Genetically encoded impairment of neuronal
            <scp>KCC</scp>
            2 cotransporter function in human idiopathic generalized epilepsy

Kahle, Kristopher T.; Merner, Nancy D.; Friedel, Perrine; Silayeva, Liliya; Liang, Bo; Khanna, Arjun; Shang, Yuze; Lachance-Touchette, Pamela; Bourassa, Cynthia V.; Levert, Annie; Dion, Patrick A.; Walcott, Brian P.; Spiegelman, Dan; Dionne‐Laporte, Alexandre; Hodgkinson, Alan; Awadalla, Philip; Nikbakht, Hamid; Majewski, Jacek; Cossette, Patrick; Deeb, Tarek Z.; Moss, Stephen J.; Medina, Igor; Rouleau, Guy

doi:10.15252/embr.201438840

Cited by 166 publications

(201 citation statements)

References 43 publications

(69 reference statements)

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“…Moreover, it is tempting to speculate that inhibitory KCC2 Thr 906 and Thr 1007 phosphorylation might also be pathologically increased in mature neurons, accounting for the documented decrease in KCC2-mediated Cl − extrusion capacity and GABAergic disinhibition in diseases, such as temporal lobe epilepsy (60) and neuropathic pain (61). Mutations in KCC2 associated with human disease, severe idiopathic generalized epilepsy in a large French Canadian patient cohort (43) and febrile seizures in an Australian family (62), are clustered in the C-terminal region and reside in residues close to the Thr 906 and Thr 1007 motifs, which would likely alter KCC2 activity. These subjects will be rich topics of future investigations with potential clinical relevance.…”

Section: Discussionmentioning

confidence: 99%

WNK1-regulated inhibitory phosphorylation of the KCC2 cotransporter maintains the depolarizing action of GABA in immature neurons

et al. 2015

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Activation of Cl(-)-permeable γ-aminobutyric acid type A (GABAA) receptors elicits synaptic inhibition in mature neurons but excitation in immature neurons. This developmental "switch" in the GABA function depends on a postnatal decrease in intraneuronal Cl(-) concentration mediated by KCC2, a Cl(-)-extruding K(+)-Cl(-) cotransporter. We showed that the serine-threonine kinase WNK1 [with no lysine (K)] forms a physical complex with KCC2 in the developing mouse brain. Dominant-negative mutation, genetic depletion, or chemical inhibition of WNK1 in immature neurons triggered a hyperpolarizing shift in GABA activity by enhancing KCC2-mediated Cl(-) extrusion. This increase in KCC2 activity resulted from reduced inhibitory phosphorylation of KCC2 at two C-terminal threonines, Thr(906) and Thr(1007). Phosphorylation of both Thr(906) and Thr(1007) was increased in immature versus mature neurons. Together, these data provide insight into the mechanism regulating Cl(-) homeostasis in immature neurons, and suggest that WNK1-regulated changes in KCC2 phosphorylation contribute to the developmental excitatory-to-inhibitory GABA sequence.

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Section: Discussionmentioning

confidence: 99%

WNK1-regulated inhibitory phosphorylation of the KCC2 cotransporter maintains the depolarizing action of GABA in immature neurons

et al. 2015

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“…Importantly, recent genetic studies of patients suffering from idiopathic epilepsy identified mutations within KCC2 (31,32) that decrease S940 phosphorylation and compromise transporter activity. Thus, changes in S940 phosphorylation may contribute to epileptogenesis in humans.…”

Section: Positive Modulation Of Kcc2 Function Upon Inhibition Of Proteinmentioning

confidence: 99%

KCC2 activity is critical in limiting the onset and severity of status epilepticus

Silayeva

Deeb

Hines

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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The K + /Cl -cotransporter (KCC2) allows adult neurons to maintain low intracellular Cl -levels, which are a prerequisite for efficient synaptic inhibition upon activation of γ-aminobutyric acid receptors. Deficits in KCC2 activity are implicated in epileptogenesis, but how increased neuronal activity leads to transporter inactivation is ill defined. In vitro, the activity of KCC2 is potentiated via phosphorylation of serine 940 (S940). Here we have examined the role this putative regulatory process plays in determining KCC2 activity during status epilepticus (SE) using knockin mice in which S940 is mutated to an alanine (S940A). In wild-type mice, SE induced by kainate resulted in dephosphorylation of S940 and KCC2 internalization. S940A homozygotes were viable and exhibited comparable basal levels of KCC2 expression and activity relative to WT mice. However, exposure of S940A mice to kainate induced lethality within 30 min of kainate injection and subsequent entrance into SE. We assessed the effect of the S940A mutation in cultured hippocampal neurons to explore the mechanisms underlying this phenotype. Under basal conditions, the mutation had no effect on neuronal Cl -extrusion. However, a selective deficit in KCC2 activity was seen in S940A neurons upon transient exposure to glutamate. Significantly, whereas the effects of glutamate on KCC2 function could be ameliorated in WT neurons with agents that enhance S940 phosphorylation, this positive modulation was lost in S940A neurons. Collectively our results suggest that phosphorylation of S940 plays a critical role in potentiating KCC2 activity to limit the development of SE.F ast synaptic inhibition in the adult brain is largely mediated via the activation of γ-aminobutyric acid receptors (GABA A Rs). The ability of neurons to maintain low intracellular Cl -is dependent upon the activity of the K + /Cl -cotransporter KCC2. KCC2 expression in the rodent brain is developmentally regulated with low levels evident before birth that then dramatically increase from postnatal day 7 (P7) onwards and correlate with the appearance of hyperpolarizing GABA A R currents (1). KCC2 null mice die shortly after birth, exhibit high levels of intracellular Cl -and anomalous excitatory actions of GABA and glycine, highlighting the vital role of KCC2 (2). In addition to regulating Cl -transport, KCC2 exhibits transporter-independent properties that affect glutamate receptors and dendritic spines (3-6).Status epilepticus (SE) is a state of continuous seizures that is highly lethal and leads to long-term neurological deficits in survivors. A key feature of SE is impaired GABAergic inhibition (7) that manifests clinically as resistance to the GABA A modulator diazepam (8). The prevailing hypothesis of impaired GABAergic signaling during SE is displacement of GABA A receptors from the synapse and a reduction in the number of receptors on the cell surface (9). However, an additional component that could contribute to the compromised inhibition during SE is a buildup of intracellular C...

