Clustering of Neuronal K+-Cl? Cotransporters in Lipid Rafts by Tyrosine Phosphorylation

The neuronal K⁺-Cl⁻ cotransporter (KCC2) is a membrane transport protein that extrudes Cl⁻ from neurons and helps maintain low intracellular [Cl⁻] and hyperpolarizing GABAergic synaptic potentials. Depolarizing γ-aminobutyric acid (GABA) responses in neonatal neurons and following various forms of neuronal injury are associated with reduced levels of KCC2 expression. Despite the importance for plasticity of inhibitory transmission, less is known about cellular mechanisms involved in more dynamic changes in KCC2 function. In this study, we investigated the role of tyrosine phosphorylation in KCC2 localization and function in hippocampal neurons and in cultured GT1-7 cells. Mutation to the putative tyrosine phosphorylation site within the long intracellular carboxyl terminus of KCC2(Y1087D) or application of the tyrosine kinase inhibitor genistein shifted the GABA reversal potential (E_GABA) to more depolarized values, indicating reduced KCC2 function. This was associated with a change in the expression pattern of KCC2 from a punctate distribution to a more uniform distribution, suggesting that functional tyrosine-phosphorylated KCC2 forms clusters in restricted membrane domains. Sodium vanadate, a tyrosine phosphatase inhibitor, increased the proportion of KCC2 associated with lipid rafts membrane domains. Loss of tyrosine phosphorylation also reduced oligomerization of KCC2. A loss of the punctuate distribution and oligomerization of KCC2 and a more depolarized E_GABA were seen when the 28-amino-acid carboxyl terminus of KCC2 was deleted. These results indicate that direct tyrosine phosphorylation of KCC2 results in membrane clusters and functional transport activity, suggesting a mechanism by which intracellular Cl⁻ concentrations and GABA responses can be rapidly modulated.The inhibitory neurotransmitters GABA² and glycine activate ionotropic Cl⁻ channels, typically leading to membrane hyperpolarization in the adult central nervous system. The neuronal K⁺-Cl⁻ cotransporter (KCC2) is the principal membrane transport protein that maintains low intracellular [Cl⁻] ([Cl⁻]_i) in mature and healthy neurons to allow such Cl⁻ influx and hyperpolarization. However, in immature neurons and in neurons following various forms of neuronal injury, [Cl⁻]_i is elevated and GABA and glycine can cause membrane depolarization and neuronal excitation (1–3). A reduced expression of KCC2 protein in immature neurons (4) and a decrease of KCC2 expression in response to various pathophysiological conditions, e.g. axotomy (5, 6) and global ischemia (7), are primarily responsible for this increased [Cl⁻]_i and for the depolarizing GABA response.In addition to changes in the expression levels of KCC2 protein, the function of KCC2 can be more dynamically and rapidly modulated by the availability of transport substrates and by various forms of kinase activity. Cl⁻ extrusion is quantitatively regulated by the K⁺ driving force across the membrane (8). Protein kinase C can down-regulate both KCC2 function (9) and surface expression (10). Staurosporine, a broad spectrum kinase inhibitor, produces a rapid up-regulation of KCC2 function in immature neurons (11). Brain-type creatinine kinase binding to KCC2 may also regulate its function (12). Finally, WNK3, by interacting with Ste20-related proline-alanine-rich kinase, prevents the cell swelling-induced activation of KCC2 in Xenopus oocytes (13, 14).KCC2 contains one tyrosine protein kinase phosphorylation consensus site (Tyr-1087) within the long carboxyl terminus in the intracellular region (15). Tyr-1087 is not present in KCC1, another family of KCCs, suggesting that direct tyrosine phosphorylation may uniquely regulate KCC2. The receptor tyrosine kinase, IGF-1, and the soluble tyrosine kinase, Src kinase, activate KCC2 during maturation of hippocampal neurons (16). Oxidative stress decreases the tyrosine phosphorylation of KCC2 and reduces KCC2 function (17). However, just how tyrosine phosphorylation regulates KCC2 function under more physiological conditions is unclear, although modulation of KCC2 has important implications for inhibitory synaptic transmission and neuronal excitability. Furthermore, although KCC2 is uniquely expressed in neurons and may be influenced by the neuronal microenvironment, many of the studies on modulation of KCC function have been done in non-neuronal cell lines, e.g. HEK293 cells, and Xenopus oocytes. In this study, we therefore examined the role and mechanisms of tyrosine phosphorylation in the regulation of KCC2 function in cultured hippocampal neurons and in GT1-7 cells, a brain-derived cell line that possesses many neuronal characteristics but does not express endogenous KCC2 (18, 19) (also, see “Experimental Procedures”). The present study proposes that tyrosine phosphorylation of KCC2 results in clustering within lipid rafts via interactions within the carboxyl terminus of KCC2 and that this clustering results in efficient extrusion of Cl⁻.     

