A kainate receptor subunit promotes the recycling of the neuron-specific K+-Cl− co-transporter KCC2 in hippocampal neurons

Abstract

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...

Exciting Times: New Advances Towards Understanding the Regulation and Roles of Kainate Receptors

“…KCC2 is crucial because it establishes the electrochemical chloride gradient for postsynaptic inhibition through GABA A Rs [121,122] and its dysregulation is implicated in autism spectrum disorder (ASD; [123]) and epilepsy [124]. KCC2 interacts with both Neto2 [125] and GluK2 [126], which increase the total abundance and enhance surface expression of KCC2 [14]. KCC2 and GluK2 form macromolecular assemblies that traffic together, and this complex regulates intraneuronal chloride homeostasis to support GABA A R-mediated transmission [126].…”

“…In contrast, postsynaptic metabotropic KAR signalling is much more widespread than ionotropic KAR signalling [12,13] and recent discoveries have highlighted their previously unsuspected roles in neuromodulation. For example, they regulate inhibitory transmission by controlling surface expression of the chloride transporter KCC2 [14] and mediate certain forms of synaptic plasticity [15,16]. For extensive reviews of metabotropic KARs see [4,17].…”

Ionotropic and metabotropic kainate receptor signalling regulates Cl− homeostasis and GABAergic inhibition

“…, Pressey et al . ). However, whether KAR‐mediated ionotropic current and/or metabotropic signalling can also regulate KCC2 function is unknown.…”

“…KCC2 interacts with the KAR subunit GluK2 and this physical interaction is required to maintain normal surface expression and oligomerization of the KCC2 protein (Mahadevan et al 2014, Pressey et al 2017. However, whether KAR-mediated ionotropic current and/or metabotropic signalling can also regulate KCC2 function is unknown.…”

Native KCC2 interactome reveals PACSIN1 as a critical regulator of synaptic inhibition

“…293identified KCC2 interactors, including: Neto2(Ivakine et al, 2013;Mahadevan et al, 2015), GluK2 294Mahadevan et al, 2014;Pressey et al, 2017), 4.1N(Li et al, 2007), beta-pix(Chevy et al, 2015; Llano et 295 al., 2015), RCC1(Garbarini and Delpire, 2008), or signaling molecules PKC(Lee et al, 2007), WNK, SPAK 296…”