Cation-chloride Cotransporters Research Articles

Effects of α7 Nicotinic Acetylcholine Receptor Positive Allosteric Modulator on BDNF, NKCC1 and KCC2 Expression in the Hippocampus following Lipopolysaccharide-Induced Allodynia and Hyperalgesia in a Mouse Model of Inflammatory Pain.

Hyperalgesia and allodynia are frequent symptoms of inflammatory pain. Neuronal excitability induced by the Brain-Derived Neurotrophic Factor (BDNF)-tyrosine receptor kinase B (TrkB) cascade has a role in the modulation of inflammatory pain. The effects of 3a,4,5,9b-tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c]quinoline-8-sulfonamide (TQS), an α7 nicotinic Acetylcholine Receptor Positive Allosteric Modulator (nAChR PAM), on hippocampal BDNF, cation-chloride cotransporters, NKCC1 and KCC2, expression in inflammatory pain are not known. The objective of the study was to determine the effects of TQS on BDNF, NKCC1, and KCC2 expression in the hippocampus following lipopolysaccharide (LPS)-induced allodynia and hyperalgesia in a mouse model of inflammatory pain. Mice were treated with TQS followed by LPS (1 mg/kg, ip) administration. The effects of TQS on mRNA and BDNF in the hippocampus were examined using qRT-PCR and Western blot, respectively. Immunoreactivity of BDNF, NKCC1, and KCC2 in the hippocampus was measured after LPS administration using immunofluorescence assay. Allodynia and hyperalgesia were determined using von Frey filaments and hot plate, respectively. The LPS (1 mg/kg) upregulates mRNA of BDNF and downregulates mRNA of KCC2 in the hippocampus and pretreatment of TQS (4 mg/kg) reversed the effects induced by LPS. In addition, the TQS decreased LPS-induced upregulation of BDNF and p-NKCC1 immunoreactivity in the dentate gyrus and CA1 region of the hippocampus. BDNF receptor (TrkB) antagonist, ANA12 (0.50 mg/kg), and NKCC1 inhibitor bumetanide (30 mg/kg) reduced LPS-induced allodynia and hyperalgesia. Blockade of TrkB with ANA12 (0.25 mg/kg) enhanced the effects of TQS (1 mg/kg) against LPS-induced allodynia and hyperalgesia. Similarly, bumetanide (10 mg/kg) enhanced the effects of TQS (1 mg/kg) against allodynia and hyperalgesia. These results suggest that antinociceptive effects of α7 nAChR PAM are associated with downregulation of hippocampal BDNF and p-NKCC1 and upregulation of KCC2 in a mouse model of inflammatory pain.

The Chloride Homeostasis of CA3 Hippocampal Neurons Is Not Altered in Fully Symptomatic Mepc2-null Mice.

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused mainly by mutations in the MECP2 gene. Mouse models of RTT show reduced expression of the cation-chloride cotransporter KCC2 and altered chloride homeostasis at presymptomatic stages. However, whether these alterations persist to late symptomatic stages has not been studied. Here we assess KCC2 and NKCC1 expressions and chloride homeostasis in the hippocampus of early [postnatal (P) day 30–35] and late (P50–60) symptomatic male Mecp2-null (Mecp2–/y) mice. We found (i) no difference in the relative amount, but an over-phosphorylation, of KCC2 and NKCC1 between wild-type (WT) and Mecp2–/y hippocampi and (ii) no difference in the inhibitory strength, nor reversal potential, of GABAA-receptor-mediated responses in Mecp2–/y CA3 pyramidal neurons compared to WT at any stages studied. Altogether, these data indicate the presence of a functional chloride extrusion mechanism in Mecp2–/y CA3 pyramidal neurons at symptomatic stages.

Melatonin modulates nitric oxide-regulated WNK-SPAK/OSR1-NKCC1 signaling in dorsal raphe nucleus of rats.

