Abstract
Synaptic inhibition depends on a transmembrane gradient of chloride, which is set by the neuron-specific K+-Cl- co-transporter KCC2. Reduced KCC2 levels in the neuronal membrane contribute to the generation of epilepsy, neuropathic pain, and autism spectrum disorders; thus, it is important to characterize the mechanisms regulating KCC2 expression. In the present study, we determined the role of KCC2-protein interactions in regulating total and surface membrane KCC2 expression. Using quantitative immunofluorescence in cultured mouse hippocampal neurons, we discovered that the kainate receptor subunit GluK2 and the auxiliary subunit Neto2 significantly increase the total KCC2 abundance in neurons but that GluK2 exclusively increases the abundance of KCC2 in the surface membrane. Using a live cell imaging assay, we further determined that KCC2 recycling primarily occurs within 1-2 h and that GluK2 produces an ∼40% increase in the amount of KCC2 recycled to the membrane during this time period. This GluK2-mediated increase in surface recycling translated to a significant increase in KCC2 expression in the surface membrane. Moreover, we found that KCC2 recycling is enhanced by protein kinase C-mediated phosphorylation of the GluK2 C-terminal residues Ser-846 and Ser-868. Lastly, using gramicidin-perforated patch clamp recordings, we found that the GluK2-mediated increase in KCC2 recycling to the surface membrane translates to a hyperpolarization of the reversal potential for GABA (EGABA). In conclusion, our results have revealed a mechanism by which kainate receptors regulate KCC2 expression in the hippocampus.
Highlights
The classic fast hyperpolarizing inhibition of the mature brain results primarily from the activation of GABAA receptors
In this study we determined that both Neto2 and GluK2 increase total KCC2 abundance in hippocampal neurons, GluK2 itself plays a unique role in promoting KCC2 recycling and surface abundance
Through the use of phosphodeficient GluK2, we revealed that PKC-mediated phosphorylation of GluK2 increases KCC2 recycling and surface expression
Summary
To determine whether Neto and/or GluK2 can regulate total and surface KCC2 abundance, we used heterologous cells. To express this phospho-deficient mutant in the absence of endogenous GluK2 and KCC2, we performed these experiments in COS-7 cells This system allowed us to test the sufficiency of GluK2 to regulate KCC2 surface expression. If PKC activation increases KCC2 recycling and surface abundance via phosphorylation of GluK2, we should see a significantly higher KCC2 transport activity (determined as a hyperpolarization of EGABA) in wild type neurons compared with GluK1/2Ϫ/Ϫ neurons. Based on the results from our recycling assay, which indicated that the phosphorylation of GluK2-Ser-846/868 promotes KCC2 recycling, we predicted that in the absence of GluK2-Ser-846/868 phosphorylation, PMA would not hyperpolarize EGABA To test this prediction, we expressed the phospho-deficient GluK2-S846A/S868A in GluK1/2Ϫ/Ϫ neurons and repeated the above electrophysiology experiments. The PKC-mediated phosphorylation of GluK2, which in turn promotes KCC2 recycling and surface abundance, increases the capacity for ClϪ extrusion, which in turn hyperpolarizes EGABA
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