Research ArticleNEURODEVELOPMENT

Developmentally regulated KCC2 phosphorylation is essential for dynamic GABA-mediated inhibition and survival

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Science Signaling  15 Oct 2019:
Vol. 12, Issue 603, eaaw9315
DOI: 10.1126/scisignal.aaw9315

KCC2 in neuronal maturity

High intracellular concentrations of Cl ions in neurons interfere with synaptic signaling, particularly of the inhibitory neurotransmitter of the central nervous system (CNS), γ-aminobutyric acid (GABA), and are implicated in several neurological diseases, such as epilepsy and schizophrenia. By extruding Cl ions, the K+/Cl cotransporter KCC2 (encoded by SLC12A5) helps maintain Cl homeostasis. Watanabe et al. and Pisella et al. (see also the Focus by Zamponi) developed two knockin mouse models of constitutive KCC2 phosphorylation at two threonine sites and examined the consequential neurodevelopmental effects. Their findings show that dephosphorylation of these sites in KCC2 during CNS development in the mouse contributes to the GABA excitatory-to-inhibitory switch that promotes the neurocircuitry that underlies cognition, respiration, and other critical neurological physiology, thereby elucidating the causes of KCC2 (SLC12A5)–related pathologies.

Abstract

Despite its importance for γ-aminobutyric acid (GABA) inhibition and involvement in neurodevelopmental disease, the regulatory mechanisms of the K+/Cl cotransporter KCC2 (encoded by SLC12A5) during maturation of the central nervous system (CNS) are not entirely understood. Here, we applied quantitative phosphoproteomics to systematically map sites of KCC2 phosphorylation during CNS development in the mouse. KCC2 phosphorylation at Thr906 and Thr1007, which inhibits KCC2 activity, underwent dephosphorylation in parallel with the GABA excitatory-inhibitory sequence in vivo. Knockin mice expressing the homozygous phosphomimetic KCC2 mutations T906E/T1007E (Kcc2E/E), which prevented the normal developmentally regulated dephosphorylation of these sites, exhibited early postnatal death from respiratory arrest and a marked absence of cervical spinal neuron respiratory discharges. Kcc2E/E mice also displayed disrupted lumbar spinal neuron locomotor rhythmogenesis and touch-evoked status epilepticus associated with markedly impaired KCC2-dependent Cl extrusion. These data identify a previously unknown phosphorylation-dependent KCC2 regulatory mechanism during CNS development that is essential for dynamic GABA-mediated inhibition and survival.

INTRODUCTION

Type A γ-aminobutyric acid (GABA) receptors (GABAARs) are ligand-gated anion channels that allow bidirectional flux of Cl ions. The direction of net Cl flux, governed by the transmembrane electrochemical gradient for Cl, is determined by regulation of the intracellular concentration of Cl ions [Cl]i and the post-synaptic membrane potential (1). GABAAR activation in the adult central nervous system (CNS) triggers hyperpolarizing phasic and tonic inhibitory neurotransmission. In contrast, GABAAR activation in the immature CNS elicits inhibitory shunting of membrane conductance or even increased membrane excitability, which plays an important role in brain development via effects on neuronal proliferation, migration, and synaptogenesis (27).

The developmental “switch” in GABA function from excitatory to inhibitory has been attributed to a transition from the immature neuronal [Cl]i of ~15 to 20 mM to the mature neuronal value of ~4 mM (8). This transition requires increased neuronal Cl extrusion dependent on the K-Cl cotransporter KCC2 (SLC12A5), beginning in rodents during the first postnatal week and progressing through the brain in a caudal-to-rostral direction (810). KCC2-null mice with increased levels of neuronal [Cl]i die perinatally and exhibit anomalous GABA-mediated neuronal excitation (11). Neuronal hyperexcitability induced by KCC2 hypofunction promotes epilepsy, autism, and other neurodevelopmental pathologies (12).

The critical role of KCC2 in GABA signaling and neurological disease has generated considerable interest in its mechanisms of regulation. Although the total abundance of KCC2 increases along with the GABA excitation-to-inhibition transition, the relative contributions to the developmental increase in KCC2 activity of altered KCC2 polypeptide abundance and altered regulation of KCC2 activity remain unclear. Moreover, some neurons of the brainstem, spinal cord, and suprachiasmatic nucleus dynamically modulate [Cl]i such that GABA rhythmogenically cycles from excitatory to inhibitory effects in respiratory (13), locomotor (14), and circadian networks (15).

KCC2 phosphorylation alters its activity, regulates neuronal [Cl]i, and strongly affects the GABA reversal potential (EGABA) (8, 1622). Nonetheless, the sites of regulated phosphorylation in KCC2 have not been systematically identified or functionally examined during CNS development in vivo. Among identified phosphosites, the dual phosphorylation of Thr906/Thr1007 (“pThr906/pThr1007”) is a particularly potent switch of KCC2 activity in vitro (19, 2325). Constitutive dephosphorylation of KCC2 Thr906/Thr1007, achieved via alanine mutagenesis at these sites, potently stimulates KCC2 activity >10-fold (24). KCC2 pThr906/pThr1007 maintains the GABA-dependent depolarization of cultured immature neurons by reducing neuronal Cl extrusion capacity (24, 26, 27). However, the function of the KCC2 pThr906/pThr1007 phospho-switch has not been examined during CNS development in vivo.

In this study, we tested the hypothesis that regulated phosphorylation of KCC2, specifically at the WNK/SPAK kinase–regulated Thr906/Thr1007 phosphorylation sites of KCC2, is essential for normal CNS function and organismal survival. To test this hypothesis, we performed unbiased quantitative phosphoproteomics to systematically map sites of KCC2 phosphorylation during development. We also performed detailed biochemical and electrophysiological characterization of a novel mouse model of KCC2 that mimics constitutive phosphorylation at pThr906/pThr1007 through glutamic acid mutagenesis.

RESULTS

Systematic identification of KCC2 phosphorylation sites during development

KCC2 expression is limited to neurons in the CNS (28). To map potential sites of KCC2-regulated phosphorylation in vivo during CNS development, we immunoprecipitated endogenous KCC2 from gestational day 18.5 (E18.5), postnatal day 0 (P0), P20, and adult mouse brains using specific anti-KCC2 antibodies and fractionated the purified immune complexes by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) (see Materials and Methods; Fig. 1A). KCC2 samples were then digested with trypsin, and the resulting peptides were identified by liquid chromatography–dual mass spectroscopy (LC-MS/MS) (Fig. 1B). Phosphopeptides and specific phosphorylation sites were assigned by precise match of predicted and observed mass/charge ratio (m/z) ratios of precursor ions and their product fragment ions using MaxQuant (29, 30).

Fig. 1 Identification of KCC2 phosphorylation sites regulated during CNS development.