The sleep-wake cycle is regulated by the alternating activity of sleep- and wake-promoting neurons. The dorsal raphe nucleus (DRN) secretes 5-hydroxytryptamine (5-HT, serotonin), promoting wakefulness. Melatonin secreted from the pineal gland also promotes wakefulness in rats. Our laboratory recently demonstrated that daily changes in nitric oxide (NO) production regulates a signaling pathway involving with-no-lysine kinase (WNK), Ste20-related proline alanine rich kinase (SPAK)/oxidative stress response kinase 1 (OSR1), and cation-chloride co-transporters (CCC) in rat DRN serotonergic neurons. This study was designed to investigate the effect of melatonin on NO-regulated WNK-SPAK/OSR1-CCC signaling in wake-inducing DRN neurons to elucidate the mechanism underlying melatonin’s wake-promoting actions in rats. Ex vivo treatment of DRN slices with melatonin suppressed neuronal nitric oxide synthase (nNOS) expression and increased WNK4 expression without altering WNK1, 2, or 3. Melatonin increased phosphorylation of OSR1 and the expression of sodium-potassium-chloride co-transporter 1 (NKCC1), while potassium-chloride co-transporter 2 (KCC2) remained unchanged. Melatonin increased the expression of tryptophan hydroxylase 2 (TPH2, serotonin-synthesizing enzyme). The present study suggests that melatonin may promote its wakefulness by modulating NO-regulated WNK-SPAK/OSR1-KNCC1 signaling in rat DRN serotonergic neurons.

Cation chloride cotransporter modulation of the seizure phenotype in rat cortical MEA

Cation chloride cotransporter modulation of the seizure phenotype in rat cortical MEA

CCT and CCT-Like Modular Protein Interaction Domains in WNK Signaling.

The WNK [with no lysine (K)] kinases and their downstream effector kinases, oxidative stress responsive 1 (OSR1) and SPS/STE20-related proline-alanine-rich kinase (SPAK), have well established functions in the maintenance of cell volume and ion homeostasis. Mutations in these kinases have been linked to an inherited form of hypertension, neurologic defects, and other pathologies. A rapidly expanding body of evidence points to the involvement of WNKs in regulating multiple diverse cellular processes as well as the progression of some forms of cancer. How OSR1 and SPAK contribute to these processes is well understood in some cases but completely unknown in others. OSR1 and SPAK are targeted to both WNKs and substrates via their conserved C-terminal (CCT) protein interaction domains. Considerable effort has been put forth to understand the structure, function, and interaction specificity of the CCT domains in relation to WNK signaling, and multiple inhibitors of WNK signaling target these domains. The domains bind RFxV and RxFxV protein sequence motifs with the consensus sequence R-F-x-V/I or R-x-F-x-V/I, but residues outside the core motif also contribute to specificity. CCT interactions are required for OSR1 and SPAK activation and deactivation as well as cation-chloride cotransporter substrate phosphorylation. All four WNKs also contain CCT-like domains that have similar structures and conserved binding residues when compared with CCT domains, but their functions and interaction specificities are mostly unknown. A better understanding of the varied actions of these domains and their interactions will better define the known signaling mechanisms of the WNK pathway as well as uncover new ones. SIGNIFICANCE STATEMENT: WNK [with no lysine (K)] kinases and their downstream effector kinases, oxidative stress responsive 1 (OSR1) and SPS/STE20-related proline-alanine-rich kinase (SPAK), have been shown to be involved in an array of diverse cellular processes. Here we review the function of modular protein interaction domains found in OSR1 and SPAK as well as related domains found in WNKs.

All-Atom Simulations Uncover the Molecular Terms of the NKCC1 Transport Mechanism.

