We have previously shown that unstimulated granular duct cells of mouse mandibular gland contain a hyperpolarization-activated Cl- conductance with characteristics resembling the hyperpolarization-activated volume-sensitive Cl- channel (ClC-2). We now show that stimulation of these cells with forskolin, but not 1,9-dideoxyforskolin, activates a second whole cell Cl- conductance with properties resembling the cystic fibrosis transmembrane conductance regulator (CFTR). This conductance has a linear current-voltage relation and is not voltage activated. Its anion permeability sequence is Br- (1.96)>> NO3- (1.36)>> Cl- (1)>> I- (0.44), and its conductance sequence is Cl- (1)>> NO3- (0.66)>> Br- (0.34)>> I- (0.21). The current carried by this conductance is attenuated 65% by 1 mmol/l diphenylamine-2-carboxylate but is not affected by 0.1 mmol/l4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid or 0.1 mmol/l glibenclamide. The current can be activated by norepinephrine (1 mumol/l), evidently acting via beta-adrenergic receptors, since the effect of norepinephrine is inhibited by propranolol (1 mumol/l). We conclude that this adrenergically evoked conductance is due to CFTR, which has previously been shown to be expressed in salivary duct cells, and suggest that it may form part of the mechanism by which beta-adrenergic agonists modulate NaCl absorption by salivary ducts.

The common gating of CLC-1 has been shown to be inhibited by intracellular adenosine triphosphate (ATP) in acidic pH conditions. Such modulation is thought to be mediated by direct binding of ATP to the cystathionine β-synthase (CBS) domains at the C-terminal cytoplasmic region of CLC-1. Guided by the crystal structure of the C-terminal domain of CLC-5, we constructed a homology model of CLC-1's C terminus and mutated critical amino acid residues lining the potential ATP-binding site. The CLC-1 mutations V634A and E865A completely abolished the ATP inhibition of CLC-1, consistent with the loss of ATP binding seen with the corresponding mutations in CLC-5. Mutating two other residues, V613 and V860, also disrupted the ATP modulation of CLC-1. However, placing aromatic amino acids at position 634 increases the apparent ATP affinity. Mutant cycle analyses showed that the modulation effects of ATP and cytidine triphosphate on wild-type CLC-1 and the V634F mutant were nonadditive, suggesting that the side chain of amino acid at position 634 interacts with the base moiety of the nucleotide. The mutation effects of V634F and V613A on the ATP modulation were also nonadditive, which is consistent with the assertion suggested from the homology model that these two residues may both interact with the bound nucleotide. These results provide evidence for a direct ATP binding for modulating the function of CLC-1 and suggest an overall conserved architecture of the ATP-binding sites in CLC-1 and CLC-5. This study also demonstrates that CLC-1 is a convenient experimental model for studying the interaction of nucleotides/nucleosides with the CBS domain.

Recently, it was observed that the acetylcholine analogue carbachol induces a transient stimulation of an apical Cl(-) conductance in basolaterally depolarized rat distal colonic epithelium (Schultheiss et al., 2003). The further characterization of this conductance was the aim of the present study. All experiments were performed at basolaterally depolarized tissues (111.5 mmol.l(-1) KCl buffer at the serosal side); in the absence of a K(+) gradient, a Cl(-) current was driven across the apical membrane (107 mmol.l(-1) K gluconate/4.5 mmol.l(-1) KCl buffer on the mucosal side). Under these conditions, carbachol evoked an atropine-sensitive biphasic change in short-circuit current (I(SC)), consisting of a transient increase followed by a long-lasting decrease, suggesting a stimulation of apical Cl(-) conductance followed by an inhibition. This conductance was inhibited by SITS, but was resistant against glibenclamide, a blocker of CFTR. The carbachol-induced I(SC) was dependent on the presence of mucosal Ca(2+). Ionomycin, a Ca(2+) ionophore, mimicked the effect of carbachol. An antibody against bovine Ca(2+)-activated Cl(-) channel ClCa 1 stained rat colonic epithelial cells both at the cell membrane as well as intracellularly, suggesting that the action of Ca(2+) may be caused by a stimulation of a ClC a-type anion channel. The activation of apical Cl(-) conductance by carbachol was resistant against any blockers of the phospholipase C/IP3/protein kinase C pathway tested (e.g., U-73122, 2-ABP, Li(+), staurosporine), but was inhibited by the NO-synthase blocker L: -NNA. Vice versa, NO-donating compounds such as GEA 3162 or sodium nitroprusside evoked a transient increase of I(SC). Consequently, NO seems to be involved in the transient stimulation of apical Ca(2+)-dependent Cl(-) conductance after muscarinic receptor stimulation.