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“…The loss of KCC2 membrane expression underlies neurological and psychiatric disorders including epilepsy (41)(42)(43)(44), stress (45), autism spectrum disorder (46,47), schizophrenia (48), motor spasticity (49), and neuropathic pain (50). Thus, it is essential to elucidate the pathways and mechanisms regulating KCC2 function to develop therapeutic strategies to restore KCC2 expression and synaptic inhibition.…”

Section: Gluk2 Regulates Kcc2 Surface Recyclingmentioning

confidence: 99%

A kainate receptor subunit promotes the recycling of the neuron-specific K+-Cl− co-transporter KCC2 in hippocampal neurons

Pressey

Mahadevan

Khademullah

et al. 2017

Journal of Biological Chemistry

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Edited by Roger J. ColbranSynaptic inhibition depends on a transmembrane gradient of chloride, which is set by the neuron-specific K ؉ -Cl ؊ co-transporter KCC2. Reduced KCC2 levels in the neuronal membrane contribute to the generation of epilepsy, neuropathic pain, and autism spectrum disorders; thus, it is important to characterize the mechanisms regulating KCC2 expression. In the present study, we determined the role of KCC2-protein interactions in regulating total and surface membrane KCC2 expression. Using quantitative immunofluorescence in cultured mouse hippocampal neurons, we discovered that the kainate receptor subunit GluK2 and the auxiliary subunit Neto2 significantly increase the total KCC2 abundance in neurons but that GluK2 exclusively increases the abundance of KCC2 in the surface membrane. Using a live cell imaging assay, we further determined that KCC2 recycling primarily occurs within 1-2 h and that GluK2 produces an ϳ40% increase in the amount of KCC2 recycled to the membrane during this time period. This GluK2-mediated increase in surface recycling translated to a significant increase in KCC2 expression in the surface membrane. Moreover, we found that KCC2 recycling is enhanced by protein kinase C-mediated phosphorylation of the GluK2 C-terminal residues Ser-846 and Ser-868. Lastly, using gramicidin-perforated patch clamp recordings, we found that the GluK2-mediated increase in KCC2 recycling to the surface membrane translates to a hyperpolarization of the reversal potential for GABA (E GABA ). In conclusion, our results have revealed a mechanism by which kainate receptors regulate KCC2 expression in the hippocampus.The classic fast hyperpolarizing inhibition of the mature brain results primarily from the activation of GABA A receptors.These receptors are Cl Ϫ -permeable ion channels, and inhibition results from the influx of Cl Ϫ into the neuron (1). This inward gradient for Cl Ϫ is set by the neuron-specific K ϩ /Cl Ϫ co-transporter KCC2 (2, 3). Despite the requirement of KCC2 for hyperpolarizing inhibition, the mechanisms that regulate KCC2 expression and function are still under intense investigation. In addition to the critical role KCC2 plays in synaptic inhibition, KCC2 is highly localized to excitatory synapses (4, 5), where it plays important roles in the development (6) and the function of glutamatergic synapses (7,8). In fact, single-particle tracking revealed that KCC2 is tightly confined to excitatory synapses (5), which may result from local protein interactions. Thus, understanding how proteins associated with excitatory synapses regulate KCC2 function may provide critical insight to the function of KCC2.KCC2 exists in a multiprotein complex and is regulated by components of excitatory synaptic transmission (9 -11). Specifically, KCC2 interacts with both the kainate-type ionotropic glutamate receptor subunit GluK2 (9) and its auxiliary subunit Neto2 (10). Neto2 regulates KCC2-mediated Cl Ϫ extrusion by binding to the active oligomeric form of KCC2 (10), whereas the GluK2-KCC2 i...

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Genetically encoded impairment of neuronal KCC 2 cotransporter function in human idiopathic generalized epilepsy

Cited by 166 publications

References 43 publications

WNK1-regulated inhibitory phosphorylation of the KCC2 cotransporter maintains the depolarizing action of GABA in immature neurons

WNK1-regulated inhibitory phosphorylation of the KCC2 cotransporter maintains the depolarizing action of GABA in immature neurons

KCC2 activity is critical in limiting the onset and severity of status epilepticus

A kainate receptor subunit promotes the recycling of the neuron-specific K+-Cl− co-transporter KCC2 in hippocampal neurons

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