KCC2 Chloride Transport Contributes to the Termination of Ictal Epileptiform Activity (original) (raw)
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Enhanced expression of potassium-chloride cotransporter KCC2 in human temporal lobe epilepsy
Brain Structure and Function, 2015
Synaptic reorganization in the epileptic hippocampus involves altered excitatory and inhibitory transmission besides the rearrangement of dendritic spines, resulting in altered excitability, ion homeostasis, and cell swelling. The potassium-chloride cotransporter-2 (KCC2) is the main chloride extruder in neurons and hence will play a prominent role in determining the polarity of GABA A receptor-mediated chloride currents. In addition, KCC2 also interacts with the actin cytoskeleton which is critical for dendritic spine morphogenesis, and for the maintenance of glutamatergic synapses and cell volume. Using immunocytochemistry, we examined the cellular and subcellular levels of KCC2 in surgically removed hippocampi of temporal lobe epilepsy (TLE) patients and compared them to control human tissue. We also studied the distribution of KCC2 in a pilocarpine mouse model of epilepsy. An overall increase in KCC2-expression was found in epilepsy and confirmed by Western blots. The cellular and subcellular distributions in control mouse and human samples were largely similar; moreover, changes affecting KCC2-expression were also alike in chronic epileptic human and mouse hippocampi. At the subcellular level, we determined the neuronal elements exhibiting enhanced KCC2 expression. In epileptic tissue, staining became more intense in the immunopositive elements detected in control tissue, and profiles with subthreshold expression of KCC2 in control samples became labelled. Positive interneuron somata and dendrites were more numerous in epileptic hippocampi, despite severe interneuron loss. Whether the elevation of KCC2-expression is ultimately a pro-or anticonvulsive change, or both-behaving differently during ictal and interictal states in a context-dependent mannerremains to be established.
Pharmacoresistant epilepsy is a chronic neurological condition in which a basal brain hyperexcitability results in paroxysmal hypersyn-chronous neuronal discharges. Human temporal lobe epilepsy has been associated with dysfunction or loss of the potassium-chloride cotransporter KCC2 in a subset of pyramidal cells in the subiculum, a key structure generating epileptic activities. KCC2 regulates intraneuronal chloride and extracellular potassium levels by extruding both ions. Absence of effective KCC2 may alter the dynamics of chloride and potassium levels during repeated activation of GABAergic synapses due to interneuron activity. In turn, such GABAergic stress may itself affect Cl regulation. Such changes in ionic homeostasis may switch GABAergic signaling from inhibitory to excitatory in affected pyramidal cells and also increase neuronal excitability. Possibly these changes contribute to periodic bursting in pyramidal cells, an essential component in the onset of ictal epileptic events. We tested this hypothesis with a computational model of a subicular network with realistic connectivity. The pyramidal cell model explicitly incorporated the cotransporter KCC2 and its effects on the internal/external chloride and potassium levels. Our network model suggested the loss of KCC2 in a critical number of pyramidal cells increased external potassium and intracellular chloride concentrations leading to seizure-like field potential oscillations. These oscillations included transient discharges leading to ictal-like field events with frequency spectra as in vitro. Restoration of KCC2 function suppressed seizure activity and thus may present a useful therapeutic option. These simulations therefore suggest that reduced KCC2 cotransporter activity alone may underlie the generation of ictal discharges.
Current view on the functional regulation of the neuronal K+-Cl− cotransporter KCC2
Frontiers in Cellular Neuroscience, 2014
In the mammalian central nervous system (CNS), the inhibitory strength of chloride (Cl − )-permeable GABA A and glycine receptors (GABA A R and GlyR) depends on the intracellular Cl − concentration ([Cl − ] i ). Lowering [Cl − ] i enhances inhibition, whereas raising [Cl − ] i facilitates neuronal activity. A neuron's basal level of [Cl − ] i , as well as its Cl − extrusion capacity, is critically dependent on the activity of the electroneutral K + -Cl − cotransporter KCC2, a member of the SLC12 cation-Cl − cotransporter (CCC) family. KCC2 deficiency compromises neuronal migration, formation and the maturation of GABAergic and glutamatergic synaptic connections, and results in network hyperexcitability and seizure activity. Several neurological disorders including multiple epilepsy subtypes, neuropathic pain, and schizophrenia, as well as various insults such as trauma and ischemia, are associated with significant decreases in the Cl − extrusion capacity of KCC2 that result in increases of [Cl − ] i and the subsequent hyperexcitability of neuronal networks. Accordingly, identifying the key upstream molecular mediators governing the functional regulation of KCC2, and modifying these signaling pathways with small molecules, might constitute a novel neurotherapeutic strategy for multiple diseases. Here, we discuss recent advances in the understanding of the mechanisms regulating KCC2 activity, and of the role these mechanisms play in neuronal Cl − homeostasis and GABAergic neurotransmission. As KCC2 mediates electroneutral transport, the experimental recording of its activity constitutes an important research challenge; we therefore also, provide an overview of the different methodological approaches utilized to monitor function of KCC2 in both physiological and pathological conditions.
Cation-Chloride Cotransporters and GABA-ergic Innervation in the Human Epileptic Hippocampus
Epilepsia, 2007
Intracellular chloride concentration, [Cl − ] i , determines the polarity of GABA A -induced neuronal Cl − currents. In neurons, [Cl − ] i is set by the activity of Na + , K + , 2Cl − cotransporters (NKCC) such as NKCC1, which physiologically accumulate Cl − in the cell, and Cl − extruding K + , Cl − cotransporters like KCC2. Alterations in the balance of NKCC1 and KCC2 activity may determine the switch from hyperpolarizing to depolarizing effects of GABA, reported in the subiculum of epileptic patients with hippocampal sclerosis. We studied the expression of NKCC (putative NKCC1) and KCC2 in human normal temporal neocortex by Western blot analysis and in normal and epileptic regions of the subiculum and the hippocampus proper using immunocytochemistry. Western blot analysis revealed NKCC and KCC2 proteins in adult human neocortical membranes similar to those in rat neocortex.