The secondary-active Na-K-Cl cotransporter 1 (NKCC1), member of the cation-chloride cotransporter (CCC) family, ensures the electroneutral movement of Cl-, Na+, and K+ ions across cellular membranes. NKCC1 regulates Cl- homeostasis and cell volume, handling a pivotal role in transepithelial water transport and neuronal excitability. Aberrant NKCC1 transport is hence implicated in a variety of human diseases (hypertension, renal disorders, neuropathies, and cancer). Building on the newly resolved NKCC1 cryo-EM structure, all-atom enhanced sampling simulations unprecedentedly unlock the mechanism of NKCC1-mediated ion transport, assessing the order and the molecular basis of its interdependent ion translocation. Our outcomes strikingly advance the understanding of the physiological mechanism of CCCs and disclose a key role of CCC-conserved asparagine residues, whose side-chain promiscuity ensures the transport of both negatively and positively charged ions along the same translocation route. This study sets a conceptual basis to devise NKCC-selective inhibitors to treat diseases linked to Cl- dishomeostasis.

Unique Actions of GABA Arising from Cytoplasmic Chloride Microdomains.

Developmental, cellular, and subcellular variations in the direction of neuronal Cl- currents elicited by GABAA receptor activation have been frequently reported. We found a corresponding variance in the GABAA receptor reversal potential (EGABA) for synapses originating from individual interneurons onto a single pyramidal cell. These findings suggest a similar heterogeneity in the cytoplasmic intracellular concentration of chloride ([Cl-]i) in individual dendrites. We determined [Cl-]i in the murine hippocampus and cerebral cortex of both sexes by (1) two-photon imaging of the Cl--sensitive, ratiometric fluorescent protein SuperClomeleon; (2) Fluorescence Lifetime IMaging (FLIM) of the Cl--sensitive fluorophore MEQ (6-methoxy-N-ethylquinolinium); and (3) electrophysiological measurements of EGABA by pressure application of GABA and RuBi-GABA uncaging. Fluorometric and electrophysiological estimates of local [Cl-]i were highly correlated. [Cl-]i microdomains persisted after pharmacological inhibition of cation-chloride cotransporters, but were progressively modified after inhibiting the polymerization of the anionic biopolymer actin. These methods collectively demonstrated stable [Cl-]i microdomains in individual neurons in vitro and in vivo and the role of immobile anions in its stability. Our results highlight the existence of functionally significant neuronal Cl- microdomains that modify the impact of GABAergic inputs.SIGNIFICANCE STATEMENT Microdomains of varying chloride concentrations in the neuronal cytoplasm are a predictable consequence of the inhomogeneous distribution of anionic polymers such as actin, tubulin, and nucleic acids. Here, we demonstrate the existence and stability of these microdomains, as well as the consequence for GABAergic synaptic signaling: each interneuron produces a postsynaptic GABAA response with a unique reversal potential. In individual hippocampal pyramidal cells, the range of GABAA reversal potentials evoked by stimulating different interneurons was >20 mV. Some interneurons generated postsynaptic responses in pyramidal cells that reversed at potentials beyond what would be considered purely inhibitory. Cytoplasmic chloride microdomains enable each pyramidal cell to maintain a compendium of unique postsynaptic responses to the activity of individual interneurons.

The structural basis of function and regulation of neuronal cotransporters NKCC1 and KCC2

NKCC and KCC transporters mediate coupled transport of Na++K++Cl− and K++Cl− across the plasma membrane, thus regulating cell Cl− concentration and cell volume and playing critical roles in transepithelial salt and water transport and in neuronal excitability. The function of these transporters has been intensively studied, but a mechanistic understanding has awaited structural studies of the transporters. Here, we present the cryo-electron microscopy (cryo-EM) structures of the two neuronal cation-chloride cotransporters human NKCC1 (SLC12A2) and mouse KCC2 (SLC12A5), along with computational analysis and functional characterization. These structures highlight essential residues in ion transport and allow us to propose mechanisms by which phosphorylation regulates transport activity.