(A) Phosphorylation site mapping. KCC2 was immunopurified from mouse brain, fractionated by SDS-PAGE, and digested with trypsin. Blot is representative of lysates from 19 mice. Schematic lays out how phosphopeptides were subjected to LC-MS/MS. MW, molecular weight. (B) Representative MS/MS spectrum assignment of peptide TLVMEQR (pThr929; presented as human KCC2B pThr906). The phosphorylated precursor ion (478.71 +2) was selected and produced the fragment ion spectrum shown. Specific y and b fragment ions allowed unambiguous identification of the precursor peptide and its phosphorylation at Thr906 (human numbering). Fragment ions with neutral loss of phosphate (−Pb/a1, −Pb/a2, −Pb/a3, etc.) are indicated. (C) Identified KCC2 phosphorylation sites are numbered as in human KCC2B (gene ID 57468). All KCC2 peptides observed at various developmental stages are listed in table S1. (D) Heatmap representation of significant KCC2 phosphorylation sites and their changes during development. Hierarchical clustering showed distinct pattern of KCC2 phosphorylation at these residues. Amino acid residue numbering is referenced to isoform 1 of mouse Slc12a5 (UniProt: Q91V14). (E) Brain lysates were subjected to immunoprecipitation (IP) by pan-KCC2 antibody (KCC2) or by phosphorylation site–specific antibodies recognizing the Thr906- or Thr1007-phosphorylated forms of KCC2, and immunoprecipitated protein was detected with pan-KCC2 antibody [immunoblotting (IB)]. Whole-cell lysates were subjected to immunoblot using antibodies recognizing the indicated proteins or phosphoproteins. D, dimeric KCC2; M, monomeric KCC2. Blot is representative of three experiments. (F) Band intensities represented in (E) were quantitated with ImageJ software. Calculation of intensity ratios was based on the calculation: (phospho-dimeric KCC2 + phospho-monomeric KCC2)/(total dimeric KCC2 + total monomeric KCC2), as described previously (24). ***P < 0.001 and **P < 0.01 by one-way ANOVA with post hoc testing (n = 6; data are means ± SEM).

Eighteen independent KCC2 phosphorylation sites were reproducibly identified at all time points in three independent experiments (Fig. 1C and table S1). Six sites were mapped to the KCC2 intracellular N-terminal domain, three were located in the large KCC2 extracellular loop, and nine were positioned in the KCC2 intracellular C-terminal domain. Sites identified (numbering standardized to the human KCC2b isoform herein) included Thr906 and Thr1007 phosphorylated by the WNK-SPAK kinases in vitro (25), Ser940 phosphorylated by protein kinase A (PKA) and protein kinase C (PKC) in vitro (21, 31), and Thr6 phosphorylated by SPAK/OSR1 in vitro (25). Candidate kinases for other identified sites in the KCC2 N terminus included PKA, PKC, CAMK2, CDK5, and GSK3 (table S2). The phosphorylation sites at Ser25, Ser31, Ser937, Ser1025, and Ser1026 did not match defined kinase specificity motifs. Three previously unknown phosphorylation sites detected in KCC2—Thr8, Thr799, and Ser812—were not previously annotated in the mouse PhosphoSitePlus database (32).

Potential developmentally regulated phosphosites were determined by label-free quantitation based on normalized peptide intensities (30). Among the 18 distinct KCC2 phosphorylation sites (table S1), phosphorylation at eight sites changed significantly during development, as assessed at E18.5, P0, P20, and adult (Fig. 1D). Hierarchical clustering analysis showed two distinct clusters of developmentally regulated phosphorylation sites (Fig. 1D). In the first cluster, Ser932, Thr999, and Ser1026 showed increased phosphorylation during development. A second cluster containing Thr6, Thr8, Thr906, Ser1022, and Ser1025 showed decreased phosphorylation during development. Among all identified sites, Thr906 showed the greatest reduction in developmental phosphorylation among all KCC2 phosphorylation sites (>65% reduction in adult brain versus E18.5 brain; Fig. 1D).

KCC2 phosphorylation sites Thr906 and Thr1007 were of particular interest, as these KCC2 residues are highly phosphorylated during early CNS development, are evolutionarily conserved from frogs to humans (fig. S1, A and B) and all human KCC paralogs (fig. S1C), and are critical regulators of KCC2 activity (23). We therefore assessed KCC2 phosphorylation at these sites in the developing brain by anti-KCC2 immunoblot of immunoprecipitates with phospho-specific antibodies recognizing pThr906 and pThr1007, as described previously (Fig. 1E) (19, 25). In wild-type (WT) mouse brains, KCC2 phosphorylation at Thr906 and Thr1007 decreased >95% during CNS development from E18.5 to adult, in parallel with a >340% increased KCC2 abundance (Fig. 1E) (24).

An in vivo genetic model to prevent the developmental dephosphorylation of KCC2 at Thr906/Thr1007

To investigate the in vivo role of regulated KCC2 pThr906/pThr1007 during CNS development, we used homologous recombination to generate knockin mice expressing the dual KCC2 glutamic acid substitutions T906E/T1007E (“T906E/T1007E”; Fig. 2A), which antagonizes KCC2-dependent Cl extrusion from neuronal cells in vitro by mimicking inhibitory phosphorylation at these sites (24, 27, 33). Southern blot analysis (fig. S2A) and DNA sequencing confirmed the presence of the T906E and T1007E mutations at their respective codons in KCC2 exons 22 and 24 (Fig. 2B).

Fig. 2 KCC2 T906E/T1007E (Kcc2E/E) phosphomimetic mice.

(A) Genomic targeting strategy depicting T906E (exon 22) and T1007E (exon 24). The intron 22 neomycin selection cassette is excised by Cre recombinase. (B) Sanger sequencing trace of KCC2 T906E/T1007E (Kcc2E/E). (C) Genotypes of surviving progeny from Kcc2+/E intercrosses at E18.5, P0, and P10. n is noted in the graph. (D) Consecutive axial brain sections revealed no gross defects in Kcc2E/E mutant mice (Hom, P0). Images are representative of 20 mice. (E) WT brain lysates at indicated ages were immunoprecipitated (IP) with site-specific phospho-antibodies recognizing KCC2 pThr906 or pThr1007. Immunoprecipitates were immunoblotted with pan-KCC2 antibody (IB). Whole-cell lysates were immunoblotted with indicated antibodies. Band intensities were quantitated with ImageJ software (shown in fig. S2C). Blot is representative of three experiments. (F to N) Percentage of WT, heterozygous (Het), and homozygous (Hom) P0 Kcc2E/E mice exhibiting seizures, type of seizure [partial (P), secondary generalized (G), tonic (T), and tonic-clonic types (T-C)], and duration of seizure (with or without opisthotonos: dark and light blue, respectively) provoked by brushing (F to H), tail pinch (I to K), and tail suspension (L to N). **P < 0.01 by χ2 test. Data are from 11 to 13 mice.