The chloride channel ClC-2 has been implicated in essential physiological functions, including cell-volume regulation and fluid secretion by specific epithelial tissues. Although ClC-2 is known to be activated by hyperpolarization and hypo-osmotic shock, the molecular basis for the regulation of this channel remains unclear. Here we show in the Xenopus oocyte expression system that the chloride-channel activity of ClC-2 is enhanced after treatment with the actin-disrupting agents cytochalasin and latrunkulin. These findings suggest that the actin cytoskeleton normally exerts an inhibitory effect on ClC-2 activity. An inhibitory domain was previously defined in the N-terminus of ClC-2, so we sought to determine whether this domain might interact directly with actin in binding assays in vitro. We found that a glutathione S-transferase fusion protein containing the inhibitory domain was capable of binding actin in overlay and co-sedimentation assays. Further, the binding of actin to this relatively basic peptide (pI 8.4) might be mediated through electrostatic interactions because binding was inhibited at high concentrations of NaCl with a half-maximal decrease in signal at 180 mM NaCl. This work suggests that electrostatic interactions between the N-terminus of ClC-2 and the actin cytoskeleton might have a role in the regulation of this channel.

ClC-1 plays an important part in the maintenance of membrane potential in the mammalian skeletal muscle. To investigate the phosphorylation sites responsible for the effect of PKC (protein kinase C) activator, we constructed 21 different ClC-1 mutants with mutations at predicted phosphorylation sites for PKC. The functional experiments were performed on both wild-type and mutant proteins (17 point mutants and 4 double mutants) expressed in Xenopus oocytes with two-electrode voltage-clamp recording. PMA (12-myristate 13-acetate), a PKC activator, caused a right shift of half-maximum activation potential (V(1/2)) significantly in the wild-type (from -42.9+/-4.4 to -13.7+/-1.7 mV; n = 8, P < 0.05) and most of the single mutants except the S892P (from -39.5+/-4.5 to -35.7+/-5.7 mV; n = 6) and S892D (from -10.2+/-4.9 to -9.6+/-3.5 mV; n = 4). S892D, a mutant mimicking PKC-mediated phosphorylation at position 892, can also mimic the effect of wild-type treated with PMA in V(1/2) value (-10.2+/-4.9 mV vs -13.7+/-1.7 mV, n = 4 - 8). However, S892A still had a significant response to PMA indicating that other sites responsible for PMA might exist. Thus double mutants are generated for the following analysis. The V(1/2) of double mutants, T891A/S892A, S892A/T893A and T891A/T893A, show no significant difference between before and after PMA treatment. We hypothesize that this structural modification results in the observed alteration of the gating properties of ClC-1 by PMA. In summary, our observations show that a C-terminal region Thr891-Ser892-Thr893, at least in part, responsible for the effect of PMA on ClC-1.