Intricacies of GABAA Receptor Function: The Critical Role of the β3 Subunit in Norm and Pathology

Neuronal intracellular chloride ([Cl−]i) is a key determinant in γ-aminobutyric acid type A (GABA)ergic signaling. γ-Aminobutyric acid type A receptors (GABAARs) mediate both inhibitory and excitatory neurotransmission, as the passive fluxes of Cl− and HCO3− via pores can be reversed by changes in the transmembrane concentration gradient of Cl−. The cation–chloride co-transporters (CCCs) are the primary systems for maintaining [Cl−]i homeostasis. However, despite extensive electrophysiological data obtained in vitro that are supported by a wide range of molecular biological studies on the expression patterns and properties of CCCs, the presence of ontogenetic changes in [Cl−]i—along with the consequent shift in GABA reversal potential—remain a subject of debate. Recent studies showed that the β3 subunit possesses properties of the P-type ATPase that participates in the ATP-consuming movement of Cl− via the receptor. Moreover, row studies have demonstrated that the β3 subunit is a key player in GABAAR performance and in the appearance of serious neurological disorders. In this review, we discuss the properties and driving forces of CCCs and Cl−, HCO3−ATPase in the maintenance of [Cl−]i homeostasis after changes in upcoming GABAAR function. Moreover, we discuss the contribution of the β3 subunit in the manifestation of epilepsy, autism, and other syndromes.

Targeting the WNK-SPAK/OSR1 Pathway and Cation-Chloride Cotransporters for the Therapy of Stroke.

Stroke is one of the major culprits responsible for morbidity and mortality worldwide, and the currently available pharmacological strategies to combat this global disease are scanty. Cation-chloride cotransporters (CCCs) are expressed in several tissues (including neurons) and extensively contribute to the maintenance of numerous physiological functions including chloride homeostasis. Previous studies have implicated two CCCs, the Na+-K+-Cl− and K+-Cl− cotransporters (NKCCs and KCCs) in stroke episodes along with their upstream regulators, the with-no-lysine kinase (WNKs) family and STE20/SPS1-related proline/alanine rich kinase (SPAK) or oxidative stress response kinase (OSR1) via a signaling pathway. As the WNK-SPAK/OSR1 pathway reciprocally regulates NKCC and KCC, a growing body of evidence implicates over-activation and altered expression of NKCC1 in stroke pathology whilst stimulation of KCC3 during and even after a stroke event is neuroprotective. Both inhibition of NKCC1 and activation of KCC3 exert neuroprotection through reduction in intracellular chloride levels and thus could be a novel therapeutic strategy. Hence, this review summarizes the current understanding of functional regulations of the CCCs implicated in stroke with particular focus on NKCC1, KCC3, and WNK-SPAK/OSR1 signaling and discusses the current and potential pharmacological treatments for stroke.

Genome-Wide Association Study (GWAS) to Identify Salt-Tolerance QTLs Carrying Novel Candidate Genes in Rice During Early Vegetative Stage

BackgroundRice is considered as a salt-sensitive plant, particularly at early vegetative stage, and its production is suffered from salinity due to expansion of salt affected land in areas under cultivation. Hence, significant increase of rice productivity on salinized lands is really necessary. Today genome-wide association study (GWAS) is a method of choice for fine mapping of QTLs involved in plant responses to abiotic stresses including salinity stress at early vegetative stage. In this study using > 33,000 SNP markers we identified rice genomic regions associated to early stage salinity tolerance. Eight salinity-related traits including shoot length (SL), root length (RL), root dry weight (RDW), root fresh weight (RFW), shoot fresh weight (SFW), shoot dry weight (SDW), relative water content (RWC) and TW, and 4 derived traits including SL-R, RL-R, RDW-R and RFW-R in a diverse panel of rice were evaluated under salinity (100 mM NaCl) and normal conditions in growth chamber. Genome-wide association study (GWAS) was applied based on MLM(+Q + K) model.ResultsUnder stress conditions 151 trait-marker associations were identified that were scattered on 10 chromosomes of rice that arranged in 29 genomic regions. A genomic region on chromosome 1 (11.26 Mbp) was identified which co-located with a known QTL region SalTol1 for salinity tolerance at vegetative stage. A candidate gene (Os01g0304100) was identified in this region which encodes a cation chloride cotransporter. Furthermore, on this chromosome two other candidate genes, Os01g0624700 (24.95 Mbp) and Os01g0812000 (34.51 Mbp), were identified that encode a WRKY transcription factor (WRKY 12) and a transcriptional activator of gibberellin-dependent alpha-amylase expression (GAMyb), respectively. Also, a narrow interval on the same chromosome (40.79–42.98 Mbp) carries 12 candidate genes, some of them were not so far reported for salinity tolerance at seedling stage. Two of more interesting genes are Os01g0966000 and Os01g0963000, encoding a plasma membrane (PM) H+-ATPase and a peroxidase BP1 protein. A candidate gene was identified on chromosome 2 (Os02g0730300 at 30.4 Mbp) encoding a high affinity K+ transporter (HAK). On chromosome 6 a DnaJ-encoding gene and pseudouridine synthase gene were identified. Two novel genes on chromosome 8 including the ABI/VP1 transcription factor and retinoblastoma-related protein (RBR), and 3 novel genes on chromosome 11 including a Lox, F-box and Na+/H+ antiporter, were also identified.ConclusionKnown or novel candidate genes in this research were identified that can be used for improvement of salinity tolerance in molecular breeding programmes of rice. Further study and identification of effective genes on salinity tolerance by the use of candidate gene-association analysis can help to precisely uncover the mechanisms of salinity tolerance at molecular level. A time dependent relationship between salt tolerance and expression level of candidate genes could be recognized.

