Cation-chloride Cotransporters Research Articles

Cervical spinal contusion alters Na+-K+-2Cl- and K+-Cl- cation-chloride cotransporter expression in phrenic motor neurons

Spinal chloride-dependent synaptic inhibition is critical in regulating breathing and requires neuronal chloride gradients established by cation-chloride cotransporters Na+-K+-2Cl− (NKCC1) and K+-Cl- (KCC2). Spinal transection disrupts NKCC1/KCC2 balance, diminishing chloride gradients in neurons below injury, contributing to spasticity and chronic pain. It is not known if similar disruptions in NKCC1/KCC2 balance occur in respiratory motor neurons after incomplete cervical contusion (C2SC). We hypothesized that C2SC disrupts NKCC1/KCC2 balance in phrenic motor neurons. NKCC1 and KCC2 immunoreactivity was assessed in CtB-positive phrenic motor neurons. Five weeks post-C2SC: 1) neither membrane-bound nor cytosolic NKCC1 expression were significantly changed, although the membrane/cytosolic ratio increased, consistent with net chloride influx; and 2) both membrane and cytosolic KCC2 expression increased, although the membrane/cytosolic ratio decreased, consistent with net chloride efflux. Thus, contrary to our original hypothesis, complex shifts in NKCC1/KCC2 balance occur post-C2SC. The functional significance of these changes remains unclear.

Insights into GABAAergic system alteration in Huntington's disease.

Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disease that is characterized by a triad of motor, psychiatric and cognitive impairments. There is still no effective therapy to delay or halt the disease progress. The striatum and cortex are two particularly affected brain regions that exhibit dense reciprocal excitatory glutamate and inhibitory gamma-amino butyric acid (GABA) connections. Imbalance between excitatory and inhibitory signalling is known to greatly affect motor and cognitive processes. Emerging evidence supports the hypothesis that disrupted GABAergic circuits underlie HD pathogenesis. In the present review, we focused on the multiple defects recently found in the GABAergic inhibitory system, including altered GABA level and synthesis, abnormal subunit composition and distribution of GABAA receptors and aberrant GABAA receptor-mediated signalling. In particular, the important role of cation–chloride cotransporters (i.e. NKCC1 and KCC2) is discussed. Recent studies also suggest that neuroinflammation contributes significantly to the abnormal GABAergic inhibition in HD. Thus, GABAA receptors and cation–chloride cotransporters are potential therapeutic targets for HD. Given the limited availability of therapeutic treatments for HD, a better understanding of GABAergic dysfunction in HD could provide novel therapeutic opportunities.

Biophysical models reveal the relative importance of transporter proteins and impermeant anions in chloride homeostasis.

Fast synaptic inhibition in the nervous system depends on the transmembrane flux of Cl- ions based on the neuronal Cl- driving force. Established theories regarding the determinants of Cl- driving force have recently been questioned. Here, we present biophysical models of Cl- homeostasis using the pump-leak model. Using numerical and novel analytic solutions, we demonstrate that the Na+/K+-ATPase, ion conductances, impermeant anions, electrodiffusion, water fluxes and cation-chloride cotransporters (CCCs) play roles in setting the Cl- driving force. Our models, together with experimental validation, show that while impermeant anions can contribute to setting [Cl-]i in neurons, they have a negligible effect on the driving force for Cl- locally and cell-wide. In contrast, we demonstrate that CCCs are well-suited for modulating Cl- driving force and hence inhibitory signaling in neurons. Our findings reconcile recent experimental findings and provide a framework for understanding the interplay of different chloride regulatory processes in neurons.

Increased expression of WNK3 in dispersed granule cells in hippocampal sclerosis of mesial temporal lobe epilepsy patients

Granule cell dispersion (GCD) is a common neuropathological feature of hippocampal sclerosis (HS) in patients with temporal lobe epilepsy (TLE). However, the underlying molecular mechanism of GCD formation remains unclear. The present study aimed to investigate the expressional changes of With No Lysine protein kinase subtype 3 (WNK3), a molecule upstream of cation-chloride cotransporters with reciprocal expression in sclerosed hippocampus of TLE patients. Using immunofluorescence staining, we analyzed WNK3 immunoreactivity in hippocampal specimens from histologically normal controls and TLE patients with HS. Our results showed that WNK3 expression was significantly increased in dispersed granule neurons in hippocampal tissues from patients with TLE compared with histologically normal hippocampus. These findings demonstrate a potential association between an increased expression of WNK3 and GCD formation during the chronic phase of epilepsy. Controlling WNK3 expression may thus be a novel therapeutic target in epileptogenesis.

