Activity-Dependent Regulation of the K/Cl Transporter KCC2 Membrane Diffusion, Clustering, and Function in Hippocampal Neurons

Abstract

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.

Regulation of neuronal chloride homeostasis by neuromodulators

“…inhibitory synapses, suggestive of stronger confinement of KCC2 to excitatory synapses (Chamma et al . 2013). The stronger confinement may result from interactions between KCC2 and proteins at excitatory synapses.…”

A Novel Regulatory Locus of Phosphorylation in the C Terminus of the Potassium Chloride Cotransporter KCC2 That Interferes with N-Ethylmaleimide or Staurosporine-mediated Activation*♦

“…Phosphorylation of human threonines Thr 906 and Thr 1007 reduced the intrinsic transport activity of KCC2 (37,38). In contrast, phosphorylated Ser 940 increased surface expression and KCC2 transport function (39) as well as membrane clustering (40). Finally, the phosphorylation status of Tyr 903 and Tyr 1087 also regulates KCC2 activity, although the functional consequences of phosphorylation appear to be context-dependent and require further investigation (5,23,36,41,42).…”

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

“…4D; p ϭ 0.02). To confirm that GluK2 increases KCC2 expression in the membrane, we performed total internal reflection microscopy (TIRF) 5 in COS-7 cells overexpressing KCC2-HA, which allows the imaging of proteins exclusive to the surface membrane. We observed a signif- Ϫ/Ϫ hippocampal neurons overexpressing KCC2-FLAG.…”

“…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.…”

“…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.…”