Potassium-chloride Cotransporter Research Articles

SNARE-dependent upregulation of potassium chloride co-transporter 2 activity after metabotropic zinc receptor activation in rat cortical neurons in vitro

The major outward chloride transporter in neurons is the potassium chloride co-transporter 2 (KCC2), critical for maintaining an inhibitory reversal potential for GABAA receptor channels. In a recent study, we showed that Zn2+ regulates GABAA reversal potentials in the hippocampus by enhancing the activity of KCC2 through an increase in its surface expression. Zn2+ initiates this process by activating the Gq-coupled metabotropic Zn2+ receptor/G protein-linked receptor 39 (mZnR/GPR39). Here, we first demonstrated that mZnR/GPR39 is functional in cortical neurons in culture, and then tested the hypothesis that the increase in KCC2 activity is mediated through a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent process. We established the presence of functional mZnR in rat cultured cortical neurons by loading cells with a Ca2+ indicator and exposing cells to Zn2+, which triggered consistent Ca2+ responses that were blocked by the Gq antagonist YM-254890, but not by the metabotropic glutamate receptor antagonist (RS)-α-methyl-4-carboxyphenylglycine (MCPG). Importantly, Zn2+ treatment under these conditions did not increase the intracellular concentrations of Zn2+ itself. We then measured KCC2 activity by monitoring both the rate and relative amount of furosemide-sensitive NH4+ influx through the co-transporter using an intracellular pH-sensitive fluorescent indicator. We observed that Zn2+ pretreatment induced a Ca2+-dependent increase in KCC2 activity. The effects of Zn2+ on KCC2 activity were also observed in wild-type mouse cortical neurons in culture, but not in neurons obtained from mZnR/GPR39−/− mice, suggesting that Zn2+ acts through mZnR/GPR39 activation to upregulate KCC2 activity. We next transfected rat cortical neurons with a plasmid encoding botulinum toxin C1 (Botox C1), which cleaves the SNARE proteins syntaxin 1 and synaptosomal-associated protein 25 (SNAP-25). Basal KCC2 activity was similar in both transfected and non-transfected neurons. Non-transfected cells, or cells transfected with marker vector alone, showed a Zn2+-dependent increase in KCC2 activity. In contrast, KCC2 activity in neurons expressing Botox C1 was unchanged by Zn2+. These results suggest that SNARE proteins are necessary for the increased activity of KCC2 after Zn2+ stimulation of mZnR/GPR39.

Regulation of spinal interneuron differentiation by the paracrine action of glycine.

Glycine and γ-aminobutyric acid (GABA) are depolarizing during early development but the purpose is unclear. We tested the effect of altering glycine signaling in zebrafish embryos by overexpressing the potassium-chloride co-transporter type 2 (KCC2) to reverse the chloride gradient or by blocking glycine receptors with strychnine or by selectively knocking down the embryonic glycine receptor (GlyR KD). Using a variety of markers we observed in all three cases a reduction of all types of spinal interneuron populations examined, indicating that glycine modulates their overall differentiation rather than choice of cell fate. Other cell populations (motor, sensory, and glial cells) were unaffected. As glycine appeared to act preceding neural and synaptic development, we examined the bandoneon (beo) mutant in which glycine receptors are functional but not clustered at synapses. Neural populations in beo embryos appeared normal, suggesting a paracrine action of circulating glycine in promoting interneuron differentiation.

K+-Cl-Cotransporter 1 (KCC1) Negatively Regulates NGF-induced Neurite Outgrowth in PC12 Cells

Potassium chloride cotransporters (KCCs) mediate electroneutrally-coupled transport of K⁺ and Cl^-, and play crucial roles in various cell functions including regulation of cell volume and homeostasis of cellular Cl^-content. Four isoforms of KCCs (KCC1, 2, 3, and 4) have been identified. KCC1 is ubiquitously expressed, whereas KCC2 is mainly expressed in neuronal cells of central nervous system. KCC3 is highly expressed in heart, skeletal muscle, kidney, lung and placenta. KCC4 is mainly expressed in epithelial cells. In this study, we investigated roles of KCCs in NGF-induced neurite outgrowth of rat pheochromocytoma PC12 cells. The most abundantly expressed isoform in PC12 cells was KCC1. Inhibition of KCCs using [(dihydronindenyl)oxy] alkanoic acid (DIOA), an inhibitor of KCCs, enhanced the NGF-induced neurite outgrowth of PC12 cells in a dose-dependent manner. Treatment of PC12 cells with NGF significantly decreased mRNA expression of KCC1, whereas other isoforms, KCC2-4, showed no changes in their mRNA expression in response to NGF treatment. Knockdown of KCC1 using small interfering RNA (siRNA) enhanced the NGF-induced neurite outgrowth. These results suggest that KCC1 negatively regulates the NGF-induced neurite outgrowth of PC12 cells.