A variant of KCC2 from patients with febrile seizures impairs neuronal Cl− extrusion and dendritic spine formation

Genetic variation in SLC12A5 which encodes KCC2, the neuron-specific cation-chloride cotransporter that is essential for hyperpolarizing GABAergic signaling and formation of cortical dendritic spines, has not been reported in human disease. Screening of SLC12A5 revealed a co-segregating variant (KCC2-R952H) in an Australian family with febrile seizures. We show that KCC2-R952H reduces neuronal Cl⁻ extrusion and has a compromised ability to induce dendritic spines in vivo and in vitro. Biochemical analyses indicate a reduced surface expression of KCC2-R952H which likely contributes to the functional deficits. Our data suggest that KCC2-R952H is a bona fide susceptibility variant for febrile seizures.     

Cytosolic chloride ion is a key factor in lysosomal acidification and function of autophagy in human gastric cancer cell

The purpose of the present study was to clarify roles of cytosolic chloride ion (Cl⁻) in regulation of lysosomal acidification [intra-lysosomal pH (pH_lys)] and autophagy function in human gastric cancer cell line (MKN28). The MKN28 cells cultured under a low Cl⁻ condition elevated pH_lys and reduced the intra-lysosomal Cl⁻ concentration ([Cl⁻]_lys) via reduction of cytosolic Cl⁻ concentration ([Cl⁻]_c), showing abnormal accumulation of LC3II and p62 participating in autophagy function (dysfunction of autophagy) accompanied by inhibition of cell proliferation via G₀/G₁ arrest without induction of apoptosis. We also studied effects of direct modification of H⁺ transport on lysosomal acidification and autophagy. Application of bafilomycin A1 (an inhibitor of V-type H⁺-ATPase) or ethyl isopropyl amiloride [EIPA; an inhibitor of Na⁺/H⁺ exchanger (NHE)] elevated pH_lys and decreased [Cl⁻]_lys associated with inhibition of cell proliferation via induction of G₀/G₁ arrest similar to the culture under a low Cl⁻ condition. However, unlike low Cl⁻ condition, application of the compound, bafilomycin A1 or EIPA, induced apoptosis associated with increases in caspase 3 and 9 without large reduction in [Cl⁻]_c compared with low Cl⁻ condition. These observations suggest that the lowered [Cl⁻]_c primarily causes dysfunction of autophagy without apoptosis via dysfunction of lysosome induced by disturbance of intra-lysosomal acidification. This is the first study showing that cytosolic Cl⁻ is a key factor of lysosome acidification and autophagy.     

Extracellular Chloride Regulates the Epithelial Sodium Channel

The extracellular domain of the epithelial sodium channel ENaC is exposed to a wide range of Cl⁻ concentrations in the kidney and in other epithelia. We tested whether Cl⁻ alters ENaC activity. In Xenopus oocytes expressing human ENaC, replacement of Cl⁻ with SO₄²⁻, H₂PO₄⁻, or SCN⁻ produced a large increase in ENaC current, indicating that extracellular Cl⁻ inhibits ENaC. Extracellular Cl⁻ also inhibited ENaC in Na⁺-transporting epithelia. The anion selectivity sequence was SCN⁻ < SO₄²⁻ < H₂PO₄⁻ < F⁻ < I⁻ < Cl⁻ < Br⁻. Crystallization of ASIC1a revealed a Cl⁻ binding site in the extracellular domain. We found that mutation of corresponding residues in ENaC (α_H418A and β_R388A) disrupted the response to Cl⁻, suggesting that Cl⁻ might regulate ENaC through an analogous binding site. Maneuvers that lock ENaC in an open state (a DEG mutation and trypsin) abolished ENaC regulation by Cl⁻. The response to Cl⁻ was also modulated by changes in extracellular pH; acidic pH increased and alkaline pH reduced ENaC inhibition by Cl⁻. Cl⁻ regulated ENaC activity in part through enhanced Na⁺ self-inhibition, a process by which extracellular Na⁺ inhibits ENaC. Together, the data indicate that extracellular Cl⁻ regulates ENaC activity, providing a potential mechanism by which changes in extracellular Cl⁻ might modulate epithelial Na⁺ absorption.The epithelial Na⁺ channel ENaC² is a heterotrimer of homologous α, β, and γ subunits (1, 2). ENaC functions as a pathway for Na⁺ absorption across epithelial cells in the kidney collecting duct, lung, distal colon, and sweat duct (reviewed in Refs. 3 and 4). Na⁺ transport is critical for the maintenance of Na⁺ homeostasis and for the control of the composition and quantity of the fluid on the apical membrane of these epithelia. ENaC mutations and defects in its regulation cause inherited forms of hypertension and hypotension (5) and may contribute to the pathogenesis of lung disease in cystic fibrosis (6).ENaC is a member of the DEG/ENaC family of ion channels. A common structural feature of these channels is a large extracellular domain that plays a critical role in channel gating. For example, in ASICs, the extracellular domain functions as a receptor for protons, which transiently activate the channel by titrating residues that form an acidic pocket (7). FaNaCh is a ligand-gated family member in Helix aspersa, activated by the peptide FMRFamide (8). In Caenorhabditis elegans MEC family members, the extracellular domain is thought to respond to mechanical signals (9).ENaC differs from other family members because it is constitutively active in the absence of a ligand/stimulus. However, a convergence of data indicate that ENaC gating is modulated by a variety of molecules that bind to or modify its extracellular domains, including proteases (10–12), Na⁺ (13–15), protons (16), and the divalent cations Zn²⁺ and Ni²⁺ (17, 18). These findings suggest that the ENaC extracellular domain might regulate epithelial Na⁺ transport by sensing and integrating diverse signals in the extracellular environment.In the current study, we tested the hypothesis that ENaC activity is regulated by changes in the extracellular Cl⁻ concentration. Several observations suggested that Cl⁻ might be a strong candidate to regulate the channel. First, transport of Na⁺ and Cl⁻ are often coupled to maintain electroneutrality. Second, ENaC is exposed to large changes in extracellular Cl⁻ concentration. For example, in the kidney collecting duct, the urine Cl⁻ concentration varies widely (19). As the predominant anion, its concentration parallels that of Na⁺ in most clinical states. However, under conditions of metabolic alkalosis and metabolic acidosis, the Na⁺ and Cl⁻ concentrations can become dissociated as a result of increased urinary bicarbonate (alkalosis) or ammonium (acidosis) (19). Thus, ENaC is well positioned to respond to changes in Cl⁻ concentration. Third, crystallization of ASIC1a revealed a binding site for a Cl⁻ ion at the base of the thumb domain (7). The Cl⁻ is coordinated by Arg-310 and Glu-314 from one subunit and Lys-212 from an adjacent subunit. Although the functional role of Cl⁻ binding to ASIC1a is unknown, it supports the hypothesis that extracellular Cl⁻ might regulate the activity of DEG/ENaC ion channels.     