WNK-SPAK/OSR1 signaling: lessons learned from an insect renal epithelium.

WNK [with no lysine (K)] kinases regulate renal epithelial ion transport to maintain homeostasis of electrolyte concentrations, extracellular volume, and blood pressure. The SLC12 cation-chloride cotransporters, including the sodium-potassium-2-chloride (NKCC) and sodium chloride cotransporters (NCC), are targets of WNK regulation via the intermediary kinases SPAK (Ste20-related proline/alanine-rich kinase) and OSR1 (oxidative stress response). The pathway is activated by low dietary potassium intake, resulting in increased phosphorylation and activity of NCC. Chloride regulates WNK kinases in vitro by binding to the active site and inhibiting autophosphorylation and has been proposed to modulate WNK activity in the distal convoluted tubule in response to low dietary potassium. WNK-SPAK/OSR1 regulation of NKCC-dependent ion transport is evolutionarily ancient, and it occurs in the Drosophila Malpighian (renal) tubule. Here, we review recent studies from the Drosophila tubule demonstrating cooperative roles for chloride and the scaffold protein Mo25 (mouse protein-25, also known as calcium-binding protein-39) in the regulation of WNK-SPAK/OSR1 signaling in a transporting renal epithelium. Insights gained from this genetically manipulable and physiologically accessible epithelium shed light on molecular mechanisms of regulation of the WNK-SPAK/OSR1 pathway, which is important in human health and disease.

Bumetanide blocks the acquisition of conditioned fear in adult rats.

Bumetanide has anxiolytic effects in rat models of conditioned fear. As a loop diuretic, bumetanide blocks cation-chloride co-transport and this property may allow bumetanide to act as an anxiolytic by modulating GABAergic synaptic transmission in the CNS. Its potential for the treatment of anxiety disorders deserves further investigation. In this study, we evaluated the possible involvement of the basolateral nucleus of the amygdala in the anxiolytic effect of bumetanide. Brain slices were prepared from Wistar rats. extracellular recording, stereotaxic surgery, fear-potentiated startle response, locomotor activity monitoring and Western blotting were applied in this study. Systemic administration of bumetanide (15.2mg·kg-1 , i.v.), 30min prior to fear conditioning, significantly inhibited the acquisition of the fear-potentiated startle response. Phosphorylation of ERK in the basolateral nucleus of amygdala was reduced after bumetanide administration. In addition, suprafusion of bumetanide (5 or 10μM) attenuated long-term potentiation in the amygdala in a dose-dependent manner. Intra-amygdala infusion of bumetanide, 15min prior to fear conditioning, also blocked the acquisition of the fear-potentiated startle response. Finally, the possible off-target effect of bumetanide on conditioned fear was excluded by side-by-side control experiments. These results suggest the basolateral nucleus of amygdala plays a critical role in the anxiolytic effects of bumetanide.

Role of nitric oxide and WNK-SPAK/OSR1-KCC2 signaling in daily changes in GABAergic inhibition in the rat dorsal raphe neurons

Serotonergic neurons in the dorsal raphe nucleus (DRN) act as wake-inducing neurons in the sleep-wake cycle and are controlled by gamma-aminobutyric acid (GABA) synaptic inputs. We investigated daily changes in GABAergic inhibition of the rat DRN neurons and the role of nitric oxide (NO) and cation-chloride co-transporters in the GABAergic action. Neuronal NO synthase (nNOS) was co-expressed in 74% of serotonergic DRN neurons and nNOS expression was higher during daytime (the sleep cycle) than that during nighttime (the wake cycle). GABAergic hyperpolarization of DRN neurons produced by GABAA receptor agonist muscimol was greater and the equilibrium potential of muscimol showed a hyperpolarizing shift during daytime compared to that during nighttime. Expression levels of potassium-chloride co-transporter 2 (KCC2) were higher during daytime than that during nighttime, whereas there were no changes in sodium-potassium-chloride co-transporter 1 (NKCC1) expression. With-no-lysine kinase (WNK) isoform 1 was more highly expressed during daytime than that during nighttime. Total Ste20-related proline alanine rich kinase (SPAK) and oxidative stress response kinase 1 (OSR1) were also higher during daytime than during nighttime, while there were no changes in phosphorylated SPAK and OSR1. Consistent with the findings during the sleep-wake cycle, ex vivo treatment of DRN slices with a NO donor sodium nitroprusside (SNP) increased the expression of KCC2, WNK1, WNK2, WNK3, SPAK, and OSR1, whilst decreasing phosphorylated SPAK. These results suggest that GABAergic synaptic inhibition of DRN serotonergic neurons shows daily changes during the sleep-wake cycle, which might be regulated by daily changes in nNOS-derived NO and WNK-SPAK/OSR1-KCC2 signaling.

