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International audience; A plethora of neurological disorders are associated with alterations in the expression and localization of potassium-chloride cotransporter type 2 (KCC2), making KCC2 a critical player in neuronal function and an attractive target for therapeutic treatment. The activity of KCC2 is determined primarily by the rates of its surface insertion and internalization. Currently the domains of KCC2 dictating its trafficking and endocytosis are unknown. Here, using live-cell immunolabeling and biotinylation of KCC2 proteins expressed in murine neuroblastoma N2a cells, human embryonic kidney 293 cells, or primary cultures of rat hippocampal neurons, we identified a novel role for the intracellular N and C termini in differentially regulating KCC2 surface expression. We report that the N terminus is required for KCC2 insertion into the plasma membrane, whereas the C terminus is critical for the membrane stability of KCC2. Our results provide novel insights into the structure-function role of specific KCC2 domains and open perspectives in exploring structural organization of this protein.

International audience; Background: Cation-chloride cotransporters (CCCs) are indispensable for maintaining chloride homeostasis in multiple cell types, but K-Cl cotransporter KCC2 is the only CCC member with an exclusively neuronal expression in mammals. KCC2 is critical for rendering fast hyperpolarizing responses of ionotropic γ-aminobutyric acid and glycine receptors in adult neurons, for neuronal migration in the developing central nervous system, and for the formation and maintenance of small dendritic protrusions-dendritic spines. Deficit in KCC2 expression and/or activity is associated with epilepsy and neuropathic pain, and effective strategies are required to search for novel drugs augmenting KCC2 function. Results: We revised current methods to develop a noninvasive optical approach for assessing KCC2 transport activity using a previously characterized genetically encoded chloride sensor. Our protocol directly assesses dynamics of KCC2-mediated chloride efflux and allows measuring genuine KCC2 activity with good spatial and temporal resolution. As a proof of concept, we used this approach to compare transport activities of the two known KCC2 splice isoforms, KCC2a and KCC2b, in mouse neuronal Neuro-2a cells. Conclusions: Our noninvasive optical protocol proved to be efficient for assessment of furosemide-sensitive chloride fluxes. Transport activities of the N-terminal splice isoforms KCC2a and KCC2b obtained by the novel approach matched to those reported previously using standard methods for measuring chloride fluxes.

Functional activation of the neuronal K+-Cl- co-transporter KCC2 (also known as SLC12A5) is a prerequisite for shifting GABAA responses from depolarizing to hyperpolarizing during development. Here, we introduce transforming growth factor beta 2 (TGF-beta 2) as a new regulator of KCC2 membrane trafficking and functional activation. TGF-beta 2 controls membrane trafficking, surface expression and activity of KCC2 in developing and mature mouse primary hippocampal neurons, as determined by immunoblotting, immunofluorescence, biotinylation of surface proteins and KCC2-mediated Cl- extrusion. We also identify the signaling pathway from TGF-beta 2 to cAMP-response-element-binding protein (CREB) and Ras-associated binding protein 11b (Rab11b) as the underlying mechanism for TGF-beta 2-mediated KCC2 trafficking and functional activation. TGF-beta 2 increases colocalization and interaction of KCC2 with Rab11b, as determined by 3D stimulated emission depletion (STED) microscopy and co-immunoprecipitation, respectively, induces CREB phosphorylation, and enhances Rab11b gene expression. Loss of function of either CREB1 or Rab11b suppressed TGF-beta 2-dependent KCC2 trafficking, surface expression and functionality. Thus, TGF-beta 2 is a new regulatory factor for KCC2 functional activation and membrane trafficking, and a putative indispensable molecular determinant for the developmental shift of GABAergic transmission.

International audience; Temporal lobe epilepsy (TLE) is the most common type of epilepsy in adults where 20–30% of the patients are refractory to currently available anti-epileptic drugs. The RhoA/Rho-kinase signaling pathway activation has been involved in inflammatory responses, neurite outgrowth and neuronal death under pathological conditions such as epileptic insults. Acute preventive administration of ROCK inhibitor has been reported to have beneficial outcomes in Status Epilepticus (SE) epilepsy. In the present study, we evaluate the effect of chronic post SE treatment with the ROCK inhibitor Y-27632 in a rat pilocarpine model of TLE. We used chronic i.p. injections of Y-27632 for 5 days in 6 week old control rats or rats subjected to pilocarpine treatment as a model of TLE. Surprisingly, our findings demonstrate that a systemic administration of Y-27632 in pilocarpine-treated rats increases neuronal death in the CA3 region and ectopic recurrent mossy fiber sprouting (rMFS) in the dentate gyrus of the hippocampal formation. Interestingly, we found that chronic treatment with Y-27632 exacerbates the down-regulation and pathological distribution of the K +-Cl − cotransporter KCC2, thus providing a putative mechanism for post SE induced neuronal death. The involvement of astrogliosis in this mechanism appears to be intricate as ROCK inhibition reduces reactive astrogliosis in pilocarpine rats. Conversely, in control rats, chronic Y-27632 treatment increases astrogliosis. Together, our findings suggest that Y-27632 has a detrimental effect when chronically used post SE in a rat pilocarpine model of TLE.