Barttin, a gene product of BSND, is one of four genes responsible for Bartter syndrome. Coexpression of barttin with ClC-K chloride channels dramatically induces the expression of ClC-K current via insertion of ClC-K-barttin complexes into plasma membranes. We previously showed that stably expressed R8L barttin, a disease-causing missense mutant, is retained in the endoplasmic reticulum (ER) of Madin-Darby canine kidney (MDCK) cells, with the barttin β-subunit remaining bound to ClC-K α-subunits (Hayama A, Rai T, Sasaki S, Uchida S. Histochem Cell Biol 119: 485-493, 2003). However, transient expression of R8L barttin in MDCK cells was reported to impair ClC-K channel function without affecting its subcellular localization. To investigate the pathogenesis in vivo, we generated a knockin mouse model of Bartter syndrome that carries the R8L mutation. These mice display disease-like phenotypes (hypokalemia, metabolic alkalosis, and decreased NaCl reabsorption in distal tubules) under a low-salt diet. Immunofluorescence and immunoelectron microscopy revealed that the plasma membrane localization of both R8L barttin and the ClC-K channel was impaired in these mice, and transepithelial chloride transport in the thin ascending limb of Henle's loop (tAL) as well as thiazide-sensitive chloride clearance were significantly reduced. This reduction in transepithelial chloride transport in tAL, which is totally dependent on ClC-K1/barttin, correlated well with the reduction in the amount of R8L barttin localized to plasma membranes. These results suggest that the major cause of Bartter syndrome type IV caused by R8L barttin mutation is its aberrant intracellular localization.

Chloride channel-3 (ClC-3) is suggested to be a component and/or a regulator of the volume-activated Cl(-) channel in the plasma membrane. However, ClC-3 is predominantly located inside cells and the role of intracellular ClC-3 in tumor growth is unknown. In this study, we found that the subcellular distribution of endogenous ClC-3 varied in a cell cycle-dependent manner in HeLa cells. During interphase, ClC-3 was distributed throughout the cell and it accumulated at various positions in different stages. In early G1, ClC-3 was mainly located in the nucleus. In middle G1, ClC-3 gathered around the nuclear periphery as a ring. In late G1, ClC-3 moved back into the nucleus, where it remained throughout S phase. In G2, ClC-3 was concentrated in the cytoplasm. When cells progressed from G2 to the prophase of mitosis, ClC-3 from the cytoplasm translocated into the nucleus. During metaphase and anaphase, ClC-3 was distributed throughout the cell except for around the chromosomes and was aggregated at the spindle poles and in between two chromosomes, respectively. ClC-3 was then again concentrated in the nucleus upon the progression from telophase to cytokinesis. These results reveal a cell cycle-dependent change of the subcellular distribution of ClC-3 and strongly suggest that ClC-3 has nucleocytoplasmic shuttling dynamics that may play key regulatory roles during different stages of the cell cycle in tumor cells.

A sensitive and reliable RP-HPLC method was developed using a C18 CLC-ODS (M) - 4.6x250 mm(2)column and gradient elution for the analysis of phenolic compounds in propolis raw material and its products. A procedure for the extraction of phenolic compounds using aqueous ethanol (90%) with the addition of veratraldehyde as the internal standard (IS) was developed allowing to quantify ten compounds: caffeic acid, coumaric acid, ferulic acid, cinnamic acid, aromadendrin-4'-methyl ether (AME), isosakuranetin, drupanin, artepellin C, baccharin, and 2,2-dimethyl-6-carboxyethenyl-2H-1-benzopyran acid (DCBEN). The developed method gave good detection response and linearity in the range of 20.83-533.33 microg/mL.

ClC-2 is a broadly expressed member of the voltage-gated ClC chloride channel family. In this study, we aimed to evaluate the role of the membrane lipid environment in ClC-2 function, and in particular the effect of cholesterol and ClC-2 distribution in membrane microdomains. Detergent-resistant and detergent-soluble microdomains (DSM) were isolated from stably transfected HEK293 cells by a discontinuous OptiPrep gradient. ClC-2 was found concentrated in detergent-insoluble membranes in basal conditions and relocalized to DSM upon cholesterol depletion by methyl-beta-cyclodextrin. As assessed by patch clamp recordings, relocalization was accompanied by acceleration of the activation kinetics of the channel. A similar distribution and activation pattern were obtained when cells were treated with the oxidant tert-butyl hydroperoxide and after ATP depletion. In both cases activation was prevented by cholesterol enrichment of cells. We conclude that the cholesterol environment regulates ClC-2 activity, and we provide evidence that the increase in ClC-2 activity in response to acute oxidative or metabolic stress involves relocalization of this channel to DSM.