Heterozygous KCC2 T906E/T1007E+/wt mutants were viable and fertile and survived to adulthood without apparent abnormalities, as true also for heterozygous and homozygous KCC2 T906A/T1007A mice (34). Among progeny from T906E/T1007E+/wt intercrosses, homozygous T906E/T1007E+/+ mutant mice (herein termed as “Kcc2E/E” mice) developed in utero at the expected Mendelian ratio, whereas all Kcc2E/E died within 4 to 12 hours after birth because of spontaneous cessation of regular respiratory rhythm (Fig. 2C).

Pregnant mice were therefore subjected to cesarean section at E18.5. WT and heterozygous pups consistently survived this early delivery and exhibited similar postnatal courses. Kcc2E/E neonates were grossly normal, although 11.3% lower in body weight than WT or T906E/T1007E+/wt mice (fig. S2B). P0 Kcc2E/E mice had anatomically and histologically normal brain, heart, lungs, and other organs (Fig. 2D and fig. S3). Whereas T906E/T1007E+/wt and Kcc2E/E brain tissues exhibited the expected decreases in anti-KCC2 pThr906 and pThr1007 phospho-specific antibody immunoreactivity (Fig. 2E and fig. S2C) (27), the total polypeptide abundances of KCC2, NKCC1, and the NKCC1/KCC2 regulatory kinases WNK1/SPAK were unchanged (Fig. 2E and fig. S2C).

Touch-provoked recurrent generalized seizures in Kcc2E/E mice

Reduction of KCC2 activity increases neuronal [Cl]i, compromises GABAergic inhibition, and promotes spontaneous, recurrent generalized seizures in mice and humans (35). Strikingly, Kcc2E/E, but not WT or T906E/T1007E+/wt, mice exhibited severe, touch-provoked, recurrent generalized seizures triggered by (i) mild brush stroke (Fig. 2, F to H, and movie S1), (ii) tail pinch (Fig. 2, I to K, and movie S2), and (iii) tail suspension (Fig. 2, L to N, and movie S3). Brush stroke induced generalized tonic-clonic seizures of >55-s duration, including tonic convulsions with or without opisthotonus (Fig. 2, F to H). Tail pinch provoked primary or secondary generalized tonic-clonic seizures of >32-s duration and tonic convulsions with or without opisthotonus (Fig. 2, I to K). Tail suspension uniformly elicited severe generalized tonic primary or secondary convulsions with opisthotonus (Fig. 2, L to N). Kcc2E/E mice also exhibited spontaneous seizures (movie S4), increasing in frequency before death.

An anomalous neuronal distribution with normal dendritic spine morphology in Kcc2E/E brains

KCC2-mediated K+/Cl cotransport is required for GABA-dependent neuronal proliferation and migration (36). We therefore examined the neuronal distribution (reflecting both neuronal proliferation and migration) in the developing Kcc2E/E mouse brain by labeling neurons with 5-ethynyl-2′-deoxyuridine (EdU) at E14.5, followed by counting at E18.5 (Fig. 3A). Analysis revealed fewer EdU-labeled neurons in the Kcc2E/E septum, greater numbers of EdU-labeled neurons in preoptic areas (POAs), and unchanged numbers of EdU-labeled neurons in caudate putamen and deeper neocortex (Fig. 3B).

Fig. 3 Developing Kcc2E/E mouse brains exhibit anomalous distribution of proliferating neurons but normal dendritic spine morphology.

(A) Neuronal distribution in WT and homozygous Kcc2E/E E14.5 brains. Representative images of EdU-positive neurons in the septum, hypothalamus, hippocampus, and cortex of WT (n = 3) and homozygous Kcc2E/E (n = 4) mouse brains. Proliferating cells were labeled with EdU at E14.5 and then immunostained for EdU at E18.5. EdU-positive cells in each region of interest (ROI) were counted as in Materials and Methods. Images are representative of seven mice. (B) Quantitation of EdU-positive neuron density in WT versus homozygous Kcc2E/E E14.5 brains assessed in the septum, POA, caudate-putamen (CPu), hippocampus, and cortex (ROIs 1 and 2). **P < 0.01 by unpaired t test; n = 4 (Kcc2E/E) and n = 3 (WT). (C) Spine formation in WT and homozygous Kcc2E/E neurons. Representative images of EGFP-transfected DIV 26 primary cultured cortical neurons from WT and homozygous Kcc2E/E mice (each n = 3).

Independent of its Cl transport function, KCC2 regulates dendritic spine maturation and excitatory synapse development via structural interactions between KCC2 and the spine cytoskeleton (37). We therefore assessed neuronal (and dendritic spine) morphology in primary cultures of cortical neurons from Kcc2E/E mice. Soma size and shape, dendritic arborization, and spine morphogenesis were similar in day 26 primary cultured WT and Kcc2E/E neurons (Fig. 3C).

Impaired GABA-dependent Cl extrusion capacity of Kcc2E/E neurons

During development, KCC2 becomes the dominant mediator of Cl extrusion in CNS neurons (8). Deficits in KCC2-mediated Cl extrusion result in increased [Cl]i, and this facilitates pathological GABAAR-mediated depolarizing responses (38). The abundance of KCC2 at P0 is greater in the spinal cord than in the neocortex or hippocampus (17, 18, 20, 39). Therefore, we applied the gramicidin-perforated patch-clamp technique in ventral spinal cord neurons and in acute lumber spinal cord slices from P0 WT and Kcc2E/E mice (Fig. 4A) to measure the GABA reversal potential (EGABA) determined primarily by the Cl equilibrium potential [reflecting [Cl]i (9, 40) and an index of KCC2 activity] (11). EGABA was indistinguishable in WT and Kcc2E/E mice spinal cord neurons (Fig. 4B). To measure efficacy of KCC2-mediated Cl extrusion, we performed transient Cl loading by whole-cell patch-clamp recording using a low Cl (12 mM) pipette solution. Ventral spinal cord neurons Cl loaded by GABA during depolarizing voltage-clamp underwent current-clamp recording of GABA responses at 20-s intervals over a 5-min period (41). In Cl-loaded conditions, GABA-evoked potentials in WT spinal cord neurons increased more than fourfold (Fig. 4C), reflecting increased [Cl]i, and then rapidly relaxed to initial amplitudes, consistent with rapid KCC2-dependent Cl extrusion (Fig. 4C). Whereas WT peak potentials had fully recovered to initial values within 100 s after prolonged GABA exposure, Kcc2E/E peak potentials remained >20% above initial values (Fig. 4D).

Fig. 4 Kcc2E/E neurons exhibit impaired GABA-dependent Cl extrusion and disrupted rhythmogenesis.

