Functional activation of the neuronal K+-Cl−-cotransporter KCC2 is a prerequisite for shifting GABAA responses from depolarizing to hyperpolarizing during development. Here we introduce transforming growth factor beta 2 (TGF-β2) as a novel 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, respectively. We also identify the signalling pathway TGF-β2/cAMP response element-binding protein (CREB)/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 CREB or Rab11b suppressed TGF-β2-dependent KCC2 trafficking, surface expression, and functionality. Thus, TGF-β2 is a novel regulatory factor of KCC2 functional activation and membrane trafficking and putative indispensable molecular determinant for the developmental shift of GABAergic transmission.
- Received March 29, 2016.
- Accepted August 2, 2016.
- © 2016. Published by The Company of Biologists Ltd
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