Abstracts

Rationale: Deficits in GABAergic inhibition result in abnormal neuronal activation and synchronization that underlies seizures. Hyperpolarizing inhibition mediated by GABAA receptors requires the generation and maintenance of an inwardly directed Cl– gradient, a process that depends upon Cl– extrusion by the neuron-specific type 2 K+/Cl– co-transporter (KCC2). KCC2 activity causes a negative shift in the reversal potential of GABAA receptor currents (EGABA). Recently, an abundance of evidence linking KCC2 dysfunction to idiopathic and acquired epilepsy has been reported. Additionally, genetic mutations of KCC2 are now known to cause infantile epilepsy in humans, confirming the necessity of KCC2 for preventing seizures. We therefore hypothesized that increasing KCC2 function will reduce seizure activity. Due to a lack of pharmacological direct activators of KCC2, this hypothesis has yet to be addressed, and so we designed an alternative genetic approach to solve this problem. It is now evident that KCC2 activity is highly dependent upon its overall phosphorylation state, and phosphorylation of residues threonine 906 and threonine 1007 (T906/T1007) inhibit KCC2 function. Mutation of these threonine sites to alanine to prevent their phosphorylation (KCC2-T906A/T1007A) increases KCC2 function in vitro. We therefore generated a KCC2-T906A/T1007A knock-in mouse to increase KCC2 function in the brain. We have used this mouse model as a tool to investigate the impact of enhanced KCC2 function on seizure severity.  Methods: Electrophysiology: The gramicidin perforated patch clamp technique was used to measure EGABA in WT and KCC2-T906A/T1007A neurons to assess basal KCC2 function. A whole-cell patch clamp assay was used to measure  rate of Cl– extrusion under Cl– loading conditions. Epileptiform activity in vitro was assessed by recording the local field potential in the entorhinal cortex from slices exposed to various conditions that induce seizure-like activity: 0-Mg2+, 4-AP, or 4-AP/low Mg2+/low Ca2+.EEG: EEG recordings were used to monitor seizure activity induced by systemic kainate in WT and KCC2-T906A/T1007A mice. Several parameters were measured, including the onset of seizure activity and status epilepticus, as well as seizure power.  Results: KCC2-T906A/T1007A mutant neurons have more negative EGABA values, demonstrating basal KCC2 function is increased compared to WT. In three different in vitro seizure models, time spent in epileptiform activity is reduced in the KCC2-T906A/T1007A slices. Additionally, 40% of KCC2-T906A/T1007A slices exposed to 4-AP/ low Mg2+/low Ca2+ exhibited no epileptiform activity at all, compared to only 5% of WT slices. In vivo, KCC2- T906A/T1007A mice exhibited delayed onset to epileptiform activity and status epilepticus after kainate administration. The power of gamma oscillations (30-100Hz) during the first hour after kainate injection was substantially reduced in KCC2-T906A/T1007A mice compared to WT mice, indicating less severe seizure activity in the mutants.KCC2-T906A/T1007A neurons extruded Cl– at a more rapid rate than WT KCC2 under Cl– loading conditions, suggesting these mutant neurons should be able to deal more effectively with Cl– loads that occur during seizures. This is likely to be the underlying mechanism responsible for the reduced seizure burden and seizure power observed in the mutant mice.  Conclusions: We have developed a KCC2 gain-of-function knock-in mouse model, which has allowed us to assess the impact of increasing KCC2 function on seizure activity. This work provides the first direct evidence that increasing KCC2 function could be a valid therapeutic strategy to alleviate the burden of epilepsy. Funding: N/A