OBJECTIVE

Cholangiolocellular carcinoma (CLC) is an extremely rare malignant liver tumor. It is thought to originate from the ductules and/or canals of Hering, where hepatic stem cells (HpSC) are located, but there are few reports on cancer stem cell markers in CLC. Thus, we evaluated the significance of cancer stem cell markers, including CD133, CD44, and EpCAM, in CLC.

METHODS

The subjects of this study were three patients with CLC and one patient with an intermediate type of combined hepatocellular cholangiocarcinoma (CHC). The cancer cell markers, CK7, CK19, and EMA, were evaluated immunohistochemically.

RESULTS

Histological examination of the CLC revealed morphologically cholangiolar features and immunohistochemical examination revealed positivity for CD133, CD44, and EpCAM. On the other hand, in the intermediate type of CHC, only CD44 was positive, whereas CD133 and EpCAM were negative.

CONCLUSIONS

CLC may have stronger features derived from HpSCs than an intermediate type of CHC.

The constitutive reuptake of albumin from the glomerular filtrate by receptor-mediated endocytosis is a key function of the renal proximal tubules. Both the Cl- channel ClC-5 and the Na+-H+ exchanger isoform 3 are critical components of the macromolecular endocytic complex that is required for albumin uptake, and therefore the cell-surface levels of these proteins may limit albumin endocytosis. This study was undertaken to investigate the potential roles of the epithelial PDZ scaffolds, Na+-H+ exchange regulatory factors, NHERF1 and NHERF2, in albumin uptake by opossum kidney (OK) cells. We found that ClC-5 co-immunoprecipitates with NHERF2 but not NHERF1 from OK cell lysate. Experiments using fusion proteins demonstrated that this was a direct interaction between an internal binding site in the C terminus of ClC-5 and the PDZ2 module of NHERF2. In OK cells, NHERF2 is restricted to the intravillar region while NHERF1 is located in the microvilli. Silencing NHERF2 reduced both cell-surface levels of ClC-5 and albumin uptake. Conversely, silencing NHERF1 increased cell-surface levels of ClC-5 and albumin uptake, presumably by increasing the mobility of NHE3 in the membrane and its availability to the albumin uptake complex. Surface biotinylation experiments revealed that both NHERF1 and NHERF2 were associated with the plasma membrane and that NHERF2 was recruited to the membrane in the presence of albumin. The importance of the interaction between NHERF2 and the cytoskeleton was demonstrated by a significant reduction in albumin uptake in cells overexpressing an ezrin binding-deficient mutant of NHERF2. Thus NHERF1 and NHERF2 differentially regulate albumin uptake by mechanisms that ultimately alter the cell-surface levels of ClC-5.

Chloride channels in the kidney are involved in important physiological functions such as cell volume regulation, acidification of intracellular vesicles, and transepithelial chloride transport. Among eight mammalian CLC chloride channels expressed in the kidney, three (CLC-K1, CLC-K2, and CLC-5) were identified to be related to kidney diseases in humans or mice. CLC-K1 mediates a transepithelial chloride transport in the thin ascending limb of Henle's loop and is essential for urinary concentrating mechanisms. CLC-K2 is a basolateral chloride channel in distal nephron segments and is necessary for chloride reabsorption. CLC-5 is a chloride channel in intracellular vesicles of proximal tubules and is involved in endocytosis. This review will cover the recent advances in research on the CLC chloride channels of the kidney with a special focus on the issues most necessary to understand their physiological roles in vivo, i.e., their intrarenal and cellular localization and their phenotypes of humans and mice that have their loss-of-function mutations.