(A) Gramicidin-perforated, voltage-clamped currents (9) recorded at −50-mV holding potential. Two 0.5-s voltage ramps from −100 to 0 mV were applied before and during 30-s puff application of 100 μM GABA; sample I-V curves before (black) and after GABA application (red). EGABA was estimated from the voltage axis intercept (detailed further in Materials and Methods). Insets (top left) are representative GABA-evoked current traces at −50-mV holding potential in ventral spinal cord neurons of acute lumber spinal cord slices from P0 WT (left) and Kcc2E/E mice (right). Data are representative of 12 mice. (B) Neuronal EGABA from WT (−59.6 ± 2.1; n = 5) and homozygous Kcc2E/E mice (−58.7 ± 1.8 mV; n = 7). Data were not significantly different by an unpaired t test. (C) Representative traces of GABA responses in P0 ventral spinal cord neurons of acute lumber spinal cord slices from WT and Kcc2E/E mice. After current-clamp recording of basal GABA responses (3 s, 100 μM GABA puffs every 20 s) in neurons from WT and Kcc2E/E mice, neurons were Cl-loaded by prolonged (20 s) GABA puff during depolarizing voltage clamp (Vh = 0 mV). After Cl loading, responses to brief GABA puffs were again recorded in current-clamp mode, demonstrating 407 ± 78% increased peak neuronal Cl extrusion. Data are representative of 23 mice. (D) Normalized recovery of neuronal GABA responses in WT (black circles; n = 10) and Kcc2E/E mice (red squares; n = 13) after Cl loading. Cl extrusion rate was impaired in Kcc2E/E mice. Each neuronal response was normalized to the GABA pulse peak value (0%) and peak post–Cl loading GABA pulse-induced response (100%) for each neuron. WT peak potentials recovered to initial values (−3.9 ± 3.8%; n = 10), whereas Kcc2E/E peak potentials remained 23.0 ± 4.1% above initial values (n = 13). *P < 0.05 and **P < 0.01 by unpaired t test. Open symbols, single cells; filled symbols, mean values with SE. (E) Respiratory motor neuron recordings from P0 mouse cervical spinal cord ventral rootlets (C4-C5) (42). Spontaneous rhythmic activity was measured in WT (n = 6), T906E/T1007E+/wt (n = 10), and Kcc2E/E mice (n = 11). (F) Respiratory rhythm of WT (10.4 ± 1.1 min−1; n = 6), heterozygous Kcc2E/wt (11 ± 1.1 min−1; n = 10), and Kcc2E/E mice (1.3 ± 0.8 min−1; n = 9). Means ± SEM. **P < 0.01 by Kruskal-Wallis test. (G) P0 L2 ventral root spontaneous activity (upper traces) and locomotor rhythm (lower traces) were induced by perfusion of 20 μM 5-HT (45, 46) in WT (n = 8), heterozygous (n = 8), and Kcc2E/E mice (n = 7). (H) Rate of the locomotor rhythm in WT (7.1 ± 2.2 min−1; n = 8), T906E/T1007E+/wt (8.5 ± 2.7 min−1; n = 8), and Kcc2E/E mice (1.9 ± 0.1 min−1; n = 7). Data are means ± SEM. **P < 0.01 by Kruskal-Wallis test. (I) Coefficient of variation of interburst intervals in WT (0.9 ± 0.04; n = 8), T906E/T1007E+/wt (0.9 ± 0.04; n = 8), and Kcc2E/E mice (0.1 ± 0.001; n = 7). Means ± SEM. **P < 0.01 by Kruskal-Wallis test.

Lack in spontaneous respiratory discharge recordings from ventral cervical spinal cord neurons of Kcc2E/E mice

Because Kcc2E/E mice died in respiratory distress shortly after birth, we examined the effects of the KCC2 T906E/T1007E mutation on spontaneous respiratory discharge. Respiratory discharges were recorded from C4 or C5 ventral cervical spinal cord neurons in brainstem–spinal cord preparations (42); for further details, see Materials and Methods. Respiratory discharges were indistinguishable in WT and heterozygous T906E/T1007E+/wt mice (Fig. 4, E and F). In contrast, spontaneous respiratory discharges were essentially absent in almost all homozygous Kcc2E/E mice (Fig. 4, E and F). A single Kcc2E/E mouse showed abnormal bursting activity (21 min−1), and one other Kcc2E/E mouse showed infrequent breathing (1 min−1). These results suggest that the abnormal respiratory patterns preceding death in Kcc2E/E mice result from aberrant spontaneous respiratory discharges in cervical spinal cord neurons.

Altered locomotor rhythm recordings from lumbar spinal cord ventral roots of Kcc2E/E mice

Analogous to the role of GABA in respiratory rhythmogenesis, the strength of GABA inhibition is an essential factor for normal locomotor rhythm (43, 44). As impaired KCC2-dependent Cl extrusion was observed in ventral spinal cord neurons of Kcc2E/E mice (Fig. 4, C and D), we assessed the effect of in vivo KCC2 T906E/T1007E mutation on recorded rhythmic motor activity from lumbar 2 (L2) ventral roots (Fig. 4G). Perfusion of 5-hydroxytryptamine (5-HT) induced rhythmic bursts in both WT and T906E/T1007E+/wt mice, as also previously described (45, 46). In contrast, although locomotor rhythm was observed in Kcc2E/E mice, the frequency of locomotor rhythm was significantly lower in Kcc2E/E mice than in WT and T906E/T1007E+/wt heterozygous mice (Fig. 4H). The coefficient of variation of the interburst intervals was also significantly lower in Kcc2E/E mice than in WT and T906E/T1007E+/wt mice (Fig. 4I). These results confirm that regulated KCC2 Thr906/Thr1007 phosphorylation is essential for generation of normal locomotor rhythm.

DISCUSSION

We have shown that the inhibitory phosphorylation of the WNK/SPAK-regulated Thr906/Thr1007 motif in KCC2 is substantially reduced during the course of CNS development. Second, regulated KCC2 Thr906/Thr1007 phosphorylation is essential for mouse survival; antagonizing the normal developmental down-regulation of KCC2 Thr906/Thr1007 via homozygous phospho-mimetic mutagenesis of these sites causes respiratory arrest and early postnatal death. Third, corroborating and extending previous in vitro findings (24, 26), phospho-mimetic mutation of Thr906/Thr1007 in vivo prevents KCC2 from dynamically increasing its Cl extrusion capacity (such as in response to a Cl load). This results in an imbalance of neuronal excitation and inhibition that leads to impaired rhythmogenesis in respiratory and locomotor networks accompanied by profound neuronal hyperexcitability manifesting as touch-evoked generalized seizures. Fourth, Kcc2E/E mice exhibit anomalous neuronal distribution with normal dendritic spine morphology, consistent with the known importance of KCC2 function on neuronal proliferation and migration (47) and the activity-independent role of KCC2 in dendrite spine maturation (37, 48).

