Abstracts

Rationale: Fast synaptic inhibition in the adult brain is primarily mediated by the type A γ-aminobutyric acid receptors (GABAARs), which permit Cl– influx resulting in membrane hyperpolarization and the restriction of neuronal firing. Neuronal Cl– homeostasis is maintained by chloride transporters. In most adult brain neurons, the K+/Cl- co-transporter 2 (KCC2), couples Cl– efflux to the outward K+ gradient to keep cytoplasmic Cl– low, promoting hyperpolarizing GABAAR responses. KCC2 surface activity is strongly controlled by phosphorylation of KCC2 residue Ser940 by PKC and PP1. KCC2 is reduced in many CNS disease states including epilepsy, autism, and chronic stress. In this study we examined the effects of reduced KCC2 on the seizure event.Methods: We performed experiments using the KCC2 specific inhibitor VU0463271 on cultured hippocampal neurons and in acute mouse slice models of epileptiform activity (4-aminopyridine and low-magnesium).Results: We performed experiments using the KCC2 specific inhibitor VU0463271 on cultured hippocampal neurons and found that reduced transporter function resulted in neuronal Cl– loading and positive shifts in EGABA. Next, we used acute slice models of epileptiform activity (low-Mg2+ and 4-aminopyridine), and found that reduced KCC2 function by the specific inhibitor VU0463271 prolonged the duration of seizure-like events (SLEs) in the entorhinal cortex in both models. We then treated slices obtained from the point-mutant KCC2 Ser940Ala mouse, which impairs KCC2 function, and found that the mutation results in increased severity of induced SLEs and epileptiform activity in the entorhinal cortexConclusions: Our findings indicate that KCC2 function and phosphorylation of Ser940 are critical determinants in tuning the efficacy of GABAergic inhibition and SLE severity in the entorhinal cortex and validate KCC2 as a therapeutic target for seizure prevention.