The neuronal K-Cl cotransporter KCC2 influences postsynaptic AMPA receptor content and lateral diffusion in dendritic spines

Abstract

The K-Cl cotransporter KCC2 plays an essential role in neuronal chloride homeostasis, and thereby influences the efficacy and polarity of GABA signaling. Although KCC2 is expressed throughout the somatodendritic membrane, it is remarkably enriched in dendritic spines, which host most glutamatergic synapses in cortical neurons. KCC2 has been shown to influence spine morphogenesis and functional maturation in developing neurons, but its function in mature dendritic spines remains unknown. Here, we report that suppressing KCC2 expression decreases the efficacy of excitatory synapses in mature hippocampal neurons. This effect correlates with a reduced postsynaptic aggregation of GluR1-containing AMPA receptors and is mimicked by a dominant negative mutant of KCC2 interaction with cytoskeleton but not by pharmacological suppression of KCC2 function. Single-particle tracking experiments reveal that suppressing KCC2 increases lateral diffusion of the mobile fraction of AMPA receptor subunit GluR1 in spines but not in adjacent dendritic shafts. Increased diffusion was also observed for transmembrane but not membrane-anchored recombinant neuronal cell adhesion molecules. We suggest that KCC2, likely through interactions with the actin cytoskeleton, hinders transmembrane protein diffusion, and thereby contributes to their confinement within dendritic spines.T he neuronal K-Cl cotransporter KCC2 transports chloride using the electrochemical gradient of K + ions (1). In mature neurons, this action maintains a low intraneuronal chloride concentration that ensures a hyperpolarizing effect of GABA at chloride-permeable GABA A receptors. KCC2 expression, activity, and membrane traffic are tightly regulated by neuronal activity, particularly through the phosphorylation of its carboxylterminal domain (CTD) (2-4). Activation of postsynaptic glutamate receptors, for instance, reduces KCC2 activity through dephosphorylation and endocytosis within minutes (3, 5). KCC2 expression is also suppressed in pathological conditions associated with enhanced neuronal activity (6), leading to a rise in intraneuronal chloride and an alteration of GABA function (7-9). KCC2 therefore appears to mediate a functional cross-talk between synaptic excitation and inhibition in neurons.Although KCC2 function primarily influences the efficacy of GABAergic signaling, its presence in dendritic spines (10) raises the question of its role in spine morphogenesis and function. Genetic ablation of KCC2 in mice compromises spine maturation and excitatory synapse formation in immature hippocampal neurons (11). This effect appears to be independent of KCC2 function but, instead, involves KCC2 interaction with the neuronal FERM-domain protein 4.1N (12). However, KCC2 expression is up-regulated during postnatal development and is maximal in mature neurons (13), after spine formation, where its role in the maintenance and function of dendritic spines remains unknown. Here, we show that suppression of KCC2 after spine morphogenesis reduces postsynaptic glutamate recept...

Neto2 is a KCC2 interacting protein required for neuronal Cl − regulation in hippocampal neurons

“…However, these roles may be independent of KCC2's ability to extrude Cl − (31,32). In the present study, we found Neto2-deficiency did not affect basic neuronal morphology (Fig.…”

“…However, KCC2 also plays an additional role in regulating neuronal morphology (5,31,32). We tested the possibility that disrupting the Neto2-KCC2 interaction also affects neuronal morphology by performing a Scholl analysis, and found no significant difference between the number of dendritic intersections between WT and Neto2 −/− neurons ( Fig.…”

“…The ability of KCC2 to interact with the cytoskeleton-associated protein 4.1N (31,32), and the requirement for KCC2 in the morphological maturation of cortical neurons (39), indicates that KCC2 plays important roles in neuronal morphology. However, these roles may be independent of KCC2's ability to extrude Cl − (31,32).…”

Regulation of neuronal chloride homeostasis by neuromodulators

“…2015), and in mature excitatory synapses KCC2 restricts AMPAR (AMPA‐type glutamate receptor) lateral diffusion in dendritic spines (Gauvain et al . 2011). Not only is KCC2 highly enriched at excitatory synapses, but quantum dot imaging has revealed that KCC2 dwell time is 1.3‐fold longer at excitatory vs .…”

Glycine is able to induce both a motility speed in- and decrease during zebrafish neuronal migration

“…It is thought that regular depolarization via glycine and NKCC1 is necessary to maintain the proliferation of neuronal precursors [11–13]. The hyperpolarizing effect of glycine in mature neurons in contrast [14,15] depends on the co-expression of KCC2 ( slc12a5b ) [15,16]. KCC2 lowers the intracellular Cl − concentration, which causes an influx of Cl − opening of the glycine receptor, and membrane hyperpolarization.…”