Abstract
ABSTRACTFunctional 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.
Highlights
Regulation of KCC2, the neuron-specific electroneutral K+-Cl- co-transporter, is crucial for development and maturation of GABAergic neurotransmission
Functional activation of the neuronal K+-Cl--cotransporter KCC2 is a prerequisite for shifting GABAA responses from depolarizing to hyperpolarizing during development
We identify the signalling pathway transforming growth factor beta 2 (TGF- 2)/cAMP response element-binding protein (CREB)/Ras-associated binding protein 11b (Rab11b) as the underlying mechanism for TGF- 2-mediated KCC2 trafficking and functional activation
Summary
Regulation of KCC2, the neuron-specific electroneutral K+-Cl- co-transporter, is crucial for development and maturation of GABAergic neurotransmission. In immature CNS neurons, GABA produces depolarizing postsynaptic potentials, which are likely involved in stabilizing synapses during development (Ben-Ari, 2002; Blaesse et al, 2009). During maturation of most central neurons, expression of the Cl- extruder KCC2 is up-regulated resulting in an intracellular Cl- concentration below its electrochemical equilibrium, thereby shifting GABAA responses from depolarizing to hyperpolarizing (Rivera et al, 1999). BDNF has been shown to upregulate KCC2 expression in immature neurons, whereas in mature neurons BDNF/TrkB signaling results in an activity-dependent decrease of KCC2 expression (Rivera et al, 2002; Rivera et al 2004). KCC2 protein (de)phosphorylation is thought to be a crucial regulatory mechanism for KCC2 surface expression, surface stability, and trafficking
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