In mammalian nonpigmented ciliary epithelial (NPE) cells, hyposmotic stimulation leading to cell swelling activates an outwardly rectifying Cl(-) conductance (I(Cl,swell)), which, in turn, results in regulatory volume decrease. The aim of this study was to determine whether increased trafficking of intracellular ClC-3 Cl channels to the plasma membrane could contribute to the I(Cl,swell) following hyposmotic stimulation. Our results demonstrate that hyposmotic stimulation reversibly activates an outwardly rectifying Cl(-) current that is inhibited by phorbol-12-dibutyrate and niflumic acid. Transfection with ClC-3 antisense, but not sense, oligonucleotides reduced ClC-3 expression as well as I(Cl,swell). Intracellular dialysis with 2 different ClC-3 antibodies abolished activation of I(Cl,swell). Immunofluorescence microscopy showed that hyposmotic stimulation increased ClC-3 immunoreactivity at the plasma membrane. To determine whether this increased expression of ClC-3 at the plasma membrane could be due to increased vesicular trafficking, we examined membrane dynamics with the fluorescent membrane dye FM1-43. Hyposmotic stimulation rapidly increased the rate of exocytosis, which, along with ICl,swell, was inhibited by the phosphoinositide-3-kinase inhibitor wortmannin and the microtubule disrupting agent, nocodazole. These findings suggest that ClC-3 channels contribute to I(Cl,swell) following hyposmotic stimulation through increased trafficking of channels to the plasma membrane.

Bisphenol A (BPA) and nonylphenol (NP) are well known endocrine-disrupting chemicals (EDCs) ubiquitous in the aquatic environment and are an ecotoxicological risk for the health of aquatic organisms. Limited attention has been given to the immunotoxicity of these chemicals. The present study revealed a concentration-dependent increase of reactive oxygen species content and an induced expression of redox-sensitive transcription factors in zebrafish embryos after exposure to various concentrations of BPA, NP, and BPA/NP mixture for 4 h to 168 h postfertilization. Transcription of genes related to the immune response, including IFNγ, IL1β, IL10, Mx, TNFα, CC-chemokine, and CXCL-clc, were significantly up-regulated on exposure to EDCs. A significant induction of concentrations of proinflammatory mediator, nitric oxide, accompanied by an increase in the activity of nitric oxide synthase (NOS) and an upregulation of inducible NOS gene expression, was detected in zebrafish embryos on exposures to EDCs. To elucidate the potential mechanisms by which BPA and NP activate the innate immune response, expression profiles of genes related to the Toll-like receptors (TLRs) signaling pathway were examined. Expressions of TLR3, TRIF, MyD88, SARM, IRAK4, and TRAF6 were altered on exposure to EDCs. The authors' results demonstrate that exposure to BPA and NP significantly affects the expression of genes related to immune response in zebrafish embryos following oxidative stress.

ClC-3, a gene encoding a candidate protein for volume-activated chloride (C(-)) channels, may be involved in tumor development. Herein we report a study using an antisense "knock-down" strategy to investigate the mechanism by which ClC-3 affects cell proliferation in nasopharyngeal carcinoma CNE-2Z cells. With immunoblots and MTT assays we demonstrated that the expression of ClC-3 was cell cycle dependent and in a similar concentration-dependent manner, an antisense oligonucleotide specific for ClC-3 inhibited ClC-3 protein expression and cell proliferation. The expression level of ClC-3 correlated with cell proliferation. Moreover, in the cells exposed to a ClC-3 antisense oligonucleotide, the cloning efficiency was inhibited, and cells were arrested in the S phase. The ClC-3 antisense oligonucleotide inhibited the volume-activated C(-) current (I(Cl,vol)) and the regulatory volume decrease (RVD) in a concentration-dependent manner. Additionally, the I(Cl,vol) or RVD was positively correlated with cell proliferation in the treated cells. In conclusion, ClC-3 is involved in cell proliferation and cell cycle progression through a mechanism involving modulation of I(Cl,vol) and RVD. CIC-3 may represent a therapeutic target in human cancer.