P2X4 Activation Modulates Volume-sensitive Outwardly Rectifying Chloride Channels in Rat Hepatoma Cells

Volume-sensitive outwardly rectifying (VSOR) Cl⁻ channels are critical for the regulatory volume decrease (RVD) response triggered upon cell swelling. Recent evidence indicates that H₂O₂ plays an essential role in the activation of these channels and that H₂O₂ per se activates the channels under isotonic isovolumic conditions. However, a significant difference in the time course for current onset between H₂O₂-induced and hypotonicity-mediated VSOR Cl⁻ activation is observed. In several cell types, cell swelling induced by hypotonic challenges triggers the release of ATP to the extracellular medium, which in turn, activates purinergic receptors and modulates cell volume regulation. In this study, we have addressed the effect of purinergic receptor activation on H₂O₂-induced and hypotonicity-mediated VSOR Cl⁻ current activation. Here we show that rat hepatoma cells (HTC) exposed to a 33% hypotonic solution responded by rapidly activating VSOR Cl⁻ current and releasing ATP to the extracellular medium. In contrast, cells exposed to 200 μm H₂O₂ VSOR Cl⁻ current onset was significantly slower, and ATP release was not detected. In cells exposed to either 11% hypotonicity or 200 μm H₂O₂, exogenous addition of ATP in the presence of extracellular Ca²⁺ resulted in a decrease in the half-time for VSOR Cl⁻ current onset. Conversely, in cells that overexpress a dominant-negative mutant of the ionotropic receptor P2X4 challenged with a 33% hypotonic solution, the half-time for VSOR Cl⁻ current onset was significantly slowed down. Our results indicate that, at high hypotonic imbalances, swelling-induced ATP release activates the purinergic receptor P2X4, which in turn modulates the time course of VSOR Cl⁻ current onset in a extracellular Ca²⁺-dependent manner.     

Functional Characterization Improves Associations between Rare Non-Synonymous Variants in CHRNB4 and Smoking Behavior

Smoking is the leading cause of preventable death worldwide. Accordingly, effort has been devoted to determining the genetic variants that contribute to smoking risk. Genome-wide association studies have identified several variants in nicotinic acetylcholine receptor genes that contribute to nicotine dependence risk. We previously undertook pooled sequencing of the coding regions and flanking sequence of the CHRNA5, CHRNA3, CHRNB4, CHRNA6 and CHRNB3 genes and found that rare missense variants at conserved residues in CHRNB4 are associated with reduced risk of nicotine dependence among African Americans. We identified 10 low frequency (<5%) non-synonymous variants in CHRNB4 and investigated functional effects by co-expression with normal α3 or α4 subunits in human embryonic kidney cells. Voltage-clamp was used to obtain acetylcholine and nicotine concentration–response curves and qRT-PCR, western blots and cell-surface ELISAs were performed to assess expression levels. These results were used to functionally weight genetic variants in a gene-based association test. We find that there is a highly significant correlation between carrier status weighted by either acetylcholine EC₅₀ (β = −0.67, r² = 0.017, P = 2×10⁻⁴) or by response to low nicotine (β = −0.29, r² = 0.02, P = 6×10⁻⁵) when variants are expressed with the α3 subunit. In contrast, there is no significant association when carrier status is unweighted (β = −0.04, r² = 0.0009, P = 0.54). These results highlight the value of functional analysis of variants and the advantages to integrating such data into genetic studies. They also suggest that an increased sensitivity to low concentrations of nicotine is protective from the risk of developing nicotine dependence.     

