Neuronal Chloride Homeostasis Research Articles

A role for intracellular zinc in glioma alteration of neuronal chloride equilibrium.

Glioma patients commonly suffer from epileptic seizures. However, the mechanisms of glioma-associated epilepsy are far to be completely understood. Using glioma-neurons co-cultures, we found that tumor cells are able to deeply influence neuronal chloride homeostasis, by depolarizing the reversal potential of γ-aminobutyric acid (GABA)-evoked currents (EGABA). EGABA depolarizing shift is due to zinc-dependent reduction of neuronal KCC2 activity and requires glutamate release from glioma cells. Consistently, intracellular zinc loading rapidly depolarizes EGABA in mouse hippocampal neurons, through the Src/Trk pathway and this effect is promptly reverted upon zinc chelation. This study provides a possible molecular mechanism linking glioma invasion to excitation/inhibition imbalance and epileptic seizures, through the zinc–mediated disruption of neuronal chloride homeostasis.

Kainate Receptors Coexist in a Functional Complex with KCC2 and Regulate Chloride Homeostasis in Hippocampal Neurons

SUMMARYKCC2 is the neuron-specific K+-Cl− cotransporter required for maintaining low intracellular Cl−, which is essential for fast inhibitory synaptic transmission in the mature CNS. Despite the requirement of KCC2 for inhibitory synaptic transmission, understanding of the cellular mechanisms that regulate KCC2 expression and function is rudimentary. We examined KCC2 in its native protein complex in vivo to identify key KCC2-interacting partners that regulate KCC2 function. Using blue native-polyacrylamide gel electrophoresis (BN-PAGE), we determined that native KCC2 exists in a macromolecular complex with kainate-type glutamate receptors (KARs). We found that KAR subunits are required for KCC2 oligomerization and surface expression. In accordance with this finding, acute and chronic genetic deletion of KARs decreased KCC2 function and weakened synaptic inhibition in hippocampal neurons. Our results reveal KARs as regulators of KCC2, significantly advancing our growing understanding of the tight interplay between excitation and inhibition.

Commentary: Morris-Coole/Epilepsia prize.

Commentary: Morris-Coole/Epilepsia prize.

Star players sidelined in chloride homeostasis in neurons.

Star players sidelined in chloride homeostasis in neurons.

Activity-Dependent Regulation of the K/Cl Transporter KCC2 Membrane Diffusion, Clustering, and Function in Hippocampal Neurons

The neuronal K/Cl transporter KCC2 exports chloride ions and thereby influences the efficacy and polarity of GABA signaling in the brain. KCC2 is also critical for dendritic spine morphogenesis and the maintenance of glutamatergic transmission in cortical neurons. Because KCC2 plays a pivotal role in the function of central synapses, it is of particular importance to understand the cellular and molecular mechanisms underlying its regulation. Here, we studied the impact of membrane diffusion and clustering on KCC2 function. KCC2 forms clusters in the vicinity of both excitatory and inhibitory synapses. Using quantum-dot-based single-particle tracking on rat primary hippocampal neurons, we show that KCC2 is slowed down and confined at excitatory and inhibitory synapses compared with extrasynaptic regions. However, KCC2 escapes inhibitory synapses faster than excitatory synapses, reflecting stronger molecular constraints at the latter. Interfering with KCC2-actin interactions or inhibiting F-actin polymerization releases diffusion constraints on KCC2 at excitatory but not inhibitory synapses. Thus, F-actin constrains KCC2 diffusion at excitatory synapses, whereas KCC2 is confined at inhibitory synapses by a distinct mechanism. Finally, increased neuronal activity rapidly increases the diffusion coefficient and decreases the dwell time of KCC2 at excitatory synapses. This effect involves NMDAR activation, Ca(2+) influx, KCC2 S940 dephosphorylation and calpain protease cleavage of KCC2 and is accompanied by reduced KCC2 clustering and ion transport function. Thus, activity-dependent regulation of KCC2 lateral diffusion and clustering allows for a rapid regulation of chloride homeostasis in neurons.