OBJECTIVE

To assess the rate of bile duct injuries (BDI) and overall biliary complications during single-port laparoscopic cholecystectomy (SPLC) compared to conventional laparoscopic cholecystectomy (CLC).

METHODS

SPLC has recently been proposed as an innovative surgical approach for gallbladder surgery. So far, its safety with respect to bile duct injuries has not been specifically evaluated. A systematic review of the literature published between January 1990 and November 2012 was performed. Randomized controlled trials (RCT) comparing SPLC versus CLC reporting BDI rate and overall biliary complications were included. The quality of RCT was assessed using the Jadad score. Analysis was made by performing a meta-analysis, using Review Manager 5.2. This study was based on the Preferred Reporting Items for Systematic reviews and Meta-Analyses guidelines. A retrospective study including all retrospective reports on SPLC was also performed alongside.

RESULTS

From 496 publications, 11 RCT including 898 patients were selected for meta-analysis. No studies were rated as high quality (Jadad score ≥ 4). Operative indications included benign gallbladder disease operated in an elective setting in all studies, excluding all emergency cases and acute cholecystitis. The median follow-up was 1 mo (range 0.03-18 mo). The incidence of BDI was 0.4% for SPLC and 0% for CLC; the difference was not statistically different (P = 0.36). The incidence of overall biliary complication was 1.6% for SPLC and 0.5% for CLC, the difference did not reached statistically significance (P = 0.21, 95%CI: 0.66-15). Sixty non-randomized trials including 3599 patients were also analysed. The incidence of BDI reported then was 0.7%.

CONCLUSIONS

The safety of SPLC cannot be assumed, based on the current evidence. Hence, this new technology cannot be recommended as standard technique for laparoscopic cholecystectomy.

The striatum is the main basal ganglia input nucleus, receiving extensive glutamatergic inputs from cortex and thalamus. Medium spiny striatal projection neurons (MSNs) are GABAergic, and their axon collaterals synapse on other MSNs. Approximately 50% of MSNs corelease substance P (SP), but how this neurotransmitter controls MSN activity is poorly understood. We used whole-cell recordings to investigate how SP affects MSNs and their glutamatergic inputs. SP elicited slow depolarizations in 47/90 MSNs, which persisted in the presence of tetrodotoxin (TTX). SP responses were mimicked by the NK1 receptor agonist [Sar9,Met(O(2))11]-substance P (SSP), and fully blocked by the NK1 receptor antagonists L-732,138, or extracellular zinc. When intracellular chloride was altered, the polarity of SP responses depended on the sign of the chloride driving force. In voltage-clamp, SP-induced currents reversed around -68 mV and displayed marked inward rectification. These data indicate that SP increased a ClC-2-type chloride conductance in MSNs, acting through NK1 receptors. SP also strongly increased glutamatergic responses in 49/89 MSNs. Facilitation of glutamatergic responses (which was observed both in MSNs directly affected by SP and in non-affected ones) was reduced by application of L-732,138, and fully blocked by coapplication of L-732,138 and SB222200 (an NK3 receptor antagonists), showing that both NK1 and NK3 receptors were involved. SP-induced facilitation of glutamatergic responses was accompanied by a marked decrease in paired-pulse ratio, indicating a presynaptic mechanism of action. These data provide an electrophysiological correlate for the anatomically known connections between SP-positive MSN terminals and the dendrites and somata of other MSNs.

