Small-molecule screen identifies inhibitors of the neuronal K-Cl cotransporter KCC2

Delpire, Eric; Days, Emily; Lewis, Lundy; Mi, Dehui; Kim, Kwang‐Ho; Lindsley, Craig W.; Weaver, C. David

doi:10.1073/pnas.0812756106

Cited by 132 publications

(125 citation statements)

References 38 publications

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“…A recent, short study has identified that VU0240551, in addition to its inhibitory action on KCC2 (Delpire et al, 2009), might also interfere with G protein-coupled receptors (GPCRs) and ion channels (Delpire et al, 2012). We are however inclined to interpret the effects of this compound identified in our present study as resulting from a decrease in KCC2 activity since similar findings could be obtained with high concentrations of bumetanide (50 μM) that are known to block both KCC2 and NKCC1 (Löscher et al, 2013).…”

Section: Modulation Of Epileptiform Activity By Blocking Kcc2 Activitysupporting

confidence: 66%

KCC2 function modulates in vitro ictogenesis

Hamidi

Avoli

2015

Neurobiology of Disease

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GABA A receptor-mediated inhibition is active and may contribute to epileptiform synchronization. The efficacy of inhibition relies on low levels of intracellular Cl − , which are controlled by KCC2 activity. This evidence has led us to analyze with field potential recordings the effects induced by the KCC2 blockers VU0240551 (10 μM) or bumetanide (50 μM) and by the KCC2 enhancer CLP257 (100 μM) on the epileptiform discharges generated by piriform and entorhinal cortices (PC and EC, respectively) in an in vitro brain slice preparation. Ictal-and interictal-like discharges along with high-frequency oscillations (HFOs, ripples: 80-200 Hz, fast ripples: 250-500 Hz) were recorded from these two regions during application of 4-aminopyridine (4AP, 50 μM). Blocking KCC2 activity with either VU024055 or high doses of bumetanide abolished ictal discharge in both PC and EC; in addition, these experimental procedures decreased the interval of occurrence and duration of interictal discharges. In contrast, enhancing KCC2 activity with CLP257 increased ictal discharge duration in both regions. Finally, blocking KCC2 activity decreased the duration and amplitude of pharmacologically isolated synchronous GABAergic events whereas enhancing KCC2 activity led to an increase in their duration. Our data demonstrate that in vitro ictogenesis is abolished or facilitated by inhibiting or enhancing KCC2 activity, respectively. We propose that these effects may result from the reduction of GABA A receptor-dependent increases in extracellular K + that are known to rest on KCC2 function.

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Section: Modulation Of Epileptiform Activity By Blocking Kcc2 Activitysupporting

confidence: 66%

KCC2 function modulates in vitro ictogenesis

Hamidi

Avoli

2015

Neurobiology of Disease

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“…4C). Importantly, the recovery of hyperpolarizing responses after glutamate exposure was completely blocked by the selective KCC2 inhibitor VU0240551 (21), indicating that our assays measure the KCC2-dependent Cl -extrusion capacity of neurons (Fig. 4B).…”

Section: S940a Mice Exhibit a Selective Deficit In Kcc2 Activity Aftementioning

confidence: 85%

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.

118

137

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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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“…S2). To identify the mechanisms underlying these effects, we used VU0240551 (25 μM), a highly specific KCC2 blocker (23). This compound depolarized E IPSP by >10 mV (Fig.…”

Section: Resultsmentioning

confidence: 99%

Activation of 5-HT2A receptors upregulates the function of the neuronal K-Cl cotransporter KCC2

Bos

Sadlaoud

Boulenguez

et al. 2012

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

155

172

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In healthy adults, activation of γ-aminobutyric acid (GABA) A and glycine receptors inhibits neurons as a result of low intracellular chloride concentration ([Cl -] i ), which is maintained by the potassium-chloride cotransporter KCC2. A reduction of KCC2 expression or function is implicated in the pathogenesis of several neurological disorders, including spasticity and chronic pain following spinal cord injury (SCI). Given the critical role of KCC2 in regulating the strength and robustness of inhibition, identifying tools that may increase KCC2 function and, hence, restore endogenous inhibition in pathological conditions is of particular importance. We show that activation of 5-hydroxytryptamine (5-HT) type 2A receptors to serotonin hyperpolarizes the reversal potential of inhibitory postsynaptic potentials (IPSPs), E IPSP , in spinal motoneurons, increases the cell membrane expression of KCC2 and both restores endogenous inhibition and reduces spasticity after SCI in rats. Up-regulation of KCC2 function by targeting 5-HT 2A receptors, therefore, has therapeutic potential in the treatment of neurological disorders involving altered chloride homeostasis. However, these receptors have been implicated in several psychiatric disorders, and their effects on pain processing are controversial, highlighting the need to further investigate the potential systemic effects of specific 5-HT 2A R agonists, such as (4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide (TCB-2). ] i (depolarizing shift of the chloride equilibrium potential, E Cl ) dramatically compromises the inhibitory control of firing rate and excitatory inputs (5-7). Given the role of KCC2 in regulating the strength of inhibitory synaptic transmission, identifying tools that may increase KCC2 function and, hence, restore endogenous inhibition in pathological conditions is of particular importance.Spasticity is a disabling complication affecting individuals with spinal cord injury (SCI) and is characterized by a velocity-dependent increase in muscle tone resulting from hyperexcitable stretch reflexes, spasms, and hypersensitivity to normally innocuous sensory stimulations (8, 9). Down-regulation of KCC2 after SCI in rats is implicated in the development of spasticity (10) and chronic pain (11,12). Notably, the expression of KCC2 in the motoneuron membrane is reduced, and, concomitantly, the density of cytoplasmic clusters is higher, suggesting that the surface stability of the transporter is reduced in these pathological conditions (10).Mounting evidence indicates that phosphorylation of KCC2 in the C-terminal intracellular domain dynamically regulates its activity and surface expression (1). In particular, phosphorylation by protein kinase (PK)C, enhances KCC2 activity and reduces endocytosis (13). Interestingly, activation of 5-hydroxytryptamine type 2 receptors (5-HT 2 Rs) to serotonin stimulates PKC and strengthens the left-right alternation of motor bursts observed during locomotion (14-16), which rely on reciprocal inhibition (17, 18). ...

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Small-molecule screen identifies inhibitors of the neuronal K-Cl cotransporter KCC2

Cited by 132 publications

References 38 publications

KCC2 function modulates in vitro ictogenesis

KCC2 function modulates in vitro ictogenesis

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

Activation of 5-HT2A receptors upregulates the function of the neuronal K-Cl cotransporter KCC2

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