KCC2 mRNA expression in mice starts as early as E10.5 in spinal cord and brainstem, which exhibit the earliest development of KCC2-dependent Cl extrusion. Mouse KCC2 transcripts are expressed in developing motoneurons in spinal cord ventral horn and in medulla as early as E12.5 (11) and in sensory nuclei at E15.5, with progressively increasing expression during embryonic development (17). After E15.5, NKCC1 abundance decreases in motoneurons, whereas KCC2 functional expression increases and contributes to emergence of a more negative ECl so that GABA and glycine function as inhibitory neurotransmitters in the majority of mouse spinal motoneurons by E17.5 (49). This switch may correspond to the period at which locomotor networks start to generate alternating flexor and extensor motor activities concomitant to network expression of left-right alternation, indicative of functional network inhibition (18, 50). Inhibitory spinal interneurons are essential for generation of locomotor rhythms (44). Consistent with the importance of KCC2 Thr906/Thr1007 phosphorylation in the developmental regulation of KCC2-dependent Cl homeostasis, spinal cord neurons of Kcc2E/E mice exhibited a substantially impaired Cl extrusion capacity compared to their counterparts. The substantially impaired (slow) locomotor rhythms in Kcc2E/E mice suggest that regulated phosphorylation of KCC2 at Thr906/Thr1007 is essential for the generation of locomotor rhythm.

GABA also critically regulates respiratory rhythmogenesis and motor output patterning (5154) and plays essential roles in termination of both inspiratory and expiratory phases of respiration (5559). Application of the GABAAR agonist, muscimol, increased respiration-related rhythmic activities in an E17 rat brainstem–spinal cord preparation but decreased those activities in an E20 preparation (60), suggesting that GABA switches from facilitating to inhibiting respiration-related rhythmic activities during development. This GABA-induced switch in respiration-related rhythmic frequency has been attributed to a developmental decrease in [Cl]i (50). We showed that Kcc2E/E mice died from respiratory distress within hours after birth and lacked spontaneous respiratory discharge recordings from cervical ventral roots. These results suggest that KCC2-dependent Cl extrusion capacity is essential for spontaneous respiratory discharges as previously observed (11) and that KCC2 Thr906/Thr1007 phosphorylation is likely essential for developmental switches in GABA-regulated rhythmic respiration. This is compatible with the previous observation that application of a KCC2 inhibitor substantially decreased the frequency of respiration-related rhythmic activities of mice at P1 (61).

Kcc2E/E mice showed normal organogenesis and the developmental up-regulation of KCC2 total protein abundance. However, Kcc2E/E mouse brains exhibited statistically significant abnormalities in neuronal distribution in the septum, hypothalamus, hippocampus, and cortex. These results suggest that regulated KCC2 Thr906/Thr1007 phosphorylation is essential for neuronal proliferation and/or migration in developing forebrain. Considering that KCC2 is absent but NKCC1 is highly expressed in the neuroepithelium (20, 62), altered distributions of proliferating cells might be due to disturbed migration rather than to altered proliferation. GABA is involved in radial and tangential migration of cortical cells. GABAAR activation is a stop signal for radial migration in the cortical plate. In contrast, depolarizing GABA promotes tangential migration of interneurons, whereas increased KCC2 expression reduces interneuron motility (63). Depolarizing GABA responses are required in immature migrating cells, because voltage-dependent Ca2+ channel–mediated Ca2+ signaling is closely coupled with migration. Thus, perturbation of immature Cl homeostasis in Kcc2E/E mice could result in anomalous migration. Overexpression of mutant KCC2 has been shown to arrest radial migration (26).

Status epilepticus (SE) is defined as a continuous seizure of long duration or recurrent seizures occurring in close temporal proximity without full interictal recovery. An imbalance of excitatory and inhibitory neurotransmission that results in hyperexcitation of neural network activity plays a critical role in generation of SE (64, 65). GABAergic mechanisms are critical in terminating seizures (6669). Susceptibility to SE is higher in neonates than in adults, likely reflecting depolarizing GABA functions secondary to KCC2 functional immaturity. An acute Cl overload because of intense neuronal activity would also reduce efficacy of GABAergic inhibition (66). In this setting, KCC2-mediated Cl extrusion is considered crucial for preventing rundown of GABAergic inhibition.

Gramicidin patch-clamp measurements of E18.5 neurons in WT and Kcc2E/E mice showed equivalent EGABA, indicating that the transporter activity was not completely eliminated in Kcc2E/E mice. Thus, resting [Cl]i in the absence of increased network activity, particularly GABAergic interneuron activity, was not substantially altered in Kcc2E/E mice. However, an impairment of neuronal Cl extrusion velocity became evident after acute Cl loading. Previous reports showed that strong activation of GABAergic input can substantially increase [Cl]i, indicating that Cl extrusion capacity of KCC2 is transiently overwhelmed by acute massive Cl influx (66, 70, 71). Thus, rapid KCC2-mediated Cl extrusion is required for recovery from such acute increases in [Cl]i. The Cl extrusion velocity of Kcc2E/E might be lower than that of WT, explaining our observed increase in time required to reverse [Cl]i after its transient GABA-induced increase.

KCC2 is important not only for maintenance of static [Cl]i but also for recovery from transient increases in [Cl]i that occur during the course of repeated inhibitory inputs. Mice with mutations that prevent KCC2 Ser940 phosphorylation (S940A) exhibit EGABA comparable to that of WT but show a deficit in Cl extrusion after Cl loading, with increased susceptibility to kainate-induced seizures (16). Kcc2E/E mice are similarly prone to stimulus-evoked seizures. All Kcc2E/E mice exhibited SE-like tonic spasms provoked by mild sensory stimulation such as brushing (tactile), tail pinch (pain), and tail suspension (proprioceptive and vestibular). Thus, “robust” GABAergic inhibition is required to prevent recruitment of excitation culminating in seizure propagation and extending to development of SE. Therefore, the impairment in Cl extrusion resulting from mild KCC2 hypofunction in Kcc2E/E mice contributes to epileptogenesis induced by activation of afferent pathways that include GABAergic inputs. Heterologous expression of two KCC2 mutants, causative for epilepsy of infancy with migrating focal seizures mimicking the patient status, resulted in [Cl]i statistically significantly higher than that associated with WT KCC2 but lower than in the absence of KCC2. These findings indicate that even mildly impaired neuronal Cl extrusion can substantially compromise the robustness of GABAergic inhibition in SE (40).

Our findings are also consistent with the recent demonstration that genetic mutation of KCC2 Thr906/Thr1007 to alanine (Ala), modeling constitutive dephosphorylation, elicited enhanced KCC2 activity while limiting onset and severity of seizures in homozygous mice (34). These KCC2 T906E(A)/T1007E(A) transgenic animals together represent valuable models to study the in vivo roles of regulated KCC2 phosphorylation and provide important genetic evidence that drug development targeting the KCC2 Thr906/Thr1007 phospho-switch is a compelling novel strategy to modulate GABA-mediated neurotransmission. Our study also identified several previously unknown candidate regulators of KCC2 Thr906, such as GSK3 kinase β, implicated in regulation of the WNK-SPAK signaling pathway (72). Our ongoing systematic identification of additional novel KCC2 phosphorylation sites, kinases, and kinase regulators should drive future work on the roles of regulated KCC2 phosphorylation during CNS development and their contributions to GABA neurophysiology.