Plant Cation-Chloride Cotransporters (CCC): Evolutionary Origins and Functional Insights.

Genomes of unicellular and multicellular green algae, mosses, grasses and dicots harbor genes encoding cation-chloride cotransporters (CCC). CCC proteins from the plant kingdom have been comparatively less well investigated than their animal counterparts, but proteins from both plants and animals have been shown to mediate ion fluxes, and are involved in regulation of osmotic processes. In this review, we show that CCC proteins from plants form two distinct phylogenetic clades (CCC1 and CCC2). Some lycophytes and bryophytes possess members from each clade, most land plants only have members of the CCC1 clade, and green algae possess only the CCC2 clade. It is currently unknown whether CCC1 and CCC2 proteins have similar or distinct functions, however they are both more closely related to animal KCC proteins compared to NKCCs. Existing heterologous expression systems that have been used to functionally characterize plant CCC proteins, namely yeast and Xenopus laevis oocytes, have limitations that are discussed. Studies from plants exposed to chemical inhibitors of animal CCC protein function are reviewed for their potential to discern CCC function in planta. Thus far, mutations in plant CCC genes have been evaluated only in two species of angiosperms, and such mutations cause a diverse array of phenotypes—seemingly more than could simply be explained by localized disruption of ion transport alone. We evaluate the putative roles of plant CCC proteins and suggest areas for future investigation.

Role of WNK Kinases in the Modulation of Cell Volume.

Ion Transport across the cell membrane is required to maintain cell volume homeostasis. In response to changes in extracellular osmolarity, most cells activate specific metabolic or membrane-transport pathways to respond to cell swelling or shrinkage and return their volume to its normal resting state. This process involves the rapid adjustment of the activities of channels and transporters that mediate flux of K+, Na+, Cl-, and small organic osmolytes. Cation chloride cotransporters (CCCs) NKCCs and KCCs are a family of membrane proteins modulated by changes in cell volume and/or in the intracellular chloride concentration ([Cl-]i). Cell swelling triggers regulatory volume decrease (RVD), promoting solute and water efflux to restore normal cell volume. Swelling-activated KCCs mediate RVD in most cell types. In contrast, cell shrinkage triggers regulatory volume increase (RVI), which involves the activation of the NKCC1 cotransporter of the CCC family. Regulation of the CCCs during RVI and RVD by protein phosphorylation is a well-characterized mechanism, where WNK kinases and their downstream kinase substrates, SPAK and OSR1 constitute the essential phospho-regulators. WNKs-SPAK/OSR1-CCCs complex is required to regulate cell shrinkage-induced RVI or cell swelling-induced RVD via activating or inhibitory phosphorylation of NKCCs or KCCs, respectively. WNK1 and WNK4 kinases have been established as [Cl-]i sensors/regulators, while a role for WNK3 kinase as a cell volume-sensing kinase has emerged and is proposed in this chapter.

Inactivation of SPAK kinase reduces body weight gain in mice fed a high-fat diet by improving energy expenditure and insulin sensitivity.

The STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) controls the activity of the electroneutral cation-chloride cotransporters (SLC12 family) and thus physiological processes such as modulation of cell volume, intracellular chloride concentration [Cl-]i, and transepithelial salt transport. Modulation of SPAK kinase activity may have an impact on hypertension and obesity, as STK39, the gene encoding SPAK, has been suggested as a hypertension and obesity susceptibility gene. In fact, the absence of SPAK activity in mice in which the activating threonine in the T loop was substituted by alanine (SPAK-KI mice) is associated with decreased blood pressure; however its consequences in metabolism have not been explored. Here, we fed wild-type and homozygous SPAK-KI mice a high-fat diet for 17 wk to evaluate weight gain, circulating substrates and hormones, energy expenditure, glucose tolerance, and insulin sensitivity. SPAK-KI mice exhibit resistance to HFD-induced obesity and hepatic steatosis associated with increased energy expenditure, higher thermogenic activity in brown adipose tissue, increased mitochondrial activity in skeletal muscle, and reduced white adipose tissue hypertrophy mediated by augmented whole body insulin sensitivity and glucose tolerance. Our data reveal a previously unrecognized role for the SPAK kinase in the regulation of energy balance, thermogenesis, and insulin sensitivity, suggesting that this kinase could be a new drug target for the treatment of obesity and the metabolic syndrome.