Pre- and post-synaptic switches of GABA actions associated with Cl− homeostatic changes are induced in the spinal nucleus of the trigeminal nerve in a rat model of trigeminal neuropathic pain

Although trigeminal neuropathic pain is one of the most common chronic pain syndromes, the etiology is still unknown. Here, a rat model was generated using chronic constrictive injury (CCI) with ligation of the infraorbital nerve to test the hypothesis that collapse of chloride homeostasis in trigeminal neurons causes impairment of γ-aminobutyric acid-ergic (GABAergic) inhibition and induces trigeminal allodynia. Rats showed a reduction and increase in pain threshold and pain response scores, respectively, to mechanical stimulation, 1 and 3weeks after CCI. In situ hybridization and immunohistochemical analysis showed that inward-directed Na+, K+–2Cl− cotransporter (NKCC1) mRNA and protein were upregulated in the small-sized and large-sized primary neurons in the injured side of the trigeminal ganglion and in the peripherin-positive terminal, respectively, for the first 2weeks, while outward-directed K+–Cl− cotransporter (KCC2) mRNA and protein were downregulated in secondary relay neurons on the injured side of the spinal trigeminal nucleus caudalis (Sp5C). Optical imaging of evoked synaptic responses using a voltage-sensitive dye revealed that pre- and post-synaptic GABA actions were disinhibited and excitatory in the injured side, respectively, but inhibited in the sham-operated side of the Sp5C. This downregulation of KCC2 in the Sp5C may result in an excitatory switch by impairing postsynaptic GABA inhibition. GABA-mediated presynaptic disinhibition was attenuated by bumetanide, suggesting that NKCC1 upregulation in primary neurons may facilitate pain transmission by presynaptic GABAergic depolarization. Such Cl− homeostatic disruption resulting in perturbation of the inhibitory system possibly increases pain transmission, which may underlie the pathophysiology of trigeminal neuropathic pain.

The neuronal K-Cl cotransporter KCC2 influences postsynaptic AMPA receptor content and lateral diffusion in dendritic spines

The K-Cl cotransporter KCC2 plays an essential role in neuronal chloride homeostasis, and thereby influences the efficacy and polarity of GABA signaling. Although KCC2 is expressed throughout the somatodendritic membrane, it is remarkably enriched in dendritic spines, which host most glutamatergic synapses in cortical neurons. KCC2 has been shown to influence spine morphogenesis and functional maturation in developing neurons, but its function in mature dendritic spines remains unknown. Here, we report that suppressing KCC2 expression decreases the efficacy of excitatory synapses in mature hippocampal neurons. This effect correlates with a reduced postsynaptic aggregation of GluR1-containing AMPA receptors and is mimicked by a dominant negative mutant of KCC2 interaction with cytoskeleton but not by pharmacological suppression of KCC2 function. Single-particle tracking experiments reveal that suppressing KCC2 increases lateral diffusion of the mobile fraction of AMPA receptor subunit GluR1 in spines but not in adjacent dendritic shafts. Increased diffusion was also observed for transmembrane but not membrane-anchored recombinant neuronal cell adhesion molecules. We suggest that KCC2, likely through interactions with the actin cytoskeleton, hinders transmembrane protein diffusion, and thereby contributes to their confinement within dendritic spines.

Synaptic Conversion of Chloride-Dependent Synapses in Spinal Nociceptive Circuits: Roles in Neuropathic Pain

Electrophysiological conversion of chloride-dependent synapses from inhibitory to excitatory function, as a result of aberrant neuronal chloride homeostasis, is a known mechanism for the genesis of neuropathic pain. This paper examines theoretically how this type of synaptic conversion can disrupt circuit logic in spinal nociceptive circuits. First, a mathematical scaling factor is developed to represent local aberration in chloride electrochemical driving potential. Using this mathematical scaling factor, electrophysiological symbols are developed to represent the magnitude of synaptic conversion within nociceptive circuits. When inserted into a nociceptive circuit diagram, these symbols assist in understanding the generation of neuropathic pain associated with the collapse of transmembrane chloride gradients. A more generalized scaling factor is also derived to represent the interplay of chloride and bicarbonate driving potentials on the function of GABAergic and glycinergic synapses. These mathematical and symbolic representations of synaptic conversion help illustrate the critical role that anion driving potentials play in the transduction of pain. Using these representations, we discuss ramifications of glial-mediated synaptic conversion in the genesis, and treatment, of neuropathic pain.

