GABAA receptor dependent synaptic inhibition rapidly tunes KCC2 activity via the Cl−-sensitive WNK1 kinase

Abstract

The K+–Cl− co-transporter KCC2 (SLC12A5) tunes the efficacy of GABAA receptor-mediated transmission by regulating the intraneuronal chloride concentration [Cl−]i. KCC2 undergoes activity-dependent regulation in both physiological and pathological conditions. The regulation of KCC2 by synaptic excitation is well documented; however, whether the transporter is regulated by synaptic inhibition is unknown. Here we report a mechanism of KCC2 regulation by GABAA receptor (GABAAR)-mediated transmission in mature hippocampal neurons. Enhancing GABAAR-mediated inhibition confines KCC2 to the plasma membrane, while antagonizing inhibition reduces KCC2 surface expression by increasing the lateral diffusion and endocytosis of the transporter. This mechanism utilizes Cl− as an intracellular secondary messenger and is dependent on phosphorylation of KCC2 at threonines 906 and 1007 by the Cl−-sensing kinase WNK1. We propose this mechanism contributes to the homeostasis of synaptic inhibition by rapidly adjusting neuronal [Cl−]i to GABAAR activity.

KCC2 Regulates Neuronal Excitability and Hippocampal Activity via Interaction with Task-3 Channels

“…Thus, Ca 2+ influx through postsynaptic NMDA receptors or during prolonged postsynaptic firing (Fiumelli et al, 2005) rapidly reduces KCC2 membrane expression and function through proteinphosphatase-1-dependent dephosphorylation of its Ser940 residue and protein cleavage by the calcium-activated protease calpain Lee et al, 2011;Puskarjov et al, 2012). Conversely, Cl À influx through GABAA receptors stabilizes KCC2 at the plasma membrane via chloride-mediated inhibition of the serine-threonine WNK1 kinase and its downstream effectors SPAK-OSR1, which phosphorylate KCC2 on Thr906 and Thr1007 residues (Caraiscos et al, 2004;Heubl et al, 2017;Kahle et al, 2016). Finally, KCC2 expression is also regulated by several neuromodulators acting on G-protein-coupled receptors (Mahadevan and Woodin, 2016) as well as neurotrophins, such as brain-derived neurotrophic factor (BDNF), acting via TrkB signaling (Rivera et al, 2002).…”

KCC2 regulates neuronal excitability and hippocampal activity via interaction with Task-3 channels

“…In mature neurons, KCC2 plays a prominent role in maintaining transmembrane chloride gradients, thereby influencing ion flux through the chloride-permeable GABAA receptor channels. Thus, KCC2 pharmacological blockade 45,46 , genetic ablation 61,62 or suppression 63 , or activity-dependent modulation 6,11,46,64 all result in a depolarization of the reversal potential of GABAAR-mediated currents (E GABA ). Consistent with these observations, we found that chronic KCC2 knockdown in dentate granule cells resulted in a depolarizing shift of E GABA by about 9 mV.…”

“…In mature neurons, KCC2 expression and function is rapidly regulated by neuronal activity via multiple posttranslational mechanisms 4,5 . Thus, Ca 2+ influx through postsynaptic NMDA receptors or during prolonged postsynaptic firing 6 rapidly reduces KCC2 membrane expression and function through protein phosphatase 1-dependent dephosphorylation of its Ser940 residue and protein cleavage by the calcium-activated protease calpain 7,8,9 . Conversely, chloride influx through GABAA receptors stabilizes KCC2 at the plasma membrane via chloride-mediated inhibition of the serine/threonine WNK1 kinase and its downstream effectors SPAK/OSR1, which phosphorylate KCC2 on Thr906 and Thr1007 residues 10,11,12 . Finally, KCC2 expression is also regulated by several neuromodulators acting on G-protein couples receptors 13 as well as neurotrophins such as BDNF acting via TrkB signaling 14 .…”

“…All biochemical assays were repeated at least 3 times on independent hippocampal extracts or cultures.ImmunocytochemistryNeuro-2a cells were grown on glass coverslips in DMEM GlutaMAX (Invitrogen) supplemented with 1 g/l glucose and 10% fetal bovine serum. Cells at 60-70% confluence were co-transfected using Transfectin (Biorad) according to manufacturer's instructions with plasmids expressing HA-tagged mouse Task-3 (kindly provided by Guillaume Sandoz, Nice Sophia Antipolis Univ., France) and either Flag-tagged rat KCC27,11 or a pCAG empty vector as control (with a 1:3 ratio). 48 hours after transfection, cells were washed with PBS, fixed with paraformaldehyde (4%) supplemented with 4% sucrose in PBS and permeabilized with 0.25% Triton X-100 in PBS.…”

The Expanding Therapeutic Potential of Neuronal KCC2

“…Inhibitory neural transmission in the adult nervous system is mediated by γ-aminobutyric acid (GABA) [1] and glycine, with fast synaptic inhibition occurring largely through the ionotropic GABA A receptor (GABA A R) [2,3]. As a GABA-gated chloride (Cl − ) channel, the consequence of GABA A R signaling is dependent on intracellular Cl − concentration, which determines the reversal potential for GABA A R currents (E GABA ).…”