Molecular characterization and functional analysis of a cation-chloride cotransporter gene from trifoliate orange (Poncirus trifoliata L.)

A cation-chloride cotransporter gene PtrCCC from trifoliate orange was identified, its ectopic expression confers salt tolerance in transgenic tobacco. Salinity is one of the most serious environmental stresses that limit crop growth. Cation-chloride cotransporters (CCCs) are membrane proteins that are involved in plant development and salt tolerance. Here we provide an insight into the molecular characterization and functional analysis of CCC-encoding gene (PtrCCC) from trifoliate orange (Poncirus trifoliata L.). The open-reading frame of PtrCCC is 2943 bp in length, which encodes a protein of 980 amino acids. Phylogenetic analysis revealed that PtrCCC is closely related to CCCs from its allied genera as well as woody perennials. Quantitative real-time PCR (qRT-PCR) analysis demonstrated that PtrCCC was expressed in all tested tissues, with higher expression level in root tip and leaf tip. Its expression was up-regulated in the leaves of trifoliate orange under the presence of KCl conditions. Ectopic expression of PtrCCC gene in tobacco exhibited an improvement in salt tolerance. The transgenic plants had higher dry biomass in the roots and shoots than that in wild type plants. Moreover, under KCl and NaCl + KCl stress conditions, the accumulation of shoot Cl− was remarkably decreased in the transgenic lines as compared with the wild type plants. Collectively, these results suggest that PtrCCC plays an important role in salt tolerance, which is partially attributed to its role in ions homeostasis.

Differential expression of putative sodium-dependent cation-chloride cotransporters in Aedes aegypti

The yellow fever mosquito, Aedes aegypti, has three genes that code for proteins with sequence similarity to vertebrate Na+-K+-Cl− cotransporters (NKCCs) of the solute-linked carrier 12 superfamily of cation-chloride cotransporters (CCCs). We hypothesized that these mosquito NKCC orthologues have diverged to perform distinct roles in salt secretion and absorption. In phylogenetic analyses, one protein (aeNKCC1) groups with a Drosophila melanogaster NKCC that mediates salt secretion whereas two others (aeCCC2 and aeCCC3) group with a Drosophila transporter that is not functionally characterized. The aeCCC2 and aeCCC3 genes probably result from a tandem gene duplication in the mosquito lineage; they have similar exon structures and are consecutive in genomic DNA. Predicted aeCCC2 and aeCCC3 proteins differ from aeNKCC1 and vertebrate NKCCs in residues from the third transmembrane domain known to influence ion and inhibitor binding. Quantitative PCR revealed that aeNKCC1 and aeCCC2 were approximately equally expressed in larvae and adults, whereas aeCCC3 was approximately 100-fold more abundant in larvae than in adults. In larval tissues, aeCCC2 was approximately 2-fold more abundant in Malpighian tubules compared to anal papillae. In contrast, aeCCC3 was nearly 100-fold more abundant in larval anal papillae compared to Malpighian tubules, suggesting a role in absorption. Western blots with polyclonal antibodies against isoform-specific peptides revealed stronger aeCCC2 immunoreactivity in adults versus larvae, whereas aeCCC3 immunoreactivity was stronger in larvae versus adults. The differential expression pattern of aeCCC2 and aeCCC3, and their sequence divergence in transmembrane domains, suggests that they may have different roles in transepithelial salt transport.