Disease-modifying effects of phenobarbital and the NKCC1 inhibitor bumetanide in the pilocarpine model of temporal lobe epilepsy.

Accumulating evidence suggests that changes in neuronal chloride homeostasis may be involved in the mechanisms by which brain insults induce the development of epilepsy. A variety of brain insults, including status epilepticus (SE), lead to changes in the expression of the cation-chloride cotransporters KCC2 and NKCC1, resulting in intracellular chloride accumulation and reappearance of immature, depolarizing synaptic responses to GABA(A) receptor activation, which may critically contribute to the neuronal hyperexcitability underlying epileptogenesis. In the present study, it was evaluated whether prolonged administration of the selective NKCC1 inhibitor, bumetanide, after a pilocarpine-induced SE modifies the development of epilepsy in adult female rats. The antiepileptic drug phenobarbital, either alone or in combination, was used for comparison. Based on pharmacokinetic studies with bumetanide, which showed extremely rapid elimination and low brain penetration of this drug in rats, bumetanide was administered systemically with different dosing protocols, including continuous intravenous infusion. As shown by immunohistochemistry, neuronal NKCC1 expression was markedly upregulated shortly after SE. Prophylactic treatment with phenobarbital after SE reduced the number of rats developing spontaneous seizures and decreased seizure frequency, indicating a disease-modifying effect. Bumetanide did not exert any significant effects on development of spontaneous seizures nor did it enhance the effects of phenobarbital. However, combined treatment with both drugs counteracted several of the behavioral consequences of SE, which was not observed with single drug treatment. These data do not indicate that bumetanide can prevent epilepsy after SE, but the disease-modifying effect of this drug warrants further studies with more lipophilic prodrugs of bumetanide.

Gating of human ClC‐2 chloride channels and regulation by carboxy‐terminal domains

Eukaryotic ClC channels are dimeric proteins with each subunit forming an individual protopore. Single protopores are gated by a fast gate, whereas the slow gate is assumed to control both protopores through a cooperative movement of the two carboxy-terminal domains. We here study the role of the carboxy-terminal domain in modulating fast and slow gating of human ClC-2 channels, a ubiquitously expressed ClC-type chloride channel involved in transepithelial solute transport and in neuronal chloride homeostasis. Partial truncation of the carboxy-terminus abolishes function of ClC-2 by locking the channel in a closed position. However, unlike other isoforms, its complete removal preserves function of ClC-2. ClC-2 channels without the carboxy-terminus exhibit fast and slow gates that activate and deactivate significantly faster than in WT channels. In contrast to the prevalent view, a single carboxy-terminus suffices for normal slow gating, whereas both domains regulate fast gating of individual protopores. Our findings demonstrate that the carboxy-terminus is not strictly required for slow gating and that the cooperative gating resides in other regions of the channel protein. ClC-2 is expressed in neurons and believed to open at negative potentials and increased internal chloride concentrations after intense synaptic activity. We propose that the function of the ClC-2 carboxy-terminus is to slow down the time course of channel activation in order to stabilize neuronal excitability.

Development of chloride homeostasis in albino and pigmented rat visual cortex neurons

Albinism has a profound effect on visual development and visual function. Pharmacologically significant alterations of the two most important chloride-transporters--KCC2 (outward transporter) and NKCC1 (inward transporter)--functions were found in albino visual cortex neurons, comprising a higher NKCC1 and a lower KCC2 action. In this study, we compare the early postnatal development of the reversal potential of gamma-aminobutyric acidAR-mediated currents in visual cortex neurons of albino and pigmented rats. At birth we found no differences. At the time of eye opening (second week postnatally) the reversal potential of gamma-aminobutyric acidAR-mediated currents is 15 mV more positive and intracellular Cl- concentration is higher in visual cortex neurons of albinos than of pigmented rats.