Expression and Purification of the Human Cation-chloride Cotransporter KCC1 from HEK293F Cells for Structural Studies

Cation-chloride cotransporters (CCCs) mediate the coupled, electroneutral symport of cations such as Na+ and/or K+ with chloride across membrane. Among CCCs family, K-Cl cotransporters (KCC1-KCC4) extrude intracellular Cl- by the transmembrane K+ gradient. In humans, these KCCs play vital roles in the physiology of the nervous system and kidney. However, mechanisms underlying the KCCs specific properties remain poorly understood, partly because purification of membrane proteins is challenging. Here, we present the protocol for purifying the full-length KCC1 from HEK293F cells used in our recent publication ( Liu et al., 2019 ). The procedure may be adapted for functional and structural studies.

Cation-chloride cotransport mediated by an ion pair transporter.

Ion transport mediated by an ion pair receptor 1 bearing both cation and anion binding sites is presented. The ion pair binding property was revealed by means of 1H NMR titrations in organic solutions and X-ray analysis in the solid state. Single crystal structures demonstrated that 1 and CaI2 formed a solvent-separated ion-pair complex with two iodides each interacting with a triazine ring through anion-π interactions, whereas the calcium ion is bound by a pentaethylene glycol moiety. The ion transport activity was studied by monitoring the efflux of ions from salt-loaded EYPC large unilamellar vesicles (EYPC-LUVs) using ion selective electrodes. Chloride transport across the membrane mediated by the ion pair receptor through K+/Cl- or Na+/Cl- cotransport with a selectivity towards the K+/Cl- symport was realized.

A Quest for Mechanisms of Plant Root Exudation Brings New Results and Models, 300 Years after Hales.

The review summarizes some of our current knowledge on the phenomenon of exudation from the cut surface of detached roots with emphasis on results that were mostly established over the last fifty years. The phenomenon is quantitatively documented in the 18th century (by Hales in 1727). By the 19th century, theories mainly ascribed exudation to the secretion of living root cells. The 20th century favored the osmometer model of root exudation. Nevertheless, growing insights into the mechanisms of water transport and new or rediscovered observations stimulated the quest for a more adequate exudation model. The historical overview shows how understanding of exudation changed with time following experimental opportunities and novel ideas from different areas of knowledge. Later theories included cytoskeleton-dependent micro-pulsations of turgor in root cells to explain the observed water exudation. Recent progress in experimental biomedicine led to detailed study of channels and transporters for ion transport via cellular membranes and to the discovery of aquaporins. These universal molecular entities have been incorporated to the more complex models of water transport via plant roots. A new set of ideas and explanations was based on cellular osmoregulation by mechanosensitive ion channels. Thermodynamic calculations predicted the possibility of water transport against osmotic forces based on co-transport of water with ions via cation-chloride cotransporters. Recent observations of rhizodermis exudation, exudation of roots without an external aqueous medium, segments cut from roots, pulses of exudation, a phase shifting of water uptake and exudation, and of effects of physiologically active compounds (like ion channel blockers, metabolic agents, and cytoskeletal agents) will likely refine our understanding of the phenomenon. So far, it seems that more than one mechanism is responsible for root pressure and root exudation, processes which are important for refilling of embolized xylem vessels. However, recent advances in ion and water transport research at the molecular level suggest potential future directions to understanding of root exudation and new models awaiting experimental testing.