Dual actions of brain-derived neurotrophic factor on GABAergic transmission in cerebellar Purkinje neurons

The ability to regulate inhibitory synapses is a critical feature of the nervous system and a growing body of evidence indicates that brain-derived neurotrophic factor (BDNF) acutely modulates the efficacy of GABA synaptic transmission. Although the neuronal potassium-chloride cotransporter 2 (KCC2) has been implied in this BDNF-induced ionic plasticity, the reports about actions of BDNF on GABA signaling remain conflicting. Here we show dual effects of BDNF on GABAergic synaptic transmission in Purkinje neurons in rat cerebellar slices. BDNF decreased the amplitude of evoked outward IPSCs postsynaptically. It induced a depolarizing shift in the reversal potential (EIPSC), which reduced the driving force for outward IPSCs. However, in the absence of KCC2 activity, BDNF directly potentiated rather than inhibited GABAA receptor, which was reflected by an increase in the amplitude of outward IPSCs. This action of BDNF coincided with its effect in increasing the amplitude of inward IPSCs. Furthermore, an interaction between GABAA receptor and KCC2 was revealed by co-immunoprecipitation. The effects of BDNF on both GABAA receptor and KCC2 were dependent on TrkB and also activation of cyclin-dependent kinase 5 (Cdk5). However, only the effect of BDNF on KCC2 activity was dependent on a rise of intracellular calcium. Taken together, these data highlight distinct actions of BDNF on KCC2 and GABAA receptor in the regulation of GABAergic synaptic transmission.

ENU Mutagenesis in the Mouse for Identification of Genes Regulating Erythropoiesis: a Mouse Mutant with An Activating Mutation of the KCl Cotransporter, KCC1 Causing Dehydrated Red Cells

Abstract 684Genome-wide phenotype-driven studies have emerged as a powerful tool for the identification of novel genes in a biological process of interest, new functions for existing genes and the establishment of mouse models of human disease. Recent advances in exomic and whole genome sequencing has simplified mutation detection, making genetic screens in the mouse more facile. We have employed a large-scale mutagenesis screen in mice using the chemical mutagen N-ethyl-N-nitrosourea (ENU) to identify genes regulating erythropoiesis. In this dominant screen, G1 progeny were screened at 7 weeks of age by automated hematological analysis for isolated abnormalities of red cell indices (HCT, RCC and MCV) defined as greater than three standard deviations from the wild type population. Here, we describe the RBC10 mouse line, in which homozygotes had mild anemia with microcytosis, target cells, spherocytes, reticulocytosis and increased resistance to osmotic stress. A combination of SSLP and SNP mapping isolated the genetic mutation to a 3.65 Mb region on chromosome 8. Whole genome Next Generation Sequencing of the mapped interval identified an A to T nucleotide substitution within the coding region of the potassium chloride (KCl) co-transporter, Slc12a4 (KCC1), resulting in a non-conservative amino acid substitution from methionine to lysine at position 935 within the C-terminal cytoplasmic domain. KCC1 and KCC3 function are the major erythroid KCl co-transporters that export KCl in response to hypotonic swelling. We hypothesized that the M935K was an activating mutation of KCC1, based upon the microcytosis and resistance to osmotic stress, which contrasts with the macrocytic phenotype of red cells lacking both KCC1 and KCC3. 86Rb efflux assay confirmed increased KCC1 activity of RBC mutant red cells. We propose that the M935K mutation increases activity of KCC1 by preventing phosphorylation of the nearby threonine reside, a critical regulatory target of the serine-threonine kinase, WNK1. Disclosures:No relevant conflicts of interest to declare.