MATERIALS AND METHODS

Immunoprecipitation and in-gel trypsin digestion of KCC2

Native mouse KCC2 was purified from brain lysates using 15 μg of rabbit anti-mouse KCC2 (catalog no. 07-432, Millipore) conjugated to protein A agarose. In all cases, protein extracts were mixed with immunoprecipitating antibodies and incubated at 4°C for 4 hours. The immunoprecipitates were washed, and the products were fractionated by SDS-PAGE on 4 to 20% gradient gels. Proteins of the expected molecular weights were visualized by QC colloidal Coomassie blue staining. The destained protein band was excised into 1-mm3 pieces, which were then subjected to in-gel trypsin digestion. The gel pieces were washed with 1:1 acetonitrile (ACN)/water followed by 1:1 ACN/NH4HCO3 (100 mM). Peptides produced by overnight trypsin digestion at 37°C were lyophilized for further analysis (25).

LC-MS/MS and quantitative analysis of KCC2 phosphorylation sites

Protein digests were analyzed using LC-MS/MS on a Thermo Scientific Q Exactive Plus mass spectrometer equipped with a Waters nanoAcquity UPLC system using a binary solvent system (buffer A: 100% water and 0.1% formic acid; buffer B: 100% ACN and 0.1% formic acid). Trapping was performed at 5 μl/min and 97% buffer A for 3 min using a Waters Symmetry C18 180 μm × 20 mm trap column. Peptides were separated using an ACQUITY UPLC PST (BEH) C18 nanoACQUITY column 1.7 μm, 75 μm × 250 mm (37°C) and eluted at 300 nl/min with the following gradient: 3% buffer B at initial conditions, 5 to 30% buffer B in 140 min, 30 to 50% buffer B in 15 min, and 50 to 90% B for 5 to 15 min before returning to initial conditions. Full MS scan was acquired in profile mode over the 300 to 1500 m/z scan range using 1 microscan, 70,000 resolution, automatic gain control (AGC) target of 3 × 106, and a maximum injection time (IT) of 45 ms. Data-dependent MS/MS scan was acquired in centroid mode using 1 microscan, 17,500 resolution, AGC target of 1 × 105, a maximum IT of 100 ms, 1.7 m/z isolation window, normalized collision energy of 28, and 200 to 2000 m/z scan range. Up to 20 MS/MS scans were collected per MS scan on species with an intensity threshold of 1 × 104, charge states of +2 to +6, peptide match preferred, and dynamic exclusion set to 20 s.

Raw mass spectra were searched against the mouse UniProt protein database using Andromeda search algorithm within MaxQuant 1.5.8.3 software (29, 73). Carbamidomethyl (C) was selected as a fixed modification, whereas oxidation (M), acetylation (protein N-term), and phosphorylation (STY) were selected as variable modifications. Perseus software 1.5.8.5 (74) was used for quantitative analysis of the results from MaxQuant. The raw intensity of each phosphorylation site was normalized on the basis of starting amount of proteins in the SDS-PAGE gel measured by densitometry. After removal of contaminant and reversed peptides, normalized phosphopeptide intensities were log2-transformed and filtered for valid values in three biological replicates from at least one developmental stage. Remaining missing values were imputed from the normal distribution. Analysis of variance (ANOVA) with permutation-based false discovery rate (control at 0.05) was used to detect statistically significant differences in phosphopeptide levels between developmental stages. Hierarchical clustering of the z-score transformed abundance of the statistically significant phosphorylation sites was performed using Euclidean distance and the average linkage method. Sequence logos around phosphorylated residues were created (PhosphoLogo) for subsets of statistically significant sites based on profile plots (increasing or decreasing).

Construction of the targeting vector

The Kcc2 gene-targeting vector was constructed from 129Sv mouse genomic DNA (GenOway, Lyon, France). T906E and T1007E point mutations were inserted into exon 22 and exon 24, respectively. A loxP-flanked neomycin cassette was inserted in intron 22. Thr1007 corresponds to numbers in human and rat, and the mutated residue in mouse is Thr1006. We mention this residue as Thr1007 in this paper to avoid confusion with previous studies performed using rat KCC2 and to be consistent with the mouse study by Moore et al. (34).

Production of Kcc2 double point mutant targeted ES cell clones

Linearized targeting vector was transfected into 129Sv embryonic stem (ES) cells (GenOway, Lyon, France) according to GenOway’s electroporation procedures. Polymerase chain reaction (PCR), Southern blot, and sequence analysis of G-418–resistant ES clones revealed the recombined locus in two clones. PCR across the 5′ end of the targeted locus used a forward primer hybridizing upstream of the 5′ homology arm (5′-ATAGCGTTGGCTACCCGTGATATTGC-3′) and a reverse primer hybridizing within the neomycin cassette (5′-AGGCTAGGCACAGGCTACATCCACAC-3′). Two Southern blot assays were hybridized with an internal and an external probe to assess recombination accuracy at the respective 5′ and 3′ ends of the Kcc2 locus. The absence of off-target mutations was confirmed by sequence analysis.

Generation of chimeric mice and breeding scheme

Recombined ES cell clones microinjected into C57BL/6 blastocysts gave rise to male chimeras with statistically significant ES cell contribution. These chimeras were bred with C57BL/6J mice expressing Cre recombinase to produce the Kcc2 double point mutant heterozygous line lacking the neomycin cassette. F1 genotyping was performed by PCR and Southern blot. PCR primers hybridizing upstream (5′-GTGGTTCGCCTATGGGATCTGCTACTC-3′) and downstream (5′-AGACAAGGGTTCATGTAACAGACTCGCC-3′) of the neomycin cassette allowed PCR identification of the 298–base pair (bp) Kcc2 endogenous allele amplicon, the 1946-bp double point mutant allele amplicon harboring the neomycin cassette, and the 387-bp double point mutant amplicon lacking the neomycin cassette. Southern blot hybridization with an external probe allowed identification of the 14.1-kb WT allele and the 4.6-kb double point mutant allele.

