Abstract
Neto2 is a transmembrane protein that interacts with the neuron-specific K+-Cl− cotransporter (KCC2) in the central nervous system (CNS). Efficient KCC2 transport is essential for setting the neuronal Cl− gradient, which is required for fast GABAergic inhibition. Neto2 is required to maintain the normal abundance of KCC2 in neurons, and increases KCC2 function by binding to the active oligomeric form of this cotransporter. In the present study, we characterized GABAergic inhibition and KCC2-mediated neuronal chloride homeostasis in pyramidal neurons from adult hippocampal slices. Using gramicidin perforated patch clamp recordings we found that the reversal potential for GABA (EGABA) was significantly depolarized. We also observed that surface levels of KCC2 and phosphorylation of KCC2 serine 940 (Ser940) were reduced in Neto2−/− neurons compared to wild-type controls. To examine GABAergic inhibition we recorded spontaneous inhibitory postsynaptic currents (sIPSCs) and found that Neto2−/− neurons had significant reductions in both their amplitude and frequency. Based on the critical role of Neto2 in regulating GABAergic inhibition we rationalized that Neto2-null mice would be prone to seizure activity. We found that Neto2-null mice demonstrated a decrease in the latency to pentylenetetrazole (PTZ)-induced seizures and an increase in seizure severity.
Highlights
GABAergic inhibition in the central nervous system (CNS) is primarily mediated by postsynaptic GABAA receptors (GABAARs), which are Cl−-permeable ion channels (Kaila, 1994)
KCC2-Mediated Cl−-Homeostasis is Impaired in Neurons from Neto2−/− Adult Hippocampus We previously demonstrated that Neto2-null mice have reduced KCC2-mediated Cl−-extrusion (Ivakine et al, 2013)
Because efficient Cl−-extrusion from neurons is required for hyperpolarizing GABAergic inhibition, we rationalized that hippocampal neurons from adult Neto2-null mice would have reduced inhibition that increased their susceptibility to seizures
Summary
GABAergic inhibition in the central nervous system (CNS) is primarily mediated by postsynaptic GABAA receptors (GABAARs), which are Cl−-permeable ion channels (Kaila, 1994). Dysfunctional inhibition in Neto2-null mice gradient established primarily by the Na+/K+ ATPase to extrude Cl−, thereby maintaining a low [Cl−]i in mature neurons. The low [Cl−]i generates a strong driving force for Cl− influx through GABAARs, which underlies hyperpolarizing inhibition. KCC2 is a cation-chloride cotransporter encoded by the SLC12 gene family (Rivera et al, 1999; Hartmann and Nothwang, 2014; Kaila et al, 2014). Because KCC2 is the only cotransporter that extrudes Cl− under isotonic conditions (Acton et al, 2012), it plays a crucial role in proper neurophysiological function. Chronic pain, spasticity, stress, and epileptic seizures all demonstrate impaired synaptic inhibition due to decreased KCC2 function (Coull et al, 2003; Tornberg et al, 2005; Huberfeld et al, 2007; Hewitt et al, 2009; Boulenguez et al, 2010; Puskarjov et al, 2012; Gagnon et al, 2013)
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