The potassium–chloride cotransporter 2 promotes cervical cancer cell migration and invasion by an ion transport‐independent mechanism

K(+)-Cl(-) cotransporters (KCCs) play a fundamental role in epithelial cell function, both in the context of ionic homeostasis and also in cell morphology, cell division and locomotion. Unlike other ubiquitously expressed KCC isoforms, expression of KCC2 is widely considered to be restricted to neurons, where it is responsible for maintaining a low intracellular chloride concentration to drive hyperpolarising postsynaptic responses to the inhibitory neurotransmitters GABA and glycine. Here we report a novel finding that KCC2 is widely expressed in several human cancer cell lines including the cervical cancer cell line (SiHa). Membrane biotinylation assays and immunostaining showed that endogenous KCC2 is located on the cell membrane of SiHa cells. To elucidate the role of KCC2 in cervical tumuorigenesis, SiHa cells with stable overexpression or knockdown of KCC2 were employed. Overexpression of KCC2 had no significant effect on cell proliferation but dramatically suppressed cell spreading and stress fibre organization, while knockdown of KCC2 showed opposite effects. In addition, insulin-like growth factor 1 (IGF-1)-induced cell migration and invasiveness were significantly increased by overexpression of KCC2. KCC2-induced cell migration and invasion were not dependent on KCC2 transport function since overexpression of an activity-deficient mutant KCC2 still increased IGF-1-induced cell migration and invasion. Moreover, overexpression of KCC2 significantly diminished the number of focal adhesions, while knockdown of KCC2 increased their number. Taken together, our data establish that KCC2 expression and function are not restricted to neurons and that KCC2 serves to increase cervical tumourigenesis via an ion transport-independent mechanism.

Dimethyl sulphoxide addition or withdrawal causes biphasic volume changes and its withdrawal causes t‐system vacuolation in skeletal muscle

Dimethyl sulphoxide addition or withdrawal causes biphasic volume changes and its withdrawal causes t‐system vacuolation in skeletal muscle

Altered expression of GABAA receptors (α4, γ2 subunit), potassium chloride cotransporter 2 and astrogliosis in tremor rat hippocampus

Impaired GABAergic inhibitory neurotransmission plays an essential role in the pathogenesis of epilepsy. GABAA receptor (GABAAR), potassium chloride cotransporter 2 (KCC2) and astrocytes are of particular importance to GABAergic transmission and thus involved in the development of increased seizure susceptibility.The tremor rat (TRM: tm/tm), a genetic mutant discovered in a Kyoto–Wistar colony, can manifest both absence-like seizures and tonic convulsions without any external stimuli. So far, there are no reports that can elucidate the effects of GABAAR (α4, γ2 subunit), KCC2 and astrocytes on TRMs. The present study was undertaken to detect the expressions of GABAAR α4, GABAAR γ2 and KCC2 in TRMs hippocampus at mRNA and protein levels. In this work, mRNA and protein expressions of GABAAR α4 were significantly elevated while GABAAR γ2 and KCC2 were both evidently decreased in TRMs hippocampus by real-time RT-PCR and western blot, respectively. Furthermore, a dramatic elevation of KCC2 protein level was found after cerebroventricular injection with K252a to TRMs than that in the DMSO-treated TRMs. Besides, our present study also demonstrated that GFAP (a major component of astrocyte) immunoreactivity was much more intense in TRMs hippocampal CA1, CA3 and DG regions than that in control group with immnohistochemistry and confocal microscopic analyses. The protein expression of GFAP was also markedly elevated in TRMs hippocampus, suggesting that astrogliosis appeared in the TRM model. These data demonstrate that altered expressions of GABAAR (α4, γ2) and KCC2 and astrogliosis observed in TRMs hippocampus may provide us good therapeutic targets for the treatment of genetic epilepsy.

Transit Defect of Potassium-Chloride Co-transporter 3 Is a Major Pathogenic Mechanism in Hereditary Motor and Sensory Neuropathy with Agenesis of the Corpus Callosum

Missense and protein-truncating mutations of the human potassium-chloride co-transporter 3 gene (KCC3) cause hereditary motor and sensory neuropathy with agenesis of the corpus callosum (HMSN/ACC), which is a severe neurodegenerative disease characterized by axonal dysfunction and neurodevelopmental defects. We previously reported that KCC3-truncating mutations disrupt brain-type creatine kinase-dependent activation of the co-transporter through the loss of its last 140 amino acids. Here, we report a novel and more distal HMSN/ACC-truncating mutation (3402C → T; R1134X) that eliminates only the last 17 residues of the protein. This small truncation disrupts the interaction with brain-type creatine kinase in mammalian cells but also affects plasma membrane localization of the mutant transporter. Although it is not truncated, the previously reported HMSN/ACC-causing 619C → T (R207C) missense mutation also leads to KCC3 loss of function in Xenopus oocyte flux assay. Immunodetection in Xenopus oocytes and in mammalian cultured cells revealed a decreased amount of R207C at the plasma membrane, with significant retention of the mutant proteins in the endoplasmic reticulum. In mammalian cells, curcumin partially corrected these mutant protein mislocalizations, with more protein reaching the plasma membrane. These findings suggest that mis-trafficking of mutant protein is an important pathophysiological feature of HMSN/ACC causative KCC3 mutations.