Antibodies

The following antibodies were raised in sheep and affinity-purified on appropriate antigens by the Division of Signal Transduction Therapy Unit at the University of Dundee: KCC2A phospho-Thr906 [SAYTYER(T)LMMEQRSRR (residues 975 to 989 of human KCC3A) corresponding to SAYTYEK(T)LVMEQRSQI (residues 899 to 915 of human KCC2A) (catalog no. S959C)], KCC2A phospho-Thr1007 [CYQEKVHM(T)WTKDKYM (residues 1032 to 1046 of human KCC3A) corresponding to TDPEKVHL(T)WTKDKSVA (residues 998 to 1014 of human KCC2A) (catalog no. S961C)], NKCC1 total antibody [residues 1 to 288 of human NKCC1 (catalog no. S022D)], SPAK total antibody [full-length glutathione S-transferase (GST)–tagged human SPAK protein (catalog no. S551D)], and SPAK/OSR1 (S-motif) phospho-Ser373/Ser325 antibody [residues 367 to 379 of human SPAK, RRVPGS(S)GHLHKT, which is highly similar to residues 319 to 331 of human OSR1 in which the sequence is RRVPGS(S)GRLHKT (catalog no. S670B)]. Pan-KCC2 antibody (residues 932 to 1043 of human KCC2) was from NeuroMab (catalog no. 73-013). Anti (neuronal)–β-tubulin III antibody was from Sigma-Aldrich (catalog no. T8578). Horseradish peroxidase–coupled secondary antibodies used for immunoblotting were from Pierce. Immunoglobulin G (IgG) for control immunoprecipitation experiments was affinity-purified from preimmune serum using protein G–Sepharose.

Buffers for Western blots

Buffer A contained 50 mM tris-HCl (pH 7.5) and 0.1 mM EGTA. Lysis buffer was 50 mM tris-HCl (pH 7.5), 1 mM EGTA, 1 mM EDTA, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1% (w/v) Triton X-100, 0.27 M sucrose, 0.1% (v/v) 2-mercaptoethanol, and protease inhibitors (Roche complete protease inhibitor cocktail tablets, 1 tablet per 50 ml). Tris-buffered saline–Tween buffer (TTBS) was tris-HCl (pH 7.5), 0.15 M NaCl, and 0.2% (v/v) Tween 20. SDS sample buffer was 1× NuPAGE LDS sample buffer (Invitrogen) containing 1% (v/v) 2-mercaptoethanol. Protein concentrations were determined following centrifugation of the lysate at 16,000g at 4°C for 20 min using the Bradford method with bovine serum albumin as the standard.

Immunoprecipitation with phosphorylation site–specific antibodies

KCCs phosphorylated at KCC2 Thr906 and Thr1007 equivalent residues were immunoprecipitated from clarified hippocampal and cortical culture lysates (centrifuged at 16,000g at 4°C for 20 min) using phosphorylation site–specific antibody coupled to protein G–Sepharose (19). The phosphorylation site–specific antibody was coupled with protein G–Sepharose at a ratio of 1 mg of antibody per 1 ml of beads in the presence of lysate (20 μg/ml) to which corresponding nonphosphorylated peptide had been added. Two milligrams of clarified cell lysate was incubated with 15 μg of antibody conjugated to 15 μl of protein G–Sepharose for 2 hours at 4°C with gentle agitation. Beads were washed three times with 1 ml of lysis buffer containing 0.15 M NaCl and twice with 1 ml of buffer A. Bound proteins were eluted with 1× LDS sample buffer.

Immunoblotting

Cell lysates (15 μg of protein) in SDS sample buffer were subjected to electrophoresis on SDS–polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were incubated for 30 min with TTBS containing 5% (w/v) skim milk and then immunoblotted in 5% (w/v) skim milk in TTBS with indicated primary antibodies overnight at 4°C. Antibodies prepared in sheep were used at concentrations of 1 to 2 μg/ml. The incubation with phosphorylation site–specific sheep antibodies was performed in the presence of the nonphosphorylated form of the phosphorylated peptide antigen (10 μg/ml) used to raise the antibody. The blots were then washed six times with TTBS and incubated for 1 hour at room temperature with secondary horseradish peroxidase–conjugated antibodies diluted 5000-fold in 5% (w/v) skim milk in TTBS. After repeating the washing steps, signal was detected with enhanced chemiluminescence reagent. Immunoblots were developed using a film automatic processor (SRX-101, Konica Minolta Medical), and films were scanned at 600-dpi (dots per inch) resolution (PowerLook 1000, UMAX). Figures were generated with Photoshop and Illustrator (Adobe). The relative densitometry intensities of immunoblot bands were determined with ImageJ software.

Seizure induction by mild physiological stimulation

Seizures were induced in P0 mice by brushing, tail pinch, or tail suspension. For brushing, backs were brushed 10 times with a soft brush. For tail pinch, tails were pinched with tweezers. For tail suspension, mice were suspended upside-down from their tails. Responses to stimulation were video-recorded for 300 s, and durations and types of seizures were analyzed. Seizure severity was classified according to the presence or absence of opisthotonus.

Patch-clamp recordings from spinal cord neurons

The Animal Care and Use Committee of Hamamatsu University School of Medicine approved all animal experiments. All efforts were made to minimize the number of animals used and minimize suffering. Under deep isoflurane anesthesia, lumber spinal cords were removed from P0 to P1 mice and embedded in 3% agarose. Coronal lumber spinal cord slices (350 μm) were made using the vibrating microtome 7000 (Campden) in an ice-cold oxygenated sucrose solution containing 220 mM sucrose, 120 mM NaCl, 2.5 mM KCl, 0.5 mM CaCl2, 1.25 mM NaH2PO4, 1 mM MgCl2, 26 mM NaHCO3, 30 mM glucose, and 10 mM MgSO4 (pH 7.4). The slices were maintained in standard artificial cerebrospinal fluid (ACSF) consisting of 120 mM NaCl, 2 mM KCl, 1 mM KH2PO4, 1 mM MgCl2, 2 mM CaCl2, 26 mM NaHCO3, and 10 mM glucose (pH 7.4) equilibrated with 95% O2 and 5% CO2 at room temperature before the recording. For recording, slices were transferred to a recording chamber, perfused with oxygenated ACSF in the presence of tetrodotoxin (500 nM). Electrophysiological recordings were performed using a MultiClamp 700B amplifier (Molecular Devices) and pClamp9 software (Molecular Devices). Currents were filtered at 2 kHz and digitized at 10 kHz using DigiData1322A. The data were analyzed offline using Clampfit9 (Molecular Devices).

To estimate the reversal potential of GABA-stimulated currents (EGABA) in ventral spinal cord neurons in acute lumber slices, we performed gramicidin-perforated patch-clamp recording to acquire GABA-evoked responses with native intracellular Cl concentrations (9). Patch electrode pipettes (2 to 4 megohms) were pulled from borosilicate glass capillaries on a P-97 puller (Sutter Instruments) and filled with pipette solution composed of 150 mM KCl and 10 mM Hepes (pH 7.2) supplemented with gramicidin. Gramicidin (Sigma-Aldrich) was dissolved in methanol to prepare a stock solution of 10 mg/ml and then diluted in pipette solution to a final concentration of 30 μg/ml.