Protein crystallization in human tissue rarely occurs. Charcot-Leyden crystals (CLCs) were described in various eosinophilic diseases >150 years ago, but our understanding of CLC formation still remains limited. In this study, we demonstrate that CLCs observed in varied inflamed human tissues are closely associated with eosinophil cell-free granules and nuclear envelope/plasma membrane disintegration with release of filamentous chromatin (extracellular traps), typical morphologies of a regulated pathway of extracellular trap cell death (ETosis). During the process of eosinophil ETosis, eccentrically localized cytoplasmic and perinuclear CLC protein (galectin-10) is homogeneously redistributed in the cytoplasm. Rapid (1-2 minutes) formation of intracytoplasmic CLCs was observed using time-lapse imaging. Plasma membrane rupture enabled the release of both intracellularly formed CLCs and soluble galectin-10 that further contributed to formation of CLCs extracellularly, in parallel with the expulsion of free intact granules and extracellular traps. CLC formation and galectin-10 release were dependent on nicotinamide adenine dinucleotide phosphate oxidase activation. To our knowledge, this is the first demonstration of natural formation of CLCs in association with an active physiological process (ie, ETosis). These results indicate that dynamic changes in intracellular localization and release of galectin-10 contribute to CLC formation in vivo and suggest that CLC/galectin-10 might serve as an indicator of ETosis.

Dysfunction of the proximal tubule (PT) is associated with variable degrees of solute wasting and low-molecular-weight proteinuria. We measured metabolic consequences and adaptation mechanisms in a model of inherited PT disorders using PT cells of ClC-5-deficient (Clcn5Y/-) mice, a well-established model of Dent's disease. Compared to cells taken from control mice, those from the mutant mice had increased expression of markers of proliferation (Ki67, proliferative cell nuclear antigen (PCNA), and cyclin E) and oxidative scavengers (superoxide dismutase I and thioredoxin). Transcriptome and protein analyses showed fourfold induction of type III carbonic anhydrase in a kidney-specific manner in the knockout mice located in scattered PT cells. Kidney-specific carbonic anhydrase type III (CAIII) upregulation was confirmed in other mice lacking the multiligand receptor megalin and in a patient with Dent's disease due to an inactivating CLCN5 mutation. The type III enzyme was specifically detected in the urine of mice lacking ClC-5 or megalin, patients with Dent's disease, and in PT cell lines exposed to oxidative stress. Our study shows that lack of PT ClC-5 in mice and men is associated with CAIII induction, increased cell proliferation, and oxidative stress.

In plant cells, anion channels and transporters are essential for key functions such as nutrition, ion homeostasis and resistance to biotic or abiotic stresses. We characterized AtCLCg, a member of the chloride channel (CLC) family in Arabidopsis localized in the vacuolar membrane. When grown on NaCl or KCl, atclcg knock-out mutants showed a decrease in biomass. In the presence of NaCl, these mutants overaccumulate chloride in shoots. No difference in growth was detected in response to osmotic stress by mannitol. These results suggest a physiological function of AtCLCg in the chloride homeostasis during NaCl stress. AtCLCg shares a high degree of identity (62%) with AtCLCc, another vacuolar CLC essential for NaCl tolerance. However, the atclcc atclccg double mutant is not more sensitive to NaCl than single mutants. As the effects of both mutations are not additive, gene expression analyses were performed and revealed that: (i)AtCLCg is expressed in mesophyll cells, hydathodes and phloem while AtCLCc is expressed in stomata; and (ii)AtCLCg is repressed in the atclcc mutant background, and vice versa. Altogether these results demonstrate that both AtCLCc and AtCLCg are important for tolerance to excess chloride but not redundant, and form part of a regulatory network controlling chloride sensitivity.

Osteopetrosis is a group of genetic bone disorders. There are three types of osteopetrosis: autosomal recessive osteopetrosis (ARO), autosomal dominant osteopetrosis type II (ADO II), and intermediate autosomal recessive osteopetrosis (IARO). The prevalence of ADO II is about 1:100,000, while no more than 20 cases of IARO have been reported worldwide. We present the first Chinese IARO patient with a novel homozygous variant in CLCN7 gene (p. Pro470Leu) and an ADO II patient with a heterozygous variant in CLCN7 gene (p. Arg286Trp). In addition to general osteosclerosis, the striking features of these two patients are unerupted teeth with root dysplasia. We speculate that ClC-7 in different tooth cells may contribute directly to the root development, the defect of ClC-7 may have a dose dependent effect on the phenotype of root dysplasia, and the tooth position may also affect the root phenotype with dysfunctional ClC-7.