Frog Oocytes to Unveil the Structure and Supramolecular Organization of Human Transport Proteins

Structural analyses of heterologously expressed mammalian membrane proteins remain a great challenge given that microgram to milligram amounts of correctly folded and highly purified proteins are required. Here, we present a novel method for the expression and affinity purification of recombinant mammalian and in particular human transport proteins in Xenopus laevis frog oocytes. The method was validated for four human and one murine transporter. Negative stain transmission electron microscopy (TEM) and single particle analysis (SPA) of two of these transporters, i.e., the potassium-chloride cotransporter 4 (KCC4) and the aquaporin-1 (AQP1) water channel, revealed the expected quaternary structures within homogeneous preparations, and thus correct protein folding and assembly. This is the first time a cation-chloride cotransporter (SLC12) family member is isolated, and its shape, dimensions, low-resolution structure and oligomeric state determined by TEM, i.e., by a direct method. Finally, we were able to grow 2D crystals of human AQP1. The ability of AQP1 to crystallize was a strong indicator for the structural integrity of the purified recombinant protein. This approach will open the way for the structure determination of many human membrane transporters taking full advantage of the Xenopus laevis oocyte expression system that generally yields robust functional expression.

Perirhinal cortex hyperexcitability in pilocarpine‐treated epileptic rats

The perirhinal cortex (PC), which is heavily connected with several epileptogenic regions of the limbic system such as the entorhinal cortex and amygdala, is involved in the generation and spread of seizures. However, the functional alterations occurring within an epileptic PC network are unknown. Here, we analyzed this issue by using in vitro electrophysiology and immunohistochemistry in brain tissue obtained from pilocarpine-treated epileptic rats and age-matched, nonepileptic controls (NECs). Neurons recorded intracellularly from the PC deep layers in the two experimental groups had similar intrinsic and firing properties and generated spontaneous depolarizing and hyperpolarizing postsynaptic potentials with comparable duration and amplitude. However, spontaneous and stimulus-induced epileptiform discharges were seen with field potential recordings in over one-fifth of pilocarpine-treated slices but never in NEC tissue. These network events were reduced in duration by antagonizing NMDA receptors and abolished by NMDA + non-NMDA glutamatergic receptor antagonists. Pharmacologically isolated isolated inhibitory postsynaptic potentials had reversal potentials for the early GABA(A) receptor-mediated component that were significantly more depolarized in pilocarpine-treated cells. Experiments with a potassium-chloride cotransporter 2 antibody identified, in pilocarpine-treated PC, a significant immunostaining decrease that could not be explained by neuronal loss. However, interneurons expressing parvalbumin and neuropeptide Y were found to be decreased throughout the PC, whereas cholecystokinin-positive cells were diminished in superficial layers. These findings demonstrate synaptic hyperexcitability that is contributed by attenuated inhibition in the PC of pilocarpine-treated epileptic rats and underscore the role of PC networks in temporal lobe epilepsy.

α-Lipoic acid normalizes nociceptive neuronal activity at the spinal cord of diabetic rats

To evaluate the effects of antioxidant treatment of streptozotocin (STZ)-diabetic rats with α-lipoic acid (α-LA) in neuronal and microglial activation at the spinal cord, an important relay station of nociceptive transmission. Because of the role of the potassium chloride co-transporter 2 (KCC2) in neuronal activation at the spinal cord and the influence of microglia in KCC2 expression, we also evaluated the effects of α-LA in KCC2 expression at the spinal cord. Four weeks after STZ injection, the rats received daily intraperitoneal injections of α-LA (100 mg/kg), during 2 weeks. Mechanical nociception was evaluated before and after α-LA treatment. Spinal cords were immunoreacted against 8-OH-dG (marker of oxidative stress damage), Fos (marker of neuronal activation) and CD11b (marker of microglia). KCC2 expression was evaluated by immunohistochemistry and western blotting. Treatment with α-LA decreased the 8-OH-dG and Fos expressions to controls' levels, but did not affect CD11b. Treatment with α-LA alleviated mechanical hyperalgesia and partially corrected KCC2 expression. This study shows that neuronal hyperactivity at the spinal cord of STZ-diabetic rats can be corrected by α-LA, which may account for alleviation of mechanical hyperalgesia. These effects are probably partially mediated by KCC2, but are independent from microglia.