Role of reactive oxygen species generated by NADPH oxidase in the mechanism of activation of K+-Cl--cotransport by N-ethylmaleimide in HepG2 human hepatoma cells

K⁺-Cl^--cotransport (KCC) is ubiquitously present in all cells, and plays an essential role in ion and volume regulation. In this study we investigated the role of reactive oxygen species (ROS) in regulation of KCC in HepG2 human hepatoblastoma cells. N-ethylmaleimide (NEM), a KCC activator, induced Cl^--dependent K⁺ efflux, which was markedly prevented by KCC inhibitors (calyculin-A, genistein and BaCl₂), indicating that KCC is activated by NEM in the HepG2 cells. Treatment with NEM also induced a sustained increase in the level of intracellular ROS assessed by 2′,7′-dichlorofluorescein flourescence. Antioxidants, N-acetyl cysteine or N,N′-diphenyl-p-phenylenediamine significantly inhibited both ROS generation and KCC activation induced by NEM. The NEM-induced ROS production was significantly suppressed by inhibitors of NADPH oxidase (diphenylene iodonium, apocynin and neopterine). These inhibitors also significantly inhibited the NEM-induced KCC activation. Taken together, these results suggest that ROS generated by NADPH oxidase may mediate the NEM-induced activation of KCC in human hepatoma cells.     

Interactions between permeation and gating in the TMEM16B/anoctamin2 calcium-activated chloride channel

At least two members of the TMEM16/anoctamin family, TMEM16A (also known as anoctamin1) and TMEM16B (also known as anoctamin2), encode Ca²⁺-activated Cl⁻ channels (CaCCs), which are found in various cell types and mediate numerous physiological functions. Here, we used whole-cell and excised inside-out patch-clamp to investigate the relationship between anion permeation and gating, two processes typically viewed as independent, in TMEM16B expressed in HEK 293T cells. The permeability ratio sequence determined by substituting Cl⁻ with other anions (P_X/P_Cl) was SCN⁻ > I⁻ > NO₃⁻ > Br⁻ > Cl⁻ > F⁻ > gluconate. When external Cl⁻ was substituted with other anions, TMEM16B activation and deactivation kinetics at 0.5 µM Ca²⁺ were modified according to the sequence of permeability ratios, with anions more permeant than Cl⁻ slowing both activation and deactivation and anions less permeant than Cl⁻ accelerating them. Moreover, replacement of external Cl⁻ with gluconate, or sucrose, shifted the voltage dependence of steady-state activation (G-V relation) to more positive potentials, whereas substitution of extracellular or intracellular Cl⁻ with SCN⁻ shifted G-V to more negative potentials. Dose–response relationships for Ca²⁺ in the presence of different extracellular anions indicated that the apparent affinity for Ca²⁺ at +100 mV increased with increasing permeability ratio. The apparent affinity for Ca²⁺ in the presence of intracellular SCN⁻ also increased compared with that in Cl⁻. Our results provide the first evidence that TMEM16B gating is modulated by permeant anions and provide the basis for future studies aimed at identifying the molecular determinants of TMEM16B ion selectivity and gating.     

Differences in the Large Extracellular Loop between the K+-Cl? Cotransporters KCC2 and KCC4

K⁺Cl⁻ cotransporters (KCCs) play fundamental physiological roles in processes such as inhibitory neurotransmission and cell volume regulation. Mammalian genomes encode four distinct KCC paralogs, which share basic transport characteristics but differ significantly in ion affinity, pharmacology, and relative sensitivity to cell volume. Studies to identify divergence in functional characteristics have thus far focused on the cytoplasmic termini. Here, we investigated sequence requirements of the large extracellular loop (LEL) for function in KCC2 and KCC4. Mutation of all four evolutionarily conserved cysteines abolished KCC2 transport activity. This behavior differs from that of its closest relative, KCC4, which is insensitive to this mutation. Chimeras supported the differences in the LEL of the two cotransporters, because swapping wild-type LEL resulted in functional KCC2 but rendered KCC4 inactive. Insertion of the quadruple cysteine substitution mutant of the KCC4 loop, which was functional in the parental isoform, abolished transport activity in KCC2. Dose-response curves of wild-type and chimeric KCCs revealed that the LEL contributes to the different sensitivity to loop diuretics; a KCC2 chimera containing the KCC4 LEL displayed an IC₅₀ of 396.5 μm for furosemide, which was closer to KCC4 (548.8 μm) than to KCC2 (184.4 μm). Cell surface labeling and immunocytochemistry indicated that mutations do not affect trafficking to the plasma membrane. Taken together, our results show a dramatic and unexpected difference in the sequence requirements of the LEL between the closely related KCC2 and KCC4. Furthermore, they demonstrate that evolutionarily highly conserved amino acids can have different functions within KCC members.     

