation has led us to develop the CENTRAL HYPOTHESIS: GABAA receptor function progressively decreases as SE duration increases due in part to SE-induced inhibition of CaM kinase II dependent positive modulation of GABAAR activity. The experimental aims will utilize two well-characterized models of SE: The rat pilocarpine model and the hippocampal neuronal culture model. Both models of SE show alteration of GABAAR function in a duration-dependent manner. Therefore, these two models will be utilized to elucidate the cellular and molecular mechanisms which result in SE duration-dependent decrease in GABAAR function, and accomplish the following specific aims: 1) Characterize the effect of SE duration on agonist and allosteric modulator binding and GABAAR function. 2) Determine the cellular mechanisms of SE-induced inhibition of GABAAR function. 3) Characterize the CaM kinase II-dependent modulation of GABAAR function. 4) Characterize the molecular mechanisms of CaM kinase Il-dependent phosphorylation on GABAAR function. 5) Determine the effect of direct inhibition of CaM kinase II activity on neuronal physiology and GABAAR function in the absence of SE. Accomplishment of these specific aims will significantly advance our understanding of drug resistance in SE. Since GABAergic intervention is the front line therapy for SE patients, understanding the cellular and molecular mechanisms that result in modulation of GABAAR function in SE is essential for advancing the clinical management of this neurological emergency. The significance of this research proposal is that it describes a molecular mechanism whereby prolonged seizure activity modulates the function of a major neurotransmitter receptor complex. Understanding the cellular mechanisms that result in altered GABAAR function in SE is essential to the development of new treatment strategies to manage SE in the clinical setting.