Volume regulation in mammalian skeletal muscle: the role of sodium–potassium–chloride cotransporters during exposure to hypertonic solutions

Controversy exists as to whether mammalian skeletal muscle is capable of volume regulation in response to changes in extracellular osmolarity despite evidence that muscle fibres have the required ion transport mechanisms to transport solute and water in situ. We addressed this issue by studying the ability of skeletal muscle to regulate volume during periods of induced hyperosmotic stress using single, mouse extensor digitorum longus (EDL) muscle fibres and intact muscle (soleus and EDL). Fibres and intact muscles were loaded with the fluorophore, calcein, and the change in muscle fluorescence and width (single fibres only) used as a metric of volume change. We hypothesized that skeletal muscle exposed to increased extracellular osmolarity would elicit initial cellular shrinkage followed by a regulatory volume increase (RVI) with the RVI dependent on the sodium–potassium–chloride cotransporter (NKCC). We found that single fibres exposed to a 35% increase in extracellular osmolarity demonstrated a rapid, initial 27–32% decrease in cell volume followed by a RVI which took 10-20 min and returned cell volume to 90–110% of pre-stimulus values. Within intact muscle, exposure to increased extracellular osmolarity of varying degrees also induced a rapid, initial shrinkage followed by a gradual RVI, with a greater rate of initial cell shrinkage and a longer time for RVI to occur with increasing extracellular tonicities. Furthermore, RVI was significantly faster in slow-twitch soleus than fast-twitch EDL. Pre-treatment of muscle with bumetanide (NKCC inhibitor) or ouabain (Na+,K+-ATPase inhibitor), increased the initial volume loss and impaired the RVI response to increased extracellular osmolarity indicating that the NKCC is a primary contributor to volume regulation in skeletal muscle. It is concluded that mouse skeletal muscle initially loses volume then exhibits a RVI when exposed to increases in extracellular osmolarity. The rate of RVI is dependent on the degree of change in extracellular osmolarity, is muscle specific, and is dependent on the functioning of the NKCC and Na+, K+-ATPase.

Volume regulation and KCl cotransport in reticulocyte populations of sickle and normal red blood cells

The potassium chloride co-transporter (KCC) is a member of the electroneutral cation chloride family of cotransporters found in multiple tissues that are involved in transepithelial ion transport and regulation of intracellular ion content and cell volume. We have shown previously that three of the four KCC genes – KCC1, KCC3, and KCC4 – are expressed in red blood cells (RBC) (Exp. Hem. 33:624, 2005). Functionally, the KCC mediates volume reduction of reticulocytes that establishes the higher cellular hemoglobin concentration (CHC) of mature RBC. KCC activity is higher in reticulocytes and diminishes with age. KCC activity in RBC containing sickle hemoglobin (SS RBC) is elevated compared to normal (AA RBC) in part due to reticulocytosis in SS blood. However, we have demonstrated that SS reticulocytes have abnormal regulation of KCC activity leading to increased CHC upon activation of KCC compared to AA reticulocytes (Blood 104:2954, 2004; Blood 109:1734, 2007). These findings implicate KCC as a factor in the dehydration of SS RBC, which leads to elevated Hb S concentration and enhances Hb S polymerization and hemolysis. Because KCC activity correlates with cell age, standard flux measurements on blood samples with different numbers of reticulocytes or young non-reticulocytes are not comparable. The Advia automated cell counter measures cell volume (MCV) and cellular hemoglobin concentration (CHC) in reticulocytes, an age-defined population of cells, and thus circumvents the problem of variable reticulocyte counts among SS and AA blood samples. In this study, reticulocyte CHC measurements on fresh blood demonstrated a clear difference between AA and SS cells, reflecting in vivo dehydration of SS reticulocytes, although there was significant inter-individual variation, and the CHC distributions of the two groups overlapped. After KCC activation in vitro by cell swelling using the nystatin method, the initial changes in reticulocyte MCV and CHC with time were used to estimate flux rates mediated by KCC, assuming that changes were associated with isotonic KCl movements. After 20–30 min a final steady state MCV/CHC (set point) was achieved and maintained, reflecting inactivation of the transporter. CHC set points were 26.5–29 g/dl in SS reticulocytes compared to 25–26.5 g/dl in AA reticulocytes, reflecting abnormal regulation in SS cells. These results were reproducible in the same individual over time. KCC flux derived from CHC ranged from 5 to 10.3 mmolK/kgHb/min in SS reticulocytes, compared to 2.9–7.2 mmolK/kgHb/min in AA reticulocytes. Such measures of KCC activity in red cell populations controlled for cell age will facilitate further studies correlating KCC activity with phenotypic or genetic variability in sickle cell disease.