Swelling activated Cl- channels in microglia: Biophysics, pharmacology and role in glutamate release

Microglia have a swelling-activated Cl⁻ current (which we call I_Clswell), and while some of its biophysical properties and functional roles have been elucidated, its molecular identity is unknown. To relate this current to cell functions and determine whether it is regulated by mechanisms other than cell swelling, it is important to establish both biophysical and pharmacological fingerprints. Here, we used rat microglia and a cell line derived from them (MLS-9) to study biophysical, regulatory and pharmacological properties of I_Clswell. The whole-cell current was activated in response to a hypo-osmotic bath solution, but not by voltage, and was time-independent during long voltage steps. The halide selectivity sequence was I⁻>Br⁻>Cl⁻ (Eisenman sequence I) and importantly, the excitatory amino acid, glutamate was permeant. Current activation required internal ATP, and was not affected by the guanine nucleotides, GTPγS or GDPβS, or physiological levels of internal Mg²⁺. The same current was activated by a low intracellular ionic strength solution without an osmotic gradient. I_Clswell was reversibly inhibited by known Cl⁻ channel blockers (NPP B, flufenamic acid, glibenclamide, DCPIB), and by the glutamate release inhibitor, riluzole. Cell swelling evoked glutamate release from primary microglia and MLS-9 cells, and this was inhibited by the blockers (above), and by IAA-94, but not by tamoxifen or the Na⁺/K⁺/Cl⁻ symport inhibitor, bumetanide. Together, these results confirm the similarity of I_Clswell in the two cell types, and point to a role for this channel in inflammation-mediated glutamate release in the CNS.Key words: rat microglia, MLS-9 cells, swelling-activated anion channels, VRAC, Cl⁻ channel biophysics, Cl⁻ channel pharmacology, ionic-strength, ATP-dependence, glutamate release     

Influences of chloride and hypochlorite on neutrophil extracellular trap formation

Background

The release by neutrophils of DNA-based extracellular traps (NETs) is a recently recognized innate immune phenomenon that contributes significantly to control of bacterial pathogens at tissue foci of infection. NETs have also been implicated in the pathogenesis of non-infectious diseases such as small vessel vasculitis, lupus and cystic fibrosis lung disease. Reactive oxygen species (ROS) are important mediators of NET generation (NETosis). Neutrophils with reduced ROS production, such as those from patients with chronic granulomatous disease or myeloperoxidase (MPO) deficiency, produce fewer NETs in response to inflammatory stimuli. To better understand the roles of various ROS in NETosis, we explore the role of MPO, its substrates chloride ion (Cl⁻) and hydrogen peroxide (H₂O₂), and its product hypochlorite (HOCl) in NETosis.

Findings

In human peripheral blood neutrophils, pharmacologic inhibition of MPO decreased NETosis. Absence of extracellular Cl⁻, a substrate for MPO, also reduced NETosis. While exogenous addition of H₂O₂ and HOCl stimulated NETosis, only exogenous HOCl could rescue NETosis in the setting of MPO inhibition. Neither pharmacological inhibition nor genetic deletion of MPO in murine neutrophils blocked NETosis, in contrast to findings in human neutrophils.

Conclusions

Our results pinpoint HOCl as the key ROS involved in human NETosis. This finding has implications for understanding innate immune function in diseases in which Cl⁻ homeostasis is disturbed, such as cystic fibrosis. Our results also reveal an example of significant species-specific differences in NET phenotypes, and the need for caution in extrapolation to humans from studies of murine NETosis.     

Phosphoregulation of K+‐Cl− cotransporters during cell swelling: Novel insights

The K⁺‐Cl^? cotransporters (KCCs) belong to the cation‐Cl^? cotransporter family and consist of four isoforms and many splice variants. Their main role is to promote electroneutral efflux of K⁺ and Cl^? ions across the surface of many cell types and, thereby, to regulate intracellular ion concentration, cell volume, and epithelial salt movement. These transport systems are induced by an increase in cell volume and are less active at lower intracellular [Cl^?] (Cl_i), but the mechanisms at play are still ill‐defined. In this work, we have exploited the Xenopus laevis expression system to study the role of lysine‐deficient protein kinases (WNKs), protein phosphatases 1 (PP1s), and SPS1‐related proline/alanine‐rich kinase (SPAK) in KCC4 regulation during cell swelling. We have found that WNK4 and PP1 regulate KCC4 activity as part of a common signaling module, but that they do not exert their effects through SPAK or carrier dephosphorylation. We have also found that the phosphatases at play include PP1α and PP1γ1, but that WNK4 acts directly on the PP1s instead of the opposite. Unexpectedly, however, both cell swelling and a T926A substitution in the C‐terminus of full‐length KCC4 led to higher levels of heterologous K⁺‐Cl^? cotransport and overall carrier phosphorylation. These results imply that the response to cell swelling must also involve allosteric‐sensitive kinase‐dependent phosphoacceptor sites in KCC4. They are thus partially inconsistent with previous models of KCC regulation.     

