The membrane trafficking and functionality of the K+-Cl− co-transporter KCC2 is regulated by TGF-β2

Abstract

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.

TGF‐β signaling directly regulates transcription and functional expression of the electrogenic sodium bicarbonate cotransporter 1, NBCe1 (SLC4A4), via Smad4 in mouse astrocytes

“…We have previously shown that the isoform 2 of TGF‐β (TGF‐β2) is able to increase trafficking and functional expression of K + /Cl ‐ cotransporter 2 in mouse hippocampal neurons (Roussa et al, 2016). We therefore asked whether TGF‐β2 is the isoform required for 4AP‐dependent regulation of NBCe1 in cortical astrocytes.…”

“…The phenotypes resulting from the knockout of the mammalian TGF‐β isoforms are very distinct (reviewed in Dünker & Krieglstein, 2000). We have recently shown that TGF‐β2 regulates functional expression of the neuronal K + /Cl ‐ cotransporter 2 (KCC2) (Roussa et al, 2016), a molecular component increasingly appreciated in the context of epilepsy (Kelley et al, 2016; Kourdougli, Varpula, Chazal, & Rivera, 2015). We have hypothesized that TGF‐β2 is the isoform required for 4AP‐dependent NBCe1 regulation.…”

Native KCC2 interactome reveals PACSIN1 as a critical regulator of synaptic inhibition

“…KCC2 is regulated by multiple posttranslational mechanisms including phosphoregulation by distinct kinases and phosphatases ( Lee et al, 2007 ; Kahle et al, 2013 ; Medina et al, 2014 ), lipid rafts and oligomerization ( Blaesse et al, 2006 ; Watanabe et al, 2009 ), and protease-dependent cleavage ( Puskarjov et al, 2012 ). KCC2 expression and function is also regulated by protein interactions, including creatine kinase B (CKB) ( Inoue et al, 2006 ), sodium/potassium ATPase subunit 2 (ATP1A2) ( Ikeda et al, 2004 ), chloride cotransporter interacting protein 1 (CIP1) ( Wenz et al, 2009 ), protein associated with Myc (PAM) ( Garbarini and Delpire, 2008 ), 4.1N ( Li et al, 2007 ), the glutamate receptor subunit GluK2, its auxiliary subunit Neto2 ( Ivakine et al, 2013 ; Mahadevan et al, 2014 ; Pressey et al, 2017 ), cofilin1 (CFL1) ( Chevy et al, 2015 ; Llano et al, 2015 ), the GABA B receptor subunit GABA B R1 ( Wright et al, 2017 ), metabotropic glutamate receptor subunits mGluR1/5 ( Farr et al, 2004 ; Banke and Gegelashvili, 2008 ; Mahadevan and Woodin, 2016 ; Notartomaso et al, 2017 ), and RAB11( Roussa et al, 2016 ). However, since KCC2 exists in a large multi-protein complex (MPC) ( Mahadevan et al, 2015 ), it is likely that these previously identified interactions do not represent all of the components of native-KCC2 MPCs.…”

K+-Cl−co-transporter 2 (KCC2) – a membrane trafficking perspective

“…Functional appearance of KCC2 on the cell surface could result from biosynthetic exocytic trafficking from the ER, as well as recycling back to the surface from an endosomal compartment. A very recent report found that KCC2 recycling in mouse E18.5 hippocampal neurons is regulated by Transforming growth factor b2 (TGFb2) signaling (Roussa et al, 2016). The TGFb family members are important regulators of nervous system development (Meyers & Kessler, 2017) as well as adult tissue homeostasis.…”

“…The Rab11 family small GTPases and their effectors are master regulators of the surface expression of receptors and cell adhesion molecules via endocytic recycling (Welz et al, 2014). In the case of KCC2, silencing of Rab11b abolished TGFb2-induced surface translocation and consequent shift in somatodendritic Cl À gradient (Roussa et al, 2016). This intracellular membrane compartment-to-cell surface translocation shown in cultured neurons may be reflective of the mode whereby surface expression of KCC2 is upregulated during neuronal development, which has been well-documented for mouse cerebellar Purkinje cells (Kawakita et al, 2013).…”