Knocking down of the KCC2 in rat hippocampal neurons increases intracellular chloride concentration and compromises neuronal survival

KCC2 is a neuron-specific potassium-chloride co-transporter controlling intracellular chloride homeostasis in mature and developing neurons. It is implicated in the regulation of neuronal migration, dendrites outgrowth and formation of the excitatory and inhibitory synaptic connections. The function of KCC2 is suppressed under several pathological conditions including neuronal trauma, different types of epilepsies, axotomy of motoneurons, neuronal inflammations and ischaemic insults. However, it remains unclear how down-regulation of the KCC2 contributes to neuronal survival during and after toxic stress. Here we show that in primary hippocampal neuronal cultures the suppression of the KCC2 function using two different shRNAs, dominant-negative KCC2 mutant C568A or DIOA inhibitor, increased the intracellular chloride concentration [Cl⁻]i and enhanced the toxicity induced by lipofectamine-dependent oxidative stress or activation of the NMDA receptors. The rescuing of the KCC2 activity using over-expression of the active form of the KCC2, but not its non-active mutant Y1087D, effectively restored [Cl⁻]i and enhanced neuronal resistance to excitotoxicity. The reparative effects of KCC2 were mimicked by over-expression of the KCC3, a homologue transporter. These data suggest an important role of KCC2-dependent potassium/chloride homeostasis under neurototoxic conditions and reveal a novel role of endogenous KCC2 as a neuroprotective molecule.

Neuronal chloride accumulation and excitatory GABA underlie aggravation of neonatal epileptiform activities by phenobarbital

Phenobarbital produces its anti-epileptic actions by increasing the inhibitory drive of γ-aminobutyric acid. However, following recurrent seizures, γ-aminobutyric acid excites neurons because of a persistent increase of chloride raising the important issue of whether phenobarbital could aggravate persistent seizures. Here we compared the actions of phenobarbital on initial and established ictal-like events in an in vitro model of mirror focus. Using the in vitro three-compartment chamber preparation with the two hippocampi and their commissural fibres placed in three different chambers, kainate was applied to one hippocampus and phenobarbital contralaterally, either after one ictal-like event or after many recurrent ictal-like events that produce an epileptogenic mirror focus. Field, perforated patch and single-channel recordings were used to determine the effects of γ-aminobutyric acid and their modulation by phenobarbital, and alterations of the chloride cotransporters were investigated using sodium-potassium-chloride cotransporter 1 and potassium chloride cotransporter 2 antagonists, potassium chloride cotransporter 2 immunocytochemistry and sodium-potassium-chloride cotransporter 1 knockouts. Phenobarbital reduced initial ictal-like events and prevented the formation of a mirror focus when applied from the start. In contrast, phenobarbital aggravated epileptiform activities when applied after many ictal-like events by enhancing the excitatory actions of γ-aminobutyric acid due to increased chloride. The accumulation of chloride and the excitatory actions of γ-aminobutyric acid in mirror foci neurons are mediated by the sodium-potassium-chloride cotransporter 1 chloride importer and by downregulation and internalization of the chloride-exporter potassium-chloride cotransporter 2. Finally, concomitant applications of the sodium-potassium-chloride cotransporter 1 antagonist bumetanide and phenobarbital decreased excitatory actions of γ-aminobutyric acid and prevented its paradoxical actions on mirror focus. Therefore, the history of seizures prior to phenobarbital applications determines its effects and rapid treatment of severe potentially epileptogenic-neonatal seizures is recommended to prevent secondary epileptogenesis associated with potassium chloride cotransporter 2 downregulation and acquisition of the excitatory γ-aminobutyric acid phenotype.