Chloride transporter KCC2-dependent neuroprotection depends on the N-terminal protein domain

Neurodegeneration is a serious issue of neurodegenerative diseases including epilepsy. Downregulation of the chloride transporter KCC2 in the epileptic tissue may not only affect regulation of the polarity of GABAergic synaptic transmission but also neuronal survival. Here, we addressed the mechanisms of KCC2-dependent neuroprotection by assessing truncated and mutated KCC2 variants in different neurotoxicity models. The results identify a threonine- and tyrosine-phosphorylation-resistant KCC2 variant with increased chloride transport activity, but they also identify the KCC2 N-terminal domain (NTD) as the relevant minimal KCC2 protein domain that is sufficient for neuroprotection. As ectopic expression of the KCC2-NTD works independently of full-length KCC2-dependent regulation of Cl⁻ transport or structural KCC2 C-terminus-dependent regulation of synaptogenesis, our study may pave the way for a selective neuroprotective therapeutic strategy that will be applicable to a wide range of neurodegenerative diseases.Neurodegeneration restricts neuron numbers during development but can become a serious issue in disease conditions such as temporal lobe epilepsy (TLE).¹ GABA-activated Cl⁻ channels contribute to activity-dependent refinement of neural networks by triggering the so-called giant depolarizing potentials providing developing neurons with a sense of activity essential for neuronal survival and co-regulation of excitatory glutamatergic and (inhibitory) GABAergic synapses.² By regulating transmembrane Cl⁻ gradients KCC2 plays a vital role in development and disease.³ In addition, KCC2 plays a protein structural role in spine formation through its C-terminal protein domain (CTD).^{4, 5} Hence, regulation of KCC2 expression and function is relevant for development and disease-specific plasticity of neural networks.^{6, 7, 8, 9}GlyR α3K RNA editing leads to proline-to-leucine substitution (P185L) in the ligand-binding domain and generates gain-of-function neurotransmitter receptors.^{10, 11, 12, 13} GlyR RNA editing is upregulated in the hippocampus of patients with TLE and leads to GlyR α3K^185L-dependent tonic inhibition of neuronal excitability associated with neurodegeneration.¹⁴ KCC2 expression promotes neuroprotection^{14, 15} but whether this involves regulation of transmembrane Cl⁻ gradient or protein structural role is a matter of debate.^{14, 15}Here, we assessed neuroprotection through several KCC2 variants in two different models of neurodegeneration including chronic neuronal silencing (α3K^185L model) and acute neuronal overexcitation (NMDA model).^{14, 15} The results identify a threonine- and tyrosine-phosphorylation-resistant KCC2 variant with increased Cl⁻ transport activity, but they also demonstrate that the N-terminal KCC2 protein domain (NTD) is sufficient for neuroprotection.     

Slc26a9 Is Inhibited by the R-region of the Cystic Fibrosis Transmembrane Conductance Regulator via the STAS Domain

SLC26 proteins function as anion exchangers, channels, and sensors. Previous cellular studies have shown that Slc26a3 and Slc26a6 interact with the R-region of the cystic fibrosis transmembrane conductance regulator (CFTR), (R)CFTR, via the Slc26-STAS (sulfate transporter anti-sigma) domain, resulting in mutual transport activation. We recently showed that Slc26a9 has both nCl⁻-HCO₃⁻ exchanger and Cl⁻ channel function. In this study, we show that the purified STAS domain of Slc26a9 (a9STAS) binds purified (R)CFTR. When Slc26a9 and (R)CFTR fragments are co-expressed in Xenopus oocytes, both Slc26a9-mediated nCl⁻-HCO₃⁻ exchange and Cl⁻ currents are almost fully inhibited. Deletion of the Slc26a9 STAS domain (a9-ΔSTAS) virtually eliminated the Cl⁻ currents with only a modest affect on nCl⁻-HCO₃⁻ exchange activity. Co-expression of a9-ΔSTAS and the (R)CFTR fragment did not alter the residual a9-ΔSTAS function. Replacing the Slc26a9 STAS domain with the Slc26a6 STAS domain (a6-a9-a6) does not change Slc26a9 function and is no longer inhibited by (R)CFTR. These data indicate that the Slc26a9-STAS domain, like other Slc26-STAS domains, binds CFTR in the R-region. However, unlike previously reported data, this binding interaction inhibits Slc26a9 ion transport activity. These results imply that Slc26-STAS domains may all interact with (R)CFTR but that the physiological outcome is specific to differing Slc26 proteins, allowing for dynamic and acute fine tuning of ion transport for various epithelia.Slc26 genes and proteins have attracted the attention of physiologists and geneticists. Why? Slc26a1 (Sat-1) was characterized as a Na⁺-independent SO₄²⁻ transporter (1). Given the transport characteristics of the founding member of the gene family, Slc26 proteins were assumed to be sulfate transporters. Disease phenotypes, clone characterization, and family additions demonstrate that the Slc26 proteins are anion transporters or channels (2–4). These proteins have varied tissue expression patterns. At one extreme, Slc26a5 in mammals is found in the hair cells of the inner ear (5), whereas Slc26a2 (DTDST) is virtually ubiquitous in epithelial tissues (2).Several Slc26 proteins are found in the epithelia of the lung, intestine, stomach, pancreas, and kidney, usually in apical membranes. Interestingly these are also tissues and membranes in which the cystic fibrosis transmembrane conductance regulator (CFTR)⁵ has been found functionally or by immunohistochemistry. Ko and co-workers (6–8) examined the distribution of Slc26a3 and Slc26a6 in HCO₃⁻ secretory epithelia, and asked if an interaction might occur between these Slc26 proteins and CFTR. In particular, these studies indicate that in expression systems, there is a reciprocal-stimulatory interaction of the STAS (sulfate transporter anti-sigma) domains of Slc26a3 and Slc26a6 with the regulatory region (R-region) of CFTR. These investigators hypothesized that this stimulatory interaction could account for the differences in pancreatic insufficiency and sufficiency observed in cystic fibrosis patients. Nevertheless, knock-out Slc26a6 mouse studies reveal more complicated cell and tissue physiology (see “Discussion”).Slc26a9 has been reported to be a Cl⁻-HCO₃⁻ exchanger (9, 10) or a large Cl⁻ conductance (3, 11, 12). Loriol and co-workers (12) indicated that SLC26A9 has a Cl⁻ conductance that may be stimulated by HCO₃⁻. Two other groups have indicated that the Cl⁻ conductance is not affected by the presence of HCO₃⁻ (10, 11). We have recently demonstrated that Slc26a9 functions as both an electrogenic nCl⁻-HCO₃⁻ exchanger and a Cl⁻ channel (10). Dorwart and colleagues (11) found that WNK kinases inhibited the SLC26A9 Cl⁻ conductance but that this effect was independent of kinase activity. One group has a preliminary report indicating that WNK3 decreased Cl⁻ uptake, whereas WNK4 increased Cl⁻ uptake via Slc26a9 expressed in Xenopus oocytes (13).Slc26a9 and CFTR are also co-expressed in several tissues. Slc26a9 protein has been localized to epithelia of the stomach and lung (9, 10, 14), although mRNA is also detectable in brain, heart, kidney, small intestine, thymus, and ovary (10). The R-region of CFTR was previously shown to increase the activity of Slc26a3 and Slc26a6 by interaction with STAS domains (6, 15, 16). Because Slc26a9 displays several different modes of ion transport, we asked if the R-region of CFTR would also increase the activity of Slc26a9. Our results indicate that the R-region of CFTR does interact with the STAS domain of Slc26a9. However, in the case of Slc26a9 this apparently similar interaction results in inhibition of Slc26a9 ion transport.     

