La polarité et l'efficacité de la transmission GABAergique dépendent de la concentration intra-neuronale en chlore. Dans les neurones matures, le co-transporteur K+/Cl- KCC2 maintient la concentration intracellulaire en chlore à un niveau bas, permettant ainsi une réponse inhibitrice du GABA. En plus de son rôle dans la transmission GABAergique, KCC2 régule aussi l'efficacité de la transmission glutamatergique en contrôlant la spinogenèse, l'exocytose et la dynamique membranaire des récepteurs AMPA. Du fait de son importance aux synapses excitatrices et inhibitrices, il est crucial de comprendre les mécanismes qui régulent l'expression membranaire et la fonction de KCC2. La régulation de KCC2 par l'activité glutamatergique excitatrice ayant été bien caractérisée, il reste à déterminer si l'expression et la fonction de KCC2 sont régulées par l'activité inhibitrice GABAergique. Pendant ma thèse, j'ai montré que KCC2 est en effet directement régulé par la transmission GABAergique. J'ai trouvé que l'activation aigue des RGABAA confine KCC2 dans la membrane alors que le blocage des RGABAA augmente la dynamique membranaire et l'internalisation du transporteur. Les mécanismes moléculaires impliquent le chlore comme messager secondaire, la kinase WNK1 et la phosphorylation de KCC2 sur des résidus thréonines clés. J'ai ensuite pu montrer que cette régulation à un impact aux synapses inhibitrice et excitatrice. Mon travail propose un mécanisme nouveau de la régulation de l'homéostasie du chlore par l'inhibition GABAergique. Ainsi les neurones peuvent compenser une augmentation ou une diminution en chlore neuronale par une adaptation rapide de KCC2 à la surface cellulaire. / The polarity and efficacy of GABAergic neurotransmission depends on the intraneuronal chloride concentration. In mature neurons chloride extrusion by the K+/Cl- co-transporter KCC2 permits an inhibitory influx upon activation of GABAA receptors. In addition to its role in GABAergic transmission, KCC2 regulates also glutamatergic transmission in an ion-independent manner by controlling spinogenesis and AMPAR exocytosis and membrane diffusion in dendritic spines. Knowing its pivotal role at central synapses, it is of particular importance to understand the cellular and molecular mechanisms underlying its regulation. While regulation of KCC2 by neuronal excitation is well documented, it is still unknown whether neuronal inhibition itself can regulate the transporter’s membrane expression and/or activity. During my PhD I was able to demonstrate a direct regulation of KCC2 membrane diffusion and stability by GABAA receptor-mediated inhibition and I characterized the underlying signaling cascade. I found that activation of GABAAR decreased KCC2 lateral diffusion while GABAAR blockade led to increased membrane dynamics and internalization of the transporter. I could show that KCC2 regulation by neuronal inhibition requires chloride as second intracellular messenger and chloride-sensing WNK1 kinase that directly phosphorylate KCC2 on key Threonine residues. This regulation has a functional impact at both excitatory and inhibitory synapses. My work reports a novel and rapid mechanism of control of chloride homeostasis by GABAA receptor-mediated inhibition that allows maintaining the polarity and activity of GABAA receptors constant.

Hepatocyte growth factor (HGF) signaling drives epithelial cells to scatter by breaking cell-cell adhesions and migrating as solitary cells, a process that parallels epithelial-mesenchymal transition. HGF binds and activates the c-Met receptor tyrosine kinase, but downstream signaling required for scattering remains poorly defined. This study addresses this shortcoming in a number of ways.A high-throughput in vitro drug screen was employed to identify proteins necessary in this HGF-induced signaling. Cells were tested for reactivity to HGF stimulation in a Boyden chamber assay. This tactic yielded several small molecules that block HGF-induced scattering, including a calcium channel blocker. Patch clamping was used to determine the precise effect of HGF stimulation on Ca2+ signaling in MDCK II cells. Cell-attached patch clamping was employed to detect Ca2+ signaling patterns, and channel blockers were used in various combinations to deduce the identity of Ca2+ channels involved in EMT. The results of these experiments show that HGF stimulation results in sudden and transient increases in calcium channel influxes. These increases occur at predictable intervals and rely on proper tubulin polymerization to appear, as determined through the use of a tubulin polymerization inhibitor. Though multiple channels occur in the membranes of MDCK II cells, noticeably TRPV4 and TrpC6, it is TrpC6 that is specifically required for HGF-induced scattering. These HGF-induced calcium influxes through TrpC6 channels drive a transient increase in NFAT-dependent gene transcription which is required for HGF-induced EMT. This was determined through the use of luciferase-based NFAT reporter assays and confirmed through confocal immunofluorescence. Using a small-molecule inhibitor of WNK kinase, it was determined that loss of WNK kinase function is sufficient to prevent HGF-induced EMT. Furthermore, patch-clamp analysis demonstrated that WNK kinase significantly increases channel opening at the surface of MDCK cells, indicating a possible mechanism of action for c-Met inhibition, but leaving doubt as to whether WNK kinase is in fact normally involved in c-Met signaling, or whether it is simply permissive.