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.

The ability of Neisseria meningitidis (MC) to interact with cellular barriers is essential to its pathogenesis. With epithelial cells, this process has been modeled in two steps. The initial stage of localized adherence is mediated by bacterial pili. After this phase, MC disperse and lose piliation, thus leading to a diffuse adherence. At this stage, microvilli have disappeared, and MC interact intimately with cells and are, in places, located on pedestals of actin, thus realizing attaching and effacing (AE) lesions. The bacterial attributes responsible for these latter phenotypes remain unidentified. Considering that bacteria are nonpiliated at this stage, pili cannot be directly responsible for this effect. However, the initial phase of pilus-mediated localized adherence is required for the occurrence of diffuse adherence, loss of microvilli, and intimate attachment, because nonpiliated bacteria are not capable of such a cellular interaction. In this work, we engineered a mutation in the cytoplasmic nucleotide-binding protein PilT and showed that this mutation increased piliation and abolished the dispersal phase of bacterial clumps as well as the loss of piliation. Furthermore, no intimate attachment nor AE lesions were observed. On the other hand, PilT ؊ MC remained adherent as piliated clumps at all times. Taken together these data demonstrate that the induction of diffuse adherence, intimate attachment, and AE lesions after pilusmediated adhesion requires the cytoplasmic PilT protein.Neisseria meningitidis (MC) is a pathogen responsible for septicemia and meningitis. To reach the meninges, MC must interact with two cellular barriers, one in the nasopharynx and one in the brain, the blood-brain barrier. A multistep model has been proposed for MC interaction with cells (1). The first step corresponds to pilus-mediated adhesion. Pili emanate from the bacterial surface and are assembled from protein subunits called pilin. They initiate adherence by serving as a long-range adhesin and recruit other bacteria into a growing microcolony. This pilus-mediated adhesion phase leads to an initial phase of localized adherence. After this first step MC disperse from these microcolonies and spread at the apical surface of the cells to form a single monolayer of bacteria covering the surface. At this stage of diffuse adherence, MC have lost their pili and are involved in an intimate attachment with disappearance of microvilli and formation of pedestals with actin polymerization, thus realizing attaching and effacing (AE) lesions resembling those observed with enteropathogenic Escherichia coli (2, 3). The loss of piliation is not caused by phase variation because bacteria recovered as cellassociated colony-forming units (cfu) are piliated. The bacterial attributes responsible for the diffuse adherence phenotype, including intimate attachment and AE lesions, have not been identified yet; however, the loss of piliation suggests that pili per se are not involved in this step. Furthermore, an inoculum of nonpiliated bac...

The K+–Cl− co-transporter KCC2 (SLC12A5) tunes the efficacy of GABAA receptor-mediated transmission by regulating the intraneuronal chloride concentration [Cl−]i. KCC2 undergoes activity-dependent regulation in both physiological and pathological conditions. The regulation of KCC2 by synaptic excitation is well documented; however, whether the transporter is regulated by synaptic inhibition is unknown. Here we report a mechanism of KCC2 regulation by GABAA receptor (GABAAR)-mediated transmission in mature hippocampal neurons. Enhancing GABAAR-mediated inhibition confines KCC2 to the plasma membrane, while antagonizing inhibition reduces KCC2 surface expression by increasing the lateral diffusion and endocytosis of the transporter. This mechanism utilizes Cl− as an intracellular secondary messenger and is dependent on phosphorylation of KCC2 at threonines 906 and 1007 by the Cl−-sensing kinase WNK1. We propose this mechanism contributes to the homeostasis of synaptic inhibition by rapidly adjusting neuronal [Cl−]i to GABAAR activity.