Distal Histidine Stabilizes Bound O2 and Acts as a Gate for Ligand Entry in Both Subunits of Adult Human Hemoglobin

The role of the distal histidine in regulating ligand binding to adult human hemoglobin (HbA) was re-examined systematically by preparing His(E7) to Gly, Ala, Leu, Gln, Phe, and Trp mutants of both Hb subunits. Rate constants for O₂, CO, and NO binding were measured using rapid mixing and laser photolysis experiments designed to minimize autoxidation of the unstable apolar E7 mutants. Replacing His(E7) with Gly, Ala, Leu, or Phe causes 20–500-fold increases in the rates of O₂ dissociation from either Hb subunit, demonstrating unambiguously that the native His(E7) imidazole side chain forms a strong hydrogen bond with bound O₂ in both the α and β chains (ΔG_{His(E7)H-bond} ≈ −8 kJ/mol). As the size of the E7 amino acid is increased from Gly to Phe, decreases in k_O2′, k_NO′, and calculated bimolecular rates of CO entry (k_entry′) are observed. Replacing His(E7) with Trp causes further decreases in k_O2′, k_NO′, and k_entry′ to 1–2 μm⁻¹ s⁻¹ in β subunits, whereas ligand rebinding to αTrp(E7) subunits after photolysis is markedly biphasic, with fast k_O2′, k_CO′, and k_NO′ values ≈150 μm⁻¹ s⁻¹ and slow rate constants ≈0.1 to 1 μm⁻¹ s⁻¹. Rapid bimolecular rebinding to an open α subunit conformation occurs immediately after photolysis of the αTrp(E7) mutant at high ligand concentrations. However, at equilibrium the closed αTrp(E7) side chain inhibits the rate of ligand binding >200-fold. These data suggest strongly that the E7 side chain functions as a gate for ligand entry in both HbA subunits.     

ZnR/GPR39 upregulation of K+/Cl−-cotransporter 3 in tamoxifen resistant breast cancer cells

