Cl Conductance Research Articles

CFTR and PC2, partners in the primary cilia in autosomal dominant polycystic kidney disease.

Defects in the primary cilium are associated with autosomal dominant polycystic kidney disease (ADPKD). We used a combination of animal models, Western blotting, and confocal microscopy and discovered that CFTR and polycystin 2 (PC2) are both colocalized to the cilium in normal kidneys, with the levels of both being decreased in cystic epithelia. Cilia were longer in CFTR-null mice and in cystic cells in our ADPKD animal models. We examined septin 2, known to play a role in cilia length, to act as a diffusion barrier and to serve as an enhancer of proliferation. We found that septin 2 protein levels were upregulated and colocalized strongly with CFTR in cystic cells. Application of VX-809, the CFTR corrector, restored CFTR and PC2 toward normal in the cilia, decreased the protein levels of septin 2, and drastically reduced septin 2 colocalization with CFTR. Our data suggest that CFTR is present in the cilia and plays a role there, perhaps through its conductance of Cl-. We also postulate that septin 2 is important for localizing CFTR to the apical membrane in cystic epithelia.NEW & NOTEWORTHY CFTR is present in the primary cilia together with polycystin 2 (PC2). Ablation of CFTR makes cilia longer suggesting that CFTR plays a role there, perhaps through its conductance of Cl.

Glycine-betaine induced salinity tolerance in maize by regulating the physiological attributes, antioxidant defense system and ionic homeostasis

The plants are exposed to different abiotic stresses, including the salinity stress (SS) that negatively affect the growth, metabolism, physiological and biochemical processes. Thus, this study investigated the effect of diverse levels of foliar-applied GB (0 control, 50 mM and 100 mM) on maize growth, membrane stability, physiological and biochemical attributes, antioxidant enzymes and nutrients accumulation under different levels of SS (i.e., control, 6 dS m-1, 12 dS m-1). Salt stress diminished the root and shoot length, root and shoot biomass, chlorophyll contents, photosynthetic rate (Pn), stomatal conductance (gs), relative water contents (RWC), soluble proteins (SP) and free amino acids; (FAA); and increased activities of antioxidant enzymes, electrical conductivity (EC) and accumulation of malondialdehyde (MDA), hydrogen peroxide (H2O2), Na+ and Cl− ions. GB application significantly increased root and shoot growth, leaves per plant, shoots length, chlorophyll contents, gs, Pn and membrane stability by reducing MDA and H2O2 accumulation. Moreover, GB also increased the SP, FAA accumulation, activities of antioxidant enzymes and Na+ and Cl- exclusion by favouring Ca2+ and K+ accumulation. In conclusion, the foliar-applied GB increased Pn, gs, ant-oxidants activities, and accumulation of SP and FAA; and reduced the accretion of Na+ and Cl− by favouring the Ca2+ and K+ accretion which in turns improved growth under SS.

Effects of exogenous oxytocin and atosiban antagonist on GABA in different region of brain

Gamma amino butyric acid (GABA) is the primary inhibitory neurotransmitter in the vertebral central nervous system. It functions by altering the membrane conductance of Cl− ions, maintaining the membrane potential close to the resting potential. The hormone oxytocin (OT) has a central action where it acts as a neuromodulatory peptide and exerts its action depending upon the distribution of OT receptors (OTR) in the target site. OTRs are G-protein-coupled receptors (GPCRs) comprising different subunits (Gq, Gi, and Gs). The G- protein isoforms have the ability to activate different pathways, but specific agonists and antagonists may show different affinities to OTRs, depending on the specific G-protein isoform to which they are coupled. It is well documented that OTR distribution varies with age and species and in regions of the brain. In this study, we attempted to observe the impact of OT and atosiban (OTA), an OT antagonist, on GABA levels in different regions of the brain. Study animals were exposed intraperitoneally (i.p.) to normal saline (0.89%), OT 0.0116 mg/kg, and OTA 1 mg/kg in different combinations, for 30days. It was observed that OT and OTA administration modulated GABA levels in different regions of brain, while normal saline had no effect. It may be due to OTR receptor expression in different regions of the brain.This is significant because region-specific expression of different receptors could be important in the development of new drugs targeting specific neuropsychiatric disorders.

Physiological roles and molecular mechanisms of K+ -Cl- cotransport in the mammalian kidney and cardiovascular system: where are we?

In the early 80s, renal microperfusion studies led to the identification of a basolateral K+ -Cl- cotransport mechanism in the proximal tubule, thick ascending limb of Henle and collecting duct. More than ten years later, this mechanism was found to be accounted for by three different K+ -Cl- cotransporters (KCC1, KCC3 and KCC4) that are differentially distributed along the renal epithelium. Two of these isoforms (KCC1 and KCC3) were also found to be expressed in arterial walls, the myocardium and a variety of neurons. Subsequently, valuable insights have been gained into the molecular and physiological properties of the KCCs in both the mammalian kidney and cardiovascular system. There is now robust evidence indicating that KCC4 sustains distal renal acidification and that KCC3 regulates myogenic tone in resistance vessels. However, progress in understanding the functional significance of these transporters has been slow, probably because each of the KCC isoforms is not identically distributed among species and some of them share common subcellular localizations with other KCC isoforms or sizeable conductive Cl- pathways. In addition, the mechanisms underlying the process of K+ -Cl- cotransport are still ill defined. The present review focuses on the knowledge gained regarding the roles and properties of KCCs in renal and cardiovascular tissues.

Membrane Permeabilizing Electric Fields Disrupt Water Channel Function and Selectivity

External MV/m electric fields can permeabilize cellular membranes, allowing influx and efflux of normally-impermeant molecules across the protective lipid bilayer. Utilization of short-duration permeabilizing electric fields (PEFs) have become widespread in the food industry, in biotechnology, and in medicine, where targeted high-voltage electric fields are used to transiently enhance the transport of water and other large molecules into and out of cells. However, despite the increasing use of PEFs in clinical and biological systems, their interactions with transmembrane proteins remain largely unknown. These limitations yield an incomplete picture of membrane electropermeabilization, where multiple components of the cell are likely perturbed (and possibly denatured) under PEFs. In order to test if PEFs affect transmembrane proteins, especially those that