Mechanism of KCC 2 activation by N-ethylmaleimide 1 N-ethylmaleimide increases KCC 2 activity by modulating transporter phosphorylation

The K/Cl co-transporter (KCC2) is selectively expressed in the adult nervous system, and allows neurons to maintain low intracellular Cl – levels. Thus, KCC2 activity is an essential prerequisite for fast hyperpolarizing synaptic inhibition mediated by type A aminobutyric acid receptors (GABAA), which are Cl – permeable, ligand-gated ion channels. Consistent with this, deficits in the activity of KCC2 lead to epilepsy, and are also implicated in neurodevelopmental disorders, neuropathic pain, and schizophrenia. Accordingly, there is significant interest in developing activators of KCC2 as therapeutic agents. To provide insights into the cellular processes that determine KCC2 activity, we have investigated the mechanism by which N-ethylmaleimide (NEM) enhances transporter activity, employing a combination of biochemical and electrophysiological approaches. Our results revealed that within 15 minutes, NEM increased cell surface levels of KCC2 and modulated the phosphorylation of key regulatory residues within the large cytoplasmic domain of KCC2 in neurons. More specifically, NEM increased the phosphorylation of serine residue 940 (S940), while it decreased phosphorylation of threonine 1007 (T1007). NEM also reduced WNK phosphorylation of SPAK, a kinase that directly phosphorylates KCC2 at residue T1007. Mutational analysis revealed that T1007 dephosphorylation mediated the effects of NEM on KCC2 activity. Collectively our results suggest that compounds that either increase the surface stability of KCC2 or reduce T1007 phosphorylation may be of use as enhancers of KCC2 activity. KCC2 is a membrane K + -Cl – cotransporter that lowers intracellular Cl – concentrations by a secondary active transport mechanism (1). This process allows Cl – to passively reenter the cell upon opening of Cl – channels such as GABAA and glycine receptors, resulting in membrane hyperpolarization (2,3). Due to the lack of ligand-gated K + channels, fast synaptic inhibition in the mammalian central nervous system is mediated exclusively by GABAA and glycine receptors. KCC2 is expressed in most adult neurons (4), and expression levels correlate well with the maturation state of neurons. Specifically, KCC2 expression levels are low in immature neurons and high in mature neurons, http://www.jbc.org/cgi/doi/10.1074/jbc.M117.817841 The latest version is at JBC Papers in Press. Published on November 1, 2017 as Manuscript M117.817841 Copyright 2017 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on N ovem er 3, 2017 hp://w w w .jb.org/ D ow nladed from Mechanism of KCC2 activation by N-ethylmaleimide 2 which underlies the developmental EGABA shift (5). Dysregulation of KCC2 is associated with a number of neurological disorders, including epilepsy (6-11) and neuropathic pain (12-17). Thus, there is great interest in understanding the mechanisms that regulate the activity of this transporter. KCC2 activity can be enhanced by modulation of its phosphorylation state (18-21). Specifically, three key phosphorylation sites in the C-terminal domain of KCC2 are associated with the regulation of its transporter activity. Dephosphorylation of residues T906 and T1007 correlates with increased KCC2 activity (19), while increased levels of S940 phosphorylation correlate with both upregulated KCC2 surface levels and increased function (18). The importance of KCC2 S940 phosphorylation in the regulation of transporter activity is highlighted by studies that demonstrated an enhanced onset and severity of status epilepticus upon kainate treatment in a KCC2-S940A knock-in mouse (22). N-ethylmaleimide (NEM) has been used as a tool compound in the field for numerous years to activate KCC transport under isotonic conditions (1,23,24). NEM contains a Michael acceptor functionality that modifies the sulfhydryl group of cysteine residues through the formation of a covalent (possibly reversible) thioether bond. While the chemical properties of NEM are known, it is unclear which cysteine moieties are modified in a cellular context. Prior work has demonstrated that NEM does not likely act directly on these transporters, but rather modulates a kinase or phosphatase involved in the activation of KCCs (25-27). However, the specific mechanism by which NEM functions to activate KCCs has yet to be established. Here we investigated the precise mechanism by which NEM affects KCC2 to rapidly increase its function in both HEK293 cells and neurons. We also developed a new phospho-specific antibody to study the modulation of a known phosphorylation site of KCC2. We show that while NEM does not affect total KCC2 levels, it does modulate its surface levels and phosphorylation state in a cell-type dependent manner. We demonstrate that NEM both increased KCC2-S940 phosphorylation and decreased KCC2-T1007 phosphorylation. Further analysis with single-point mutant constructs indicated that the dephosphorylation of the T1007 residue alone mediates the effects of NEM. These studies provide a novel mechanistic understanding of the activation of KCC2 by NEM and demonstrate that posttranslational modifications of KCC2 lead to a rapid enhancement of Cl – extrusion.