OBJECTIVE

Although clinical reports have described infantile malignant autosomal recessive osteopetrosis (ARO) in Indian patients, no published data are available about the genetic causes of ARO in this population. We investigated the main genetic causes of ARO in eight Indian patients with early postnatal onset and the typical severe clinical course including visual impairment and anaemia.

METHODS

Mutation screening in the genes CLCN7 and TCIRG1 was done on genomic DNA from 8 affected individuals (diagnosed on the basis of clinical and haematological parameters and characteristic radiological changes of increased bone density) and their parents. In one family, after detection of both mutations in the proband, targeted mutation analysis was also done in chorionic villus samples for prenatal diagnosis.

RESULTS

Six patients had mutations in TCIRG1 and two patients harboured mutations in CLCN7 gene. Three of the five different TCIRG1 mutations identified and both CLCN7 mutations were novel mutations. Except for the already known mutation p.Ile720del, all TCIRG1 mutations disrupt conserved splice consensus sequences or lead to premature stop codons. In contrast, both CLCN7 mutations only lead to missense changes of conserved amino acids. In a foetus harbouring TCIRG1 mutations osteopetrosis was visible radiologically at 23 wk of gestation.

CONCLUSIONS

That the CLCN7 mutations provoke a phenotype as severe as the one caused by TCIRG1 loss of function suggests the affected residues to be crucial for the function of the ClC-7 chloride channel or chloride/proton-exchanger. Our data also show that ARO can manifest as early as in the second trimester of pregnancy.

Transmembrane proton transport is of fundamental importance for life. The list of H(+) transporting proteins has been recently expanded with the discovery that some members of the CLC gene family are stoichiometrically coupled Cl(-)/H(+) antiporters. Other CLC proteins are instead passive Cl(-) selective anion channels. The gating of these CLC channels is, however, strongly regulated by pH, likely reflecting the evolutionary relationship with CLC Cl(-)/H(+) antiporters. The role of protons in the gating of the model Torpedo channel ClC-0 is best understood. ClC-0 is a homodimer with separate pores in each subunit. Each protopore can be opened and closed independently from the other pore by a "fast gate". A common, slow gate acts on both pores simultaneously. The opening of the fast gate is controlled by a critical glutamate (E166), whose protonation state determines the fast gate's pH dependence. Extracellular protons likely can arrive directly at E166. In contrast, protonation of E166 from the inside has been proposed to be mediated by the dissociation of an intrapore water molecule. The OH(-) anion resulting from the water dissociation is stabilized in one of the anion binding sites of the channel, competing with intracellular Cl(-) ions. The pH dependence of the slow gate is less well understood. It has been shown that proton translocation drives irreversible gating transitions associated with the slow gate. However, the relationship of the fast gate's pH dependence on the proton translocation and the molecular basis of the slow gate remain to be discovered.

Primary aldosteronism is the most common and curable form of secondary arterial hypertension. We performed whole-exome sequencing in patients with early-onset primary aldosteronism and identified a de novo heterozygous c.71G>A/p.Gly24Asp mutation in the CLCN2 gene, encoding the voltage-gated ClC-2 chloride channel 1 , in a patient diagnosed at 9 years of age. Patch-clamp analysis of glomerulosa cells of mouse adrenal gland slices showed hyperpolarization-activated Cl- currents that were abolished in Clcn2-/- mice. The p.Gly24Asp variant, located in a well-conserved 'inactivation domain'2,3, abolished the voltage- and time-dependent gating of ClC-2 and strongly increased Cl- conductance at resting potentials. Expression of ClC-2Asp24 in adrenocortical cells increased expression of aldosterone synthase and aldosterone production. Our data indicate that CLCN2 mutations cause primary aldosteronism. They highlight the important role of chloride in aldosterone biosynthesis and identify ClC-2 as the foremost chloride conductor of resting glomerulosa cells.