To elucidate mechanisms that control and execute activity-dependent synaptic plasticity, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPA-Rs) with an electrophysiological tag were expressed in rat hippocampal neurons. Long-term potentiation (LTP) or increased activity of the calcium/calmodulin-dependent protein kinase II (CaMKII) induced delivery of tagged AMPA-Rs into synapses. This effect was not diminished by mutating the CaMKII phosphorylation site on the GluR1 AMPA-R subunit, but was blocked by mutating a predicted PDZ domain interaction site. These results show that LTP and CaMKII activity drive AMPA-Rs to synapses by a mechanism that requires the association between GluR1 and a PDZ domain protein.
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...
The neuronal K/Cl transporter KCC2 exports chloride ions and thereby influences the efficacy and polarity of GABA signaling in the brain. KCC2 is also critical for dendritic spine morphogenesis and the maintenance of glutamatergic transmission in cortical neurons. Because KCC2 plays a pivotal role in the function of central synapses, it is of particular importance to understand the cellular and molecular mechanisms underlying its regulation. Here, we studied the impact of membrane diffusion and clustering on KCC2 function. KCC2 forms clusters in the vicinity of both excitatory and inhibitory synapses. Using quantum-dot-based single-particle tracking on rat primary hippocampal neurons, we show that KCC2 is slowed down and confined at excitatory and inhibitory synapses compared with extrasynaptic regions. However, KCC2 escapes inhibitory synapses faster than excitatory synapses, reflecting stronger molecular constraints at the latter. Interfering with KCC2-actin interactions or inhibiting F-actin polymerization releases diffusion constraints on KCC2 at excitatory but not inhibitory synapses. Thus, F-actin constrains KCC2 diffusion at excitatory synapses, whereas KCC2 is confined at inhibitory synapses by a distinct mechanism. Finally, increased neuronal activity rapidly increases the diffusion coefficient and decreases the dwell time of KCC2 at excitatory synapses. This effect involves NMDAR activation, Ca 2ϩ influx, KCC2 S940 dephosphorylation and calpain protease cleavage of KCC2 and is accompanied by reduced KCC2 clustering and ion transport function. Thus, activity-dependent regulation of KCC2 lateral diffusion and clustering allows for a rapid regulation of chloride homeostasis in neurons.
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