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 β2 (TGF-β2) as a new regulator of KCC2 membrane trafficking and functional activation. TGF-β2 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-β2 to cAMP-response-element-binding protein (CREB) and Ras-associated binding protein 11b (Rab11b) as the underlying mechanism for TGF-β2-mediated KCC2 trafficking and functional activation. TGF-β2 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-β2-dependent KCC2 trafficking, surface expression and functionality. Thus, TGF-β2 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.

KCC2 mediates extrusion of K+ and Cl− and assuresthe developmental “switch” in GABA function during neuronal maturation. However, the molecular mechanisms underlying KCC2 regulation are not fully elucidated. We investigated the impact of transforming growth factor beta 2 (TGF-β2) on KCC2 during neuronal maturation using quantitative RT-PCR, immunoblotting, immunofluorescence and chromatin immunoprecipitation in primary mouse hippocampal neurons and brain tissue from Tgf-β2-deficient mice. Inhibition of TGF-β/activin signaling downregulates Kcc2 transcript in immature neurons. In the forebrain of Tgf-β2−/− mice, expression of Kcc2, transcription factor Ap2β and KCC2 protein is downregulated. AP2β binds to Kcc2 promoter, a binding absent in Tgf-β2−/−. In hindbrain/brainstem tissue of Tgf-β2−/− mice, KCC2 phosphorylation at T1007 is increased and approximately half of pre-Bötzinger-complex neurons lack membrane KCC2phenotypes rescued through exogenous TGF-β2. These results demonstrate that TGF-β2 regulates KCC2 transcription in immature neurons, possibly acting upstream of AP2β, and contributes to the developmental dephosphorylation of KCC2 at T1007. The present work suggests multiple and divergent roles for TGF-β2 on KCC2 during neuronal maturation and provides novel mechanistic insights for TGF-β2-mediated regulation of KCC2 gene expression, posttranslational modification and surface expression. We propose TGF-β2 as a major regulator of KCC2 with putative implications for pathophysiological conditions.

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Changes in extracellular pH (pHo) are common events associated with both physiological conditions and pathological states in brain function. Neuronal activity is associated with extracellular alkalinization and a rapid intracellular acidification whereas significant changes of pHo have also been observed during seizures and spreading depression. In the present study we have investigated regulation of V‐ATPase in hippocampal neurons following metabolic‐induced and activity‐dependent extracellular acid‐base changes. Therefore, dissociated primary hippocampal neurons and organotypic slice cultures have been used as experimental models in vitro. The results show differential regulation mechanisms of V‐ATPase depending on the cause of pathological stimulus. Metabolic acidosis caused translocation of V‐ATPase from intracellular pools towards the membrane, and increased V‐ATPase membrane expression. Acidosis‐induced V‐ATPase redistribution was mainly controlled by Rab11b, as assessed by loss‐of‐function experiments. In contrast, activity‐dependent acidosis resulted in both transcriptional and post‐translational V‐ATPase regulation in hippocampal neurons. The data suggest translolation of V‐ATPase as regulatory mechanism in neurons to cope with changes of pH and provide first evidence for a molecular mechanism underlying transporter trafficking in neurons.