Larvicidal Activity of Cinnamic Acid Derivatives: Investigating Alternative Products for Aedes aegypti L. Control.

The mosquito Aedes aegypti transmits the virus that causes dengue, yellow fever, Zika and Chikungunya viruses, and in several regions of the planet represents a vector of great clinical importance. In terms of mortality and morbidity, infections caused by Ae. aegypti are among the most serious arthropod transmitted viral diseases. The present study investigated the larvicidal potential of seventeen cinnamic acid derivatives against fourth stage Ae. aegypti larvae. The larvicide assays were performed using larval mortality rates to determine lethal concentration (LC50). Compounds containing the medium alkyl chains butyl cinnamate (7) and pentyl cinnamate (8) presented excellent larvicidal activity with LC50 values of around 0.21–0.17 mM, respectively. While among the derivatives with aryl substituents, the best LC50 result was 0.55 mM for benzyl cinnamate (13). The tested derivatives were natural compounds and in pharmacology and antiparasitic studies, many have been evaluated using biological models for environmental and toxicological safety. Molecular modeling analyses suggest that the larvicidal activity of these compounds might be due to a multi-target mechanism of action involving inhibition of a carbonic anhydrase (CA), a histone deacetylase (HDAC2), and two sodium-dependent cation-chloride co-transporters (CCC2 e CCC3).

Advances in the development of novel compounds targeting cation-chloride cotransporter physiology

For about half a century, the pharmacology of electroneutral cation-chloride cotransporters has been dominated by a few drugs that are widely used in clinical medicine. Because these diuretic drugs are so good at what they do, there has been little incentive in expanding their pharmacology. The increasing realization that cation-chloride cotransporters are involved in many other key physiological processes and the knowledge that different tissues express homologous proteins with matching transport functions have rekindled interest in drug discovery. This review summarizes the methods available to assess the function of these transporters and describe the multiple efforts that have made to identify new compounds. We describe multiple screens targeting KCC2 function and one screen designed to find compounds that discriminate between NKCC1 and NKCC2. Two of the KCC2 screens identified new inhibitors that are 3-4 orders of magnitude more potent than furosemide. Additional screens identified compounds that purportedly increase cell surface expression of the cotransporter, as well as several FDA-approved drugs that increase KCC2 transcription and expression. The technical details of each screen biased them toward specific processes in the life cycle of the transporter, making these efforts independent and complementary. In addition, each drug discovery effort contributes to our understanding of the biology of the cotransporters.

The Therapeutic Potential of Neuronal K-Cl Co-Transporter KCC2 in Huntington's Disease and Its Comorbidities.