KCC2 activity is critical in limiting the onset and severity of status epilepticus
Proceedings of the National Academy of Sciences, 2015
The K + /Clcotransporter (KCC2) allows adult neurons to maintain low intracellular Cllevels, 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 Clextrusion. 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.
Neuroscience, 2014
The normal function of GABAA receptor-mediated inhibition is governed by several factors, including release of GABA, subunit composition and density of the receptors and in particular by the appropriate ionic gradient. In the human epileptogenic neocortex an impaired chloride (Cl(-)) gradient has been proposed, due to decreases of potassium-coupled chloride transport (KCC2) and voltage-gated Cl(-) channels (ClC). Regarding sodium- and potassium-coupled Cl(-) transport (NKCC1) both up- and downregulations have been proposed. We investigated changes of Cl(-) homeostasis of human and rat neocortical neurons (layer 2/3) with intracellular recordings and iontophoretic Cl(-) loading employing selective compounds. After cessation of iontophoresis, the IPSPA amplitudes of rat neurons recovered with a time constant (τrec) of 6.5s (n=21). In human neurons, τrec averaged 17.8s (n=36; 23 resections). Application of the novel KCC2 blocker VU0240551 (1 μM) caused in rat neurons a reversible prolo...
BioMed Research International, 2019
Chloride (Cl-) homeostasis is an essential process involved in neuronal signalling and cell survival. Inadequate regulation of intracellular Cl-interferes with synaptic signalling and is implicated in several neurological diseases. The main inhibitory neurotransmitter of the central nervous system isγ-aminobutyric acid (GABA). GABA hyperpolarises the membrane potential by activating Cl-permeableGABAAreceptor channels(GABAAR). This process is reliant on Cl-extruder K+-Cl-cotransporter 2 (KCC2), which generates the neuron’s inward, hyperpolarising Cl-gradient. KCC2 is encoded by the fifth member of the solute carrier 12 family (SLC12A5)and has remained a poorly understood component in the development and severity of many neurological diseases for many years. Recent advancements in next-generation sequencing and specific gene targeting, however, have indicated that loss of KCC2 activity is involved in a number of diseases including epilepsy and schizophrenia. It has also been implicate...
Modulation of neuronal activity by phosphorylation of the K–Cl cotransporter KCC2
Trends in Neurosciences, 2013
The K-Cl cotransporter KCC2 establishes the low intraneuronal Cllevels required for the hyperpolarizing inhibitory postsynaptic potentials mediated by ionotropic g-aminobutyric acid receptors (GABA A Rs) and glycine receptors (GlyRs). Decreased KCC2-mediated Clextrusion and impaired hyperpolarizing GABA A R-and/or GlyR-mediated currents have been implicated in epilepsy, neuropathic pain, and spasticity. Recent evidence suggests that the intrinsic ion transport rate, cell surface stability, and plasmalemmal trafficking of KCC2 are rapidly and reversibly modulated by the (de)phosphorylation of critical serine, threonine, and tyrosine residues in the C terminus of this protein. Alterations in KCC2 phosphorylation have been associated with impaired KCC2 function in several neurological diseases. Targeting KCC2 phosphorylation directly or indirectly via upstream regulatory kinases might be a novel strategy to modulate GABA-and/or glycinergic signaling for therapeutic benefit.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2015
GABAA receptors form Cl(-) permeable channels that mediate the majority of fast synaptic inhibition in the brain. The K(+)/Cl(-) cotransporter KCC2 is the main mechanism by which neurons establish low intracellular Cl(-) levels, which is thought to enable GABAergic inhibitory control of neuronal activity. However, the widely used KCC2 inhibitor furosemide is nonselective with antiseizure efficacy in slices and in vivo, leading to a conflicting scheme of how KCC2 influences GABAergic control of neuronal synchronization. Here we used the selective KCC2 inhibitor VU0463271 [N-cyclopropyl-N-(4-methyl-2-thiazolyl)-2-[(6-phenyl-3-pyridazinyl)thio]acetamide] to investigate the influence of KCC2 function. Application of VU0463271 caused a reversible depolarizing shift in EGABA values and increased spiking of cultured hippocampal neurons. Application of VU0463271 to mouse hippocampal slices under low-Mg(2+) conditions induced unremitting recurrent epileptiform discharges. Finally, microinfus...
Implications of the N-Terminal Heterogeneity for the Neuronal K-Cl cotransporter KCC2 Function
Brain research, 2017
The neuron-specific K-Cl cotransporter KCC2 maintains the low intracellular chloride concentration required for the fast hyperpolarizing responses of the inhibitory neurotransmitters γ-aminobutyric acid (GABA) and glycine. The two KCC2 isoforms, KCC2a and KCC2b differ by their N-termini as a result of alternative promoter usage. Whereas the role of KCC2b in mediating the chloride transport is unequivocal, the physiological role of KCC2a in neurons has remained obscure. We show that KCC2a isoform can decrease the intracellular chloride concentration in cultured neurons and attenuate calcium responses evoked by application of the GABAA receptor agonist muscimol. While the biotinylation assay detected both KCC2 isoforms at the cell surface of cultured neurons, KCC2a was not detected at the plasma membrane in immunostainings, suggesting that the N-terminal KCC2a epitope is masked. Confirming this hypothesis, KCC2a surface expression was detected by the C-terminal KCC2pan antibody but no...