Intracellular Chloride Regulation in AVP+ and VIP+ Neurons of the Suprachiasmatic Nucleus

Several reports have described excitatory GABA transmission in the suprachiasmatic nucleus (SCN), the master pacemaker of circadian physiology. However, there is disagreement regarding the prevalence, timing, and neuronal location of excitatory GABA transmission in the SCN. Whether GABA is inhibitory or excitatory depends, in part, on the intracellular concentration of chloride ([Cl−]i). Here, using ratiometric Cl− imaging, we have investigated intracellular chloride regulation in AVP and VIP-expressing SCN neurons and found evidence suggesting that [Cl−]i is higher during the day than during the night in both AVP+ and VIP+ neurons. We then investigated the contribution of the cation chloride cotransporters to setting [Cl−]i in these SCN neurons and found that the chloride uptake transporter NKCC1 contributes to [Cl−]i regulation in SCN neurons, but that the KCCs are the primary regulators of [Cl−]i in SCN neurons. Interestingly, we observed that [Cl−]i is differentially regulated between AVP+ and VIP+ neurons-a low concentration of the loop diuretic bumetanide had differential effects on AVP+ and VIP+ neurons, while blocking the KCCs with VU0240551 had a larger effect on VIP+ neurons compared to AVP+ neurons.

Molecular cloning and biochemical characterization of two cation chloride cotransporter subfamily members of Hydra vulgaris.

Cation Chloride Cotransporters (CCCs) comprise secondary active membrane proteins mainly mediating the symport of cations (Na+, K+) coupled with chloride (Cl−). They are divided into K+-Cl− outward transporters (KCCs), the Na+-K+-Cl− (NKCCs) and Na+-Cl− (NCCs) inward transporters, the cation chloride cotransporter interacting protein CIP1, and the polyamine transporter CCC9. KCCs and N(K)CCs are established in the genome since eukaryotes and metazoans, respectively. Most of the physiological and functional data were obtained from vertebrate species. To get insights into the basal functional properties of KCCs and N(K)CCs in the metazoan lineage, we cloned and characterized KCC and N(K)CC from the cnidarian Hydra vulgaris. HvKCC is composed of 1,032 amino-acid residues. Functional analyses revealed that hvKCC mediates a Na+-independent, Cl− and K+ (Tl+)-dependent cotransport. The classification of hvKCC as a functional K-Cl cotransporter is furthermore supported by phylogenetic analyses and a similar structural organization. Interestingly, recently obtained physiological analyses indicate a role of cnidarian KCCs in hyposmotic volume regulation of nematocytes. HvN(K)CC is composed of 965 amino-acid residues. Phylogenetic analyses and structural organization suggest that hvN(K)CC is a member of the N(K)CC subfamily. However, no inorganic ion cotransport function could be detected using different buffer conditions. Thus, hvN(K)CC is a N(K)CC subfamily member without a detectable inorganic ion cotransporter function. Taken together, the data identify two non-bilaterian solute carrier 12 (SLC12) gene family members, thereby paving the way for a better understanding of the evolutionary paths of this important cotransporter family.

Expression of Sodium‐Dependent Cation‐Chloride Cotransporters in Adult and Larval Osmoregulatory Tissues of Aedes aegypti Mosquitoes

Yellow fever mosquitoes, Aedes aegypti, face diverse salt and water challenges. Larvae lose salt to the freshwater environment and gain water osmotically, whereas adults tend to lose water by evaporation. Moreover, adult females take in large quantities of salt and water during a blood meal. We hypothesize that sodium‐dependent cation‐chloride cotransporters (CCCs) contribute to the salt and water balance of Aedes aegypti by participating in epithelial salt secretion and absorption. Sequence analysis revealed three genes (AAEL006180, AAEL009888, and AAEL009886) in Aedes aegypti with similarity to vertebrate Na‐K‐Cl cotransporters. We performed multiple sequence alignment and phylogenetic analysis of the proteins encoded by the Aedes aegypti genes and orthologs in Culex and Anopheles mosquitoes, Drosophila, and other arthropods. The protein encoded by AAEL006180 groups in a clade with the human secretory Na‐K‐Cl cotransporter and a Drosophila cotransporter known to be involved in salt secretion. In contrast, AAEL009888 and AAEL009886 fall into a clade that includes sequences from other insects but not vertebrates. Moreover, both AAEL009888 and AAEL009886 have orthologs in other mosquito genera but other insects only have one ortholog, indicating that AAEL009888 and AAEL009886 probably diverged early in the evolution of mosquitoes. Amino acids that differ among the paralogs include residues known to influence ion and inhibitor binding affinities. Using qPCR, we quantified the relative gene expression of the Aedes aegypti paralogs with at least two isoform‐specific primer sets for each gene. AAEL009886 transcript is expressed at significantly greater levels in larvae compared to adults and at significantly greater levels in anal papillae compared to Malpighian tubules, suggesting that AAEL009886 may be specialized for ion absorption in larvae. In contrast, AAEL006180 and AAEL009888 transcripts are expressed at approximately the same levels in adults and larvae. To evaluate the physiological role of the proteins encoded by AAEL006180 and AAEL009888, we attempted to reduce expression of each transporter using dsRNA. Larvae exposed to dsRNA corresponding to either transporter had up to five‐fold increases in hemolymph ammonium levels compared to negative controls as well as smaller changes in other hemolymph cations. Our results point towards physiological roles for AAEL006180 and AAEL009888 in osmoregulation and ammonia balance of larval mosquitoes.Support or Funding InformationNSF‐IOS‐1557230