Intracellular chloride levels in the brain are regulated primarily through the opposing effects of two cation-chloride co-transporters (CCCs), namely K+-Cl− co-transporter-2 (KCC2) and Na+-K+-Cl− co-transporter-1 (NKCC1). These CCCs are differentially expressed throughout the course of development, thereby determining the excitatory-to-inhibitory γ-aminobutyric acid (GABA) switch. GABAergic excitation (depolarisation) is important in controlling the healthy development of the nervous system; as the brain matures, GABAergic inhibition (hyperpolarisation) prevails. This developmental switch in excitability is important, as uncontrolled regulation of neuronal excitability can have implications for health. Huntington’s disease (HD) is an example of a genetic disorder whereby the expression levels of KCC2 are abnormal due to mutant protein interactions. Although HD is primarily considered a motor disease, many other clinical manifestations exist; these often present in advance of any movement abnormalities. Cognitive change, in addition to sleep disorders, is prevalent in the HD population; the effect of uncontrolled KCC2 function on cognition and sleep has also been explored. Several mechanisms by which KCC2 expression is reduced have been proposed recently, thereby suggesting extensive investigation of KCC2 as a possible therapeutic target for the development of pharmacological compounds that can effectively treat HD co-morbidities. Hence, this review summarizes the role of KCC2 in the healthy and HD brain, and highlights recent advances that attest to KCC2 as a strong research and therapeutic target candidate.

Molecular mechanisms of Cl- transport in fishes: New insights and their evolutionary context.

With remarkably few exceptions, aquatic vertebrates maintain internal Cl- homeostasis despite strong and sometimes fluctuating Cl- concentration gradients between extracellular fluids and external environments. In this "Perspective," we discuss recent advances in the understanding of epithelial Cl- transport at the molecular level within key osmoregulatory organs in fishes. New insights into mechanisms for epithelial Cl- transport in basal lineages are highlighted to provide an evolutionary context. We describe Cl- transport processes that employ: cystic fibrosis transmembrane conductance regulator, cation-chloride cotransporters, voltage-gated chloride channels, and chloride-anion exchangers. As the collective understanding of Cl- transport processes continues to expand, investigators are equipped to more precisely characterize how endocrine factors promote hydromineral balance. We, therefore, conclude our discussion by paying special attention to recently defined roles for prolactin and corticosteroids in the regulation of Cl- transport in basal and derived clades.

Preventing neuronal edema increases network excitability after traumatic brain injury.

Edema is an important target for clinical intervention after traumatic brain injury (TBI). We used in vivo cellular resolution imaging and electrophysiological recording to examine the ionic mechanisms underlying neuronal edema and their effects on neuronal and network excitability after controlled cortical impact (CCI) in mice. Unexpectedly, we found that neuronal edema 48 hours after CCI was associated with reduced cellular and network excitability, concurrent with an increase in the expression ratio of the cation-chloride cotransporters (CCCs) NKCC1 and KCC2. Treatment with the CCC blocker bumetanide prevented neuronal swelling via a reversal in the NKCC1/KCC2 expression ratio, identifying altered chloride flux as the mechanism of neuronal edema. Importantly, bumetanide treatment was associated with increased neuronal and network excitability after injury, including increased susceptibility to spreading depolarizations (SDs) and seizures, known agents of clinical worsening after TBI. Treatment with mannitol, a first-line edema treatment in clinical practice, was also associated with increased susceptibility to SDs and seizures after CCI, showing that neuronal volume reduction, regardless of mechanism, was associated with an excitability increase. Finally, we observed an increase in excitability when neuronal edema normalized by 1 week after CCI. We conclude that neuronal swelling may exert protective effects against damaging excitability in the aftermath of TBI and that treatment of edema has the potential to reverse these effects.

Structure of the Regulatory Cytosolic Domain of a Eukaryotic Potassium-Chloride Cotransporter

Cation-chloride cotransporters (CCCs) regulate the movement of chloride across membranes, controlling physiological processes from cell volume maintenance to neuronal signaling. Human CCCs are clinical targets for existing diuretics and potentially additional indications. Here, we report the X-ray crystal structure of the soluble C-terminal regulatory domain of a eukaryotic potassium-chloride cotransporter, Caenorhabditis elegans KCC-1. We observe a core α/β fold conserved among CCCs. Using structure-based sequence alignment, we analyze similarities and differences to the C-terminal domains of other CCC family members. We find that important regulatory motifs are in less-structured regions and residues important for dimerization are not widely conserved, suggesting that oligomerization and its effects may vary within the larger family. This snapshot of a eukaryotic KCC is a valuable starting point for the rational design of studies of cellular chloride regulation.