Differential maturation of chloride homeostasis in primary afferent neurons of the somatosensory system

Recent research into the generation of hyperalgesia has revealed that both the excitability of peripheral nociceptors and the transmission of their afferent signals in the spinal cord are subject to modulation by Cl− currents. The underlying Cl− homeostasis of nociceptive neurons, in particular its postnatal maturation, is, however, poorly understood. Here we measure the intracellular Cl− concentration, [Cl−]i, of somatosensory neurons in intact dorsal root ganglia of mice. Using two-photon fluorescence-lifetime imaging microscopy, we determined [Cl−]i in newborn and adult animals. We found that the somatosensory neurons undergo a transition of Cl− homeostasis during the first three postnatal weeks that leads to a decline of [Cl−]i in most neurons. Immunohistochemistry showed that a major fraction of neurons in the dorsal root ganglia express the cation–chloride co-transporters NKCC1 and KCC2, indicating that the molecular equipment for Cl− accumulation and extrusion is present. RT-PCR analysis showed that the transcription pattern of electroneutral Cl− co-transporters does not change during the maturation process.These findings demonstrate that dorsal root ganglion neurons undergo a developmental transition of chloride homeostasis during the first three postnatal weeks. This process parallels the developmental “chloride switch” in the central nervous system. However, while most CNS neurons achieve homogeneously low [Cl−]i levels – which is the basis of GABAergic and glycinergic inhibition – somatosensory neurons maintain a heterogeneous pattern of [Cl−]i values. This suggests that Cl− currents are excitatory in some somatosensory neurons, but inhibitory in others. Our results are consistent with the hypothesis that Cl− homeostasis in somatosensory neurons is regulated through posttranslational modification of cation–chloride co-transporters.

Quaternary structure of the chloride channel ClC-2.

The chloride channel ClC-2 is thought to be essential for chloride homeostasis in neurons and critical for chloride secretion by the developing respiratory tract. In the present work, we investigated the quaternary structure of ClC-2 required to mediate chloride conduction. We found using chemical cross-linking and a novel PAGE system that tagged ClC-2 expressed in Sf9 cells exists as oligomers. Fusion of membranes from Sf9 cells expressing this protein confers double-barreled channel activity, with each pore exhibiting a unitary conductance of 32 pS. Polyhistidine-tagged ClC-2 from Sf9 cells can be purified as monomers, dimers, and tetramers. Purified, reconstituted ClC-2 monomers do not possess channel function whereas both purified ClC-2 dimers and tetramers do mediate chloride flux. In planar bilayers, reconstitution of dimeric ClC-2 leads to the appearance of a single, anion selective 32 pS pore, and tetrameric ClC-2 confers double-barreled channel activity similar to that observed in Sf9 membranes. These reconstitution studies suggest that a ClC-2 dimer is the minimum functional structure and that ClC-2 tetramers likely mediate double-barreled channel function.

Developmental expression of ClC-2 in the rat nervous system

Regulation of expression of the voltage-gated chloride channel, ClC-2, was investigated during development and adult life in rat brain. RNase protection assays demonstrated a marked increase in levels of expression of ClC-2 in brain during early postnatal development which was also detected in adult brain. In situ hybridization of E15 and E18 rat brains demonstrated ClC-2 expression in deep brain nuclei and scattered cells within the neuroepithelial layers, but not in the regions of subventricular zone that primarily give rise to glial populations. By E18 all neurons within the emerging cortical plate and its equivalent in other areas of the CNS were heavily labeled. During the first postnatal week, ClC-2 was highly expressed in most neurons. By P7 a pattern of differential expression emerged with evidence of decreased expression of ClC-2 mRNA in many neuronal populations. In adult rat brain, ClC-2 was expressed at highest levels in large neurons as found within layer V of cortex, Ammon's Horn of hippocampus, or mitral cells of the olfactory bulb and Purkinje cells within the cerebellum. Many smaller neurons within the diencephalon maintained significant levels of expression. A functional conductance was readily detected in hippocampal neurons during the first postnatal week, which had the same characteristic properties as the conductance observed in adult neurons. The observed expression and functional presence of ClC-2 suggest a widespread role in neuronal chloride homeostasis in early postnatal life, and demonstrated that cell specific shut-down resulted in the adult pattern of expression.