Reversal potential of the 100 μM GABA–induced current was measured at −50 mV holding potential (Vh), and 0.5-s voltage ramps from −100 to 0 mV were applied before and during GABA application. EGABA was estimated by measuring the voltage at which the I-V relationships before and during GABA application intersected (40). GABA puffs (30-s duration) were applied through a patch pipette approximated to the soma using an IM-300 programmable microinjector (Narishige).

To measure the efficacy of KCC2-mediated Cl extrusion, we performed transient Cl loading by whole-cell voltage-clamp recording. Patch electrodes were filled with pipette solution containing 123 mM K-gluconate, 2 mM MgCl2, 8 mM NaCl, 1 mM EGTA, 4 mM adenosine 5′-triphosphate (ATP), 0.3 mM guanosine 5′-triphosphate (GTP), and 10 mM Hepes (pH 7.2). Basal responses to 100 μM GABA puffs (3 psi and 3-s duration) were recorded every 20 s in current-clamp mode. Then, Cl was loaded by exposing the neurons to GABA for 20 s in voltage-clamp mode (Vh = 0 mV). After Cl loading, GABA responses were once again recorded every 20 s in current-clamp mode to measure the rate of Cl extrusion (41).

Extracellular recordings from spinal cord ventral roots

For recordings of respiratory discharges, brainstem–spinal cord block preparations from P0 mice were isolated under deep isoflurane anesthesia (42). The brainstem was rostrally decerebrated between the VI cranial nerve roots and the lower border of the trapezoid body. The preparation was placed in a recording chamber and perfused continuously with the following modified ACSF: 124 mM NaCl, 5 mM KCl, 1.2 mM KH2PO4, 2.4 mM CaCl2, 1.3 mM MgSO4, 26 mM NaHCO3, and 30 mM glucose (pH 7.4) equilibrated with 95% O2 and 5% CO2. Respiratory motor neuron activity was recorded extracellularly with a glass suction electrode from the proximal end of ventral spinal roots at the cervical 4 (C4) or C5 level. The neuronal activity was recorded using a patch-clamp amplifier (MultiClamp 700B), and pClamp9 software neuronal activity (bursts per minute) was calculated from the mean burst activity over a period of 3 min.

For locomotor rhythm recording, spinal cord was isolated from P0 to P1 mice under deep isoflurane anesthesia (46). The spinal cord was dissected from the mid-cervical to sacral levels, placed in a recording chamber, and perfused continuously with the following modified ACSF: 118.4 mM NaCl, 4.69 mM KCl, 1.18 mM KH2PO4, 2.52 mM CaCl2, 1.25 mM MgSO4, 25 mM NaHCO3, and 11.1 mM glucose (pH 7.4) equilibrated with 95% O2 and 5% CO2. The locomotor rhythm [induced by perfusion with 20 μM 5-HT (46)] was recorded from L2 ventral roots by suction electrode using a MultiClamp 700B amplifier. Data were sampled at 10 kHz, low pass–filtered at 3 kHz, high pass–filtered at 15 Hz, and analyzed with pClamp9 software.

EdU staining

To label dividing cells, EdU (50 mg/kg per body weight; Invitrogen) was intraperitoneally injected into pregnant mice at E14.5. Under deep anesthesia with ketamine/xylazine, embryos were dissected at E18.5 and transcardially perfused with 4% paraformaldehyde. Brains were sectioned coronally (30 μm), and EdU was visualized using the Click-iT EdU Alexa Fluor 488 Imaging Kit (Invitrogen) according to the manufacturer’s protocol. Slices were imaged using a confocal laser-scanning microscope (FV1000-D, Olympus), and EdU-positive cells were counted in coronal sections (three sections of each animal) of the septum, POA, caudate-putamen, hippocampus, and neocortex using ImageJ software (NIH).

Primary culture of neurons

E17.5 embryos (2 WT and 2 homozygotes) were removed from pregnant mice under deep ketamine/xylazine anesthesia. Cortices were papain-dissociated for 20 min at 32°C and plated on 10-mm microcoverglass (Matsunami) coated with polyethyleneimine in a Nunc 12-well dish (Thermo Fisher Scientific). One culture from each animal was maintained at 37°C in Neurobasal medium (Invitrogen) supplemented with B-27 (Invitrogen) and humidified 5% CO2/95% air. Neurons were transfected with pCMV-EGFP (enhanced green fluorescent protein) at DIV (days in vitro) 5 using Lipofectamine 3000 (Invitrogen) per the manufacturer’s instructions. After 26 days in culture, neurons were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, and blocked with 1% normal goat serum and 2% bovine serum albumin. The neurons were then incubated overnight at 4°C with chicken anti-GFP antibody (1:500; Aves Labs). Goat anti-chicken Alexa Fluor–conjugated secondary antibody (1:300; Invitrogen) was then applied for 2 hours at room temperature. Immunofluorescent images were acquired by confocal laser-scanning microscope (FV1000-D, Olympus). Spine density was analyzed from two randomly selected neurons in each culture dish from individual animals by using the Filament Tracer module (v.8.1.2, Bitplane) within Imaris software. Spine formation by WT and homozygote neurons was compared by unpaired t tests.

SUPPLEMENTARY MATERIALS

stke.sciencemag.org/cgi/content/full/12/603/eaaw9315/DC1

Fig. S1. Sequence alignments.

Fig. S2. Generation and characterization of KCC2 Thr906/Thr1007 phosphomimetic mice.

Fig. S3. Hematoxylin and eosin staining of horizontal and sagittal midline sections of P0 mice showing no gross defects of the central nervous or the musculoskeletal system.

Table S1. In vivo KCC2 phosphorylation sites identified and quantified in all four conditions (E18.5, P0, P20, and adult).

Table S2. List of candidate protein kinases.

Movie S1. Seizure triggered by mild brush stroke in a homozygous P0 Kcc2E/E mouse.

Movie S2. Seizure triggered by tail pinch in a homozygous P0 Kcc2E/E mouse.

Movie S3. Seizure triggered by tail suspension in a homozygous P0 Kcc2E/E mouse.

Movie S4. Spontaneous seizure in a homozygous P0 Kcc2E/E mouse.

Reference (75)

REFERENCES AND NOTES

Funding: K.T.K. is supported by the March of Dimes, Simons Foundation, and NIH (4K12NS080223-05, RO1NS109358, and RO1NS111029). A.F. is supported by Grants-in-Aid for Scientific Research on Innovative Areas (#15H05872) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and Grants-in-Aid for Scientific Research (B) #17H04025 from the Japan Society for the Promotion of Science. Author contributions: K.T.K., M.W., J.Z., M.S.M., and A.F. conceived and designed the experiments. M.W., J.Z., M.S.M., J.D., J.K.K., E.D., S.L.A., R.P.L., A.F., and K.T.K. performed the experiments. J.Z., M.W., M.S.M., A.F., and K.T.K. analyzed the data. J.Z., M.W., M.S.M., A.F., and K.T.K. wrote the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Materials.
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