KCC2 is the neuron-specific K+-Cl(-) cotransporter required for maintaining low intracellular Cl(-), which is essential for fast inhibitory synaptic transmission in the mature CNS. Despite the requirement of KCC2 for inhibitory synaptic transmission, understanding of the cellular mechanisms that regulate KCC2 expression and function is rudimentary. We examined KCC2 in its native protein complex in vivo to identify key KCC2-interacting partners that regulate KCC2 function. Using blue native-polyacrylamide gel electrophoresis (BN-PAGE), we determined that native KCC2 exists in a macromolecular complex with kainate-type glutamate receptors (KARs). We found that KAR subunits are required for KCC2 oligomerization and surface expression. In accordance with this finding, acute and chronic genetic deletion of KARs decreased KCC2 function and weakened synaptic inhibition in hippocampal neurons. Our results reveal KARs as regulators of KCC2, significantly advancing our growing understanding of the tight interplay between excitation and inhibition.

KCC2 is a neuron-specific K+-Cl– cotransporter essential for establishing the Cl- gradient required for hyperpolarizing inhibition in the central nervous system (CNS). KCC2 is highly localized to excitatory synapses where it regulates spine morphogenesis and AMPA receptor confinement. Aberrant KCC2 function contributes to human neurological disorders including epilepsy and neuropathic pain. Using functional proteomics, we identified the KCC2-interactome in the mouse brain to determine KCC2-protein interactions that regulate KCC2 function. Our analysis revealed that KCC2 interacts with diverse proteins, and its most predominant interactors play important roles in postsynaptic receptor recycling. The most abundant KCC2 interactor is a neuronal endocytic regulatory protein termed PACSIN1 (SYNDAPIN1). We verified the PACSIN1-KCC2 interaction biochemically and demonstrated that shRNA knockdown of PACSIN1 in hippocampal neurons increases KCC2 expression and hyperpolarizes the reversal potential for Cl-. Overall, our global native-KCC2 interactome and subsequent characterization revealed PACSIN1 as a novel and potent negative regulator of KCC2.

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.

Edited by Roger J. ColbranSynaptic 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 ϳ40% 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 (E GABA ). In conclusion, our results have revealed a mechanism by which kainate receptors regulate KCC2 expression in the hippocampus.The classic fast hyperpolarizing inhibition of the mature brain results primarily from the activation of GABA A receptors.These receptors are Cl Ϫ -permeable ion channels, and inhibition results from the influx of Cl Ϫ into the neuron (1). This inward gradient for Cl Ϫ is set by the neuron-specific K ϩ /Cl Ϫ co-transporter KCC2 (2, 3). Despite the requirement of KCC2 for hyperpolarizing inhibition, the mechanisms that regulate KCC2 expression and function are still under intense investigation. In addition to the critical role KCC2 plays in synaptic inhibition, KCC2 is highly localized to excitatory synapses (4, 5), where it plays important roles in the development (6) and the function of glutamatergic synapses (7,8). In fact, single-particle tracking revealed that KCC2 is tightly confined to excitatory synapses (5), which may result from local protein interactions. Thus, understanding how proteins associated with excitatory synapses regulate KCC2 function may provide critical insight to the function of KCC2.KCC2 exists in a multiprotein complex and is regulated by components of excitatory synaptic transmission (9 -11). Specifically, KCC2 interacts with both the kainate-type ionotropic glutamate receptor subunit GluK2 (9) and its auxiliary subunit Neto2 (10). Neto2 regulates KCC2-mediated Cl Ϫ extrusion by binding to the active oligomeric form of KCC2 (10), whereas the GluK2-KCC2 i...

12as a novel and potent negative regulator of KCC2.