A kainate receptor subunit promotes the recycling of the neuron-specific K+-Cl− co-transporter KCC2 in hippocampal neurons

Synaptic inhibition depends on a transmembrane gradient of chloride, which is set by the neuron-specific K+-Cl- co-transporter KCC2. Reduced KCC2 levels in the neuronal membrane contribute to the generation of epilepsy, neuropathic pain, and autism spectrum disorders; thus, it is important to characterize the mechanisms regulating KCC2 expression. In the present study, we determined the role of KCC2-protein interactions in regulating total and surface membrane KCC2 expression. Using quantitative immunofluorescence in cultured mouse hippocampal neurons, we discovered that the kainate receptor subunit GluK2 and the auxiliary subunit Neto2 significantly increase the total KCC2 abundance in neurons but that GluK2 exclusively increases the abundance of KCC2 in the surface membrane. Using a live cell imaging assay, we further determined that KCC2 recycling primarily occurs within 1-2 h and that GluK2 produces an ∼40% increase in the amount of KCC2 recycled to the membrane during this time period. This GluK2-mediated increase in surface recycling translated to a significant increase in KCC2 expression in the surface membrane. Moreover, we found that KCC2 recycling is enhanced by protein kinase C-mediated phosphorylation of the GluK2 C-terminal residues Ser-846 and Ser-868. Lastly, using gramicidin-perforated patch clamp recordings, we found that the GluK2-mediated increase in KCC2 recycling to the surface membrane translates to a hyperpolarization of the reversal potential for GABA (EGABA). In conclusion, our results have revealed a mechanism by which kainate receptors regulate KCC2 expression in the hippocampus.

Dysbindin Deficiency Modifies the Expression of GABA Neuron and Ion Permeation Transcripts in the Developing Hippocampus.

The neurodevelopmental factor dysbindin is required for synapse function and GABA interneuron development. Dysbindin protein levels are reduced in the hippocampus of schizophrenia patients. Mouse dysbindin genetic defects and other mouse models of neurodevelopmental disorders share defective GABAergic neurotransmission and, in several instances, a loss of parvalbumin-positive interneuron phenotypes. This suggests that mechanisms downstream of dysbindin deficiency, such as those affecting GABA interneurons, could inform pathways contributing to or ameliorating diverse neurodevelopmental disorders. Here we define the transcriptome of developing wild type and dysbindin null Bloc1s8sdy/sdy mouse hippocampus in order to identify mechanisms downstream dysbindin defects. The dysbindin mutant transcriptome revealed previously reported GABA parvalbumin interneuron defects. However, the Bloc1s8sdy/sdy transcriptome additionally uncovered changes in the expression of molecules controlling cellular excitability such as the cation-chloride cotransporters NKCC1, KCC2, and NCKX2 as well as the potassium channel subunits Kcne2 and Kcnj13. Our results suggest that dysbindin deficiency phenotypes, such as GABAergic defects, are modulated by the expression of molecules controlling the magnitude and cadence of neuronal excitability.

Neonatal seizures and disruption to neurotransmitter systems

Neonatal seizures and disruption to neurotransmitter systems

Cotransport of water and solutes in plant membranes: The molecular basis, and physiological functions

Current concepts of plant membrane transport are based on the assumption that water and solutes move across membranes via separate pathways. According to this view, coupling between the fluxes is more or less exclusively constituted via the osmotic force that solutes exert on water transport. This view is questioned here, and experimental evidence for a cotransport of water and solutes is reviewed. The overview starts with ion channels that provide pathways for both ion and water transport, as exemplified for maxi K + channels from cytoplasmic droplets of Chara corallina . Aquaporins are usually considered to be selective for water (just allowing for slippage of some other small, neutral molecules). Recently, however, a “dual function” aquaporin has been characterized from Arabidopsis thaliana (AtPIP2.1) that translocates water and at the same time conducts cations, preferentially Na + . By analogy with mammalian physiology, other candidates for solute-water flux coupling are cation-chloride cotransporters of the CCC type, and transporters of sugars and amino acids. The last part is dedicated to possible physiological functions that could rely on solute-water cotransport. Among these are the generation of root pressure, refilling of embolized xylem vessels, fast turgor-driven movements of leaves, cell elongation (growth), osmoregulation and adjustment of buoyancy in marine algae. This review will hopefully initiate further research in the field.

Lack of the effect of bumetanide, a selective NKCC1 inhibitor, in patients with schizophrenia: A double‐blind randomized trial

The excitatory function of GABA has been reported to contribute to the pathogenesis of some neuropsychological disorders, for example autism1 and epilepsy.2 The switch in GABA function, from inhibitory to excitatory, has been attributed to the alteration of cation-chloride cotransporters involved in the regulation of intercellular chloride concentration, chloride-intruder NKCC1 and chloride-extruder KCC2. A recent post-mortem study revealed that the NKCC1/KCC2 expression ratio is reduced in hippocampal formation of schizophrenic patients, which notably indicates that GABA may be excitatory in this region.3 In accordance, it has been suggested that hippocampal overactivity may underlie dopaminergic system dysregulation in cortical and sub-cortical regions.4 Bumetanide, a selective NKCC1 inhibitor, works as a potent agent for restoring the inhibitory function of GABA.5 Recently, the therapeutic effect of bumetanide in some neurological disorders, such as autism1 and epilepsy,2 has been shown. Thus, we designed a study to investigate the effect of bumetanide on patients with schizophrenia. The present study was registered in the official clinical trial registration system of the Iranian Health Ministry with registration number IRCT2014012616374N1. All of the procedures and treatments were approved by the local ethics committee of the University of Social Welfare and Rehabilitation Sciences with ethical number USWR.REC.1392.47. Twenty-six patients with schizophrenia were enrolled in this study. In summary, the diagnosis of schizophrenia, at any subtype, according to the DSM-IV, was the mainstay of patients’ enrollment and patients were excluded: (i) if they had a total score of <65 on the Positive and Negative Syndrome Scale (PANSS); (ii) if they had a documented fluctuation in symptoms; or (iii) if they had concurrent history of significant psychiatric disorder (except schizophrenia) or any other significant organ disorders. All patients and their families provided written consent. The experimental group (n = 14) received bumetanide tablet orally 1 mg twice daily while the control group (n = 12) received placebo in the same way. All patients were allowed to continue their routine antipsychotic drugs during the study. Duration of treatment was 2 months. For each group, the PANSS total score, PANSS subscores (Positive, Negative, and Cognitive), and the Brief Psychiatric Rating Scale (BPRS) total score were determined at the beginning, 1 and 2 months after treatment, and 1 month after a drug-free washout period (Table 1). Results of two-way repeated-measure analysis of variance revealed that in schizophrenic patients, treatment with bumetanide or placebo had no significant effect on: PANSS total score, F(1, 24) = 1.47, P = 0.236; PANSS Positive subscore, F(1, 24) = 0.012, P = 0.913; PANSS Negative subscore, F(1, 24) = 1.492, P = 0.234; or PANSS Cognitive subscore, F(1, 24) = 1.33, P = 0.259. Also, the effect of interaction of time and treatment on PANSS score was not significantly different, F(3, 72) = 0.098, P = 0.961. In addition, our results showed that treatment with bumetanide or placebo had no significant effect on BPRS score in patients, F(1, 24) = 0.062, P = 0.805. Similar to previous reports,2 assessment of treated patients 2 months after bumetanide intake revealed that both bumetanide and placebo had no serious side-effects based on a prepared checklist. These study data have some limitations. First, the effect of bumetanide on patients with schizophrenia was not investigated on a specific symptom. Second, the dose of bumetanide 2 mg/day may be inadequate to show biological effects. Further studies are required to address these issues and to show the actual effect of bumetanide on schizophrenia. The authors declare no conflicts of interest.