Expression of the zinc receptor, ZnR/GPR39, is increased in higher grade breast cancer tumors and cells. Zinc, its ligand, is accumulated at larger concentrations in the tumor tissue and can therefore activate ZnR/GPR39-dependent Ca²⁺ signaling leading to tumor progression. The K⁺/Cl⁻ co-transporters (KCC), activated by intracellular signaling, enhance breast cancer cell migration and invasion. We asked if ZnR/GPR39 enhances breast cancer cell malignancy by activating KCC. Activation of ZnR/GPR39 by Zn²⁺ upregulated K⁺/Cl⁻ co-transport activity, measured using NH₄⁺ as a surrogate to K⁺ while monitoring intracellular pH. Upregulation of NH₄⁺ transport was monitored in tamoxifen resistant cells with functional ZnR/GPR39-dependent Ca²⁺ signaling but not in MCF-7 cells lacking this response. The NH₄⁺ transport was Na⁺-independent, and we therefore focused on KCC family members. Silencing of KCC3, but not KCC4, expression abolished Zn²⁺-dependent K⁺/Cl⁻ co-transport, suggesting that KCC3 is mediating upregulated NH₄⁺ transport. The ZnR/GPR39-dependent KCC3 activation accelerated scratch closure rate, which was abolished by inhibiting KCC transport with [(DihydroIndenyl) Oxy] Alkanoic acid (DIOA). Importantly, silencing of either ZnR/GPR39 or KCC3 attenuated Zn²⁺-dependent scratch closure. Thus, a novel link between KCC3 and Zn²⁺, via ZnR/GPR39, promotes breast cancer cell migration and proliferation.     

The ClC-3 Cl?/H+ Antiporter Becomes Uncoupled at Low Extracellular pH

Adenovirus expressing ClC-3 (Ad-ClC-3) induces Cl⁻/H⁺ antiport current (I_ClC-3) in HEK293 cells. The outward rectification and time dependence of I_ClC-3 closely resemble an endogenous HEK293 cell acid-activated Cl⁻ current (ICl_acid) seen at extracellular pH ≤ 5.5. ICl_acid was present in smooth muscle cells from wild-type but not ClC-3 null mice. We therefore sought to determine whether these currents were related. ICl_acid was larger in cells expressing Ad-ClC-3. Protons shifted the reversal potential (E_rev) of I_ClC-3 between pH 8.2 and 6.2, but not pH 6.2 and 5.2, suggesting that Cl⁻ and H⁺ transport become uncoupled at low pH. At pH 4.0 E_rev was completely Cl⁻ dependent (55.8 ± 2.3 mV/decade). Several findings linked ClC-3 with native ICl_acid; 1) RNA interference directed at ClC-3 message reduced native ICl_acid; 2) removal of the extracellular “fast gate” (E224A) produced large currents that were pH-insensitive; and 3) wild-type I_ClC-3 and ICl_acid were both inhibited by (2-sulfonatoethyl)methanethiosulfonate (MTSES; 10–500 μm)-induced alkanethiolation at exposed cysteine residues. However, a ClC-3 mutant lacking four extracellular cysteine residues (C103_P130del) was completely resistant to MTSES. C103_P130del currents were still acid-activated, but could be distinguished from wild-type I_ClC-3 and from native ICl_acid by a much slower response to low pH. Thus, ClC-3 currents are activated by protons and ClC-3 protein may account for native ICl_acid. Low pH uncouples Cl⁻/H⁺ transport so that at pH 4.0 ClC-3 behaves as an anion-selective channel. These findings have important implications for the biology of Cl⁻/H⁺ antiporters and perhaps for pH regulation in highly acidic intracellular compartments.     

The NOD2 single nucleotide polymorphisms rs2066843 and rs2076756 are novel and common Crohn's disease susceptibility gene variants

Background

The aims were to analyze two novel NOD2 variants (rs2066843 and rs2076756) in a large cohort of patients with inflammatory bowel disease and to elucidate phenotypic consequences.

Methodology/Principal Findings

Genomic DNA from 2700 Caucasians including 812 patients with Crohn''s disease (CD), 442 patients with ulcerative colitis (UC), and 1446 healthy controls was analyzed for the NOD2 SNPs rs2066843 and rs2076756 and the three main CD-associated NOD2 variants p.Arg702Trp (rs2066844), p.Gly908Arg (rs2066847), and p.Leu1007fsX1008 (rs2066847). Haplotype and genotype-phenotype analyses were performed. The SNPs rs2066843 (p = 3.01×10⁻⁵, OR 1.48, [95% CI 1.23-1.78]) and rs2076756 (p = 4.01×10⁻⁶; OR 1.54, [95% CI 1.28-1.86]) were significantly associated with CD but not with UC susceptibility. Haplotype analysis revealed a number of significant associations with CD susceptibility with omnibus p values <10⁻¹⁰. The SNPs rs2066843 and rs2076756 were in linkage disequilibrium with each other and with the three main CD-associated NOD2 mutations (D''>0.9). However, in CD, SNPs rs2066843 and rs2076756 were more frequently observed than the other three common NOD2 mutations (minor allele frequencies for rs2066843 and rs2076756: 0.390 and 0.380, respectively). In CD patients homozygous for these novel NOD2 variants, genotype-phenotype analysis revealed higher rates of a penetrating phenotype (rs2076756: p = 0.015) and fistulas (rs2076756: p = 0.015) and significant associations with CD-related surgery (rs2076756: p = 0.003; rs2066843: p = 0.015). However, in multivariate analysis only disease localization (p<2×10⁻¹⁶) and behaviour (p = 0.02) were significantly associated with the need for surgery.

Conclusion/Significance

The NOD2 variants rs2066843 and rs2076756 are novel and common CD susceptibility gene variants.