NKCC1 cotransporters: keeping an ‘ion’ them

NKCC1 cotransporters: keeping an ‘ion’ them

Contribution of the potassium-chloride co-transporter KCC2 to the modulation of lumbar spinal networks in mice

Spontaneous activity is observed in most developing neuronal circuits, such as the retina, hippocampus, brainstem and spinal cord. In the spinal cord, spontaneous activity is important for generating embryonic movements critical for the proper development of motor axons, muscles and synaptic connections. A spontaneous bursting activity can be recorded in vitro from ventral roots during perinatal development. The depolarizing action of the inhibitory amino acids γ-aminobutyric acid and glycine is widely proposed to contribute to spontaneous activity in several immature systems. During development, the intracellular chloride concentration decreases, leading to a shift of equilibrium potential for Cl(-) ions towards more negative values, and thereby to a change in glycine- and γ-aminobutyric acid-evoked potentials from depolarization/excitation to hyperpolarization/inhibition. The up-regulation of the outward-directed Cl(-) pump, the neuron-specific potassium-chloride co-transporter type 2 KCC2, has been shown to underlie this shift. Here, we investigated whether spontaneous and locomotor-like activities are altered in genetically modified mice that express only 8-20% of KCC2, compared with wild-type animals. We show that a reduced amount of KCC2 leads to a depolarized equilibrium potential for Cl(-) ions in lumbar motoneurons, an increased spontaneous activity and a faster locomotor-like activity. However, the left-right and flexor-extensor alternating pattern observed during fictive locomotion was not affected. We conclude that neuronal networks within the spinal cord are more excitable in KCC2 mutant mice, which suggests that KCC2 strongly modulates the excitability of spinal cord networks.

Early Growth Response 4 Mediates BDNF Induction of Potassium Chloride Cotransporter 2 Transcription

A major event in the maturation of CNS GABAergic transmission is the qualitative change in GABA(A)-mediated responses from depolarizing to hyperpolarizing. In cortical regions, this is attributed to the increased expression of potassium chloride cotransporter 2b (KCC2b), the main isoform of the neuron-specific K-Cl cotransporter KCC2. We have previously shown that transcription factor early growth response 4 (Egr4) can activate the KCC2b promoter. Here we demonstrate that in immature hippocampal neurons BDNF robustly induces ERK1/2 (extracellular signal-regulated kinase 1/2)-dependent Egr4 expression and rapid Egr4-dependent activation of the KCC2b promoter. The subsequent increase in KCC2b mRNA contributes to the expression of total KCC2 protein levels. These results indicate that Egr4 is an important component in the mechanism of BDNF-dependent KCC2 gene regulation via the ERK1/2 pathway in immature neurons.

Enhanced synaptic activity and epileptiform events in the embryonic KCC2 deficient hippocampus

The neuronal potassium-chloride co-transporter 2 [indicated thereafter as KCC2 (for protein) and Kcc2 (for gene)] is thought to play an important role in the post natal excitatory to inhibitory switch of GABA actions in the rodent hippocampus. Here, by studying hippocampi of wild-type (Kcc2+/+) and Kcc2 deficient (Kcc2−/−) mouse embryos, we unexpectedly found increased spontaneous neuronal network activity at E18.5, a developmental stage when KCC2 is thought not to be functional in the hippocampus. Embryonic Kcc2−/− hippocampi have also an augmented synapse density and a higher frequency of spontaneous glutamatergic and GABA-ergic postsynaptic currents than naïve age matched neurons. However, intracellular chloride concentration ([Cl−]i) and the reversal potential of GABA-mediated currents (EGABA) were similar in embryonic Kcc2+/+ and Kcc2−/− CA3 neurons. In addition, KCC2 immunolabeling was cytoplasmic in the majority of neurons suggesting that the molecule is not functional as a plasma membrane chloride co-transporter. Collectively, our results show that already at an embryonic stage, KCC2 controls the formation of synapses and, when deleted, the hippocampus has a higher density of GABA-ergic and glutamatergic synapses and generates spontaneous and evoked epileptiform activities. These results may be explained either by a small population of orchestrating neurons in which KCC2 operates early as a chloride exporter or by transporter independent actions of KCC2 that are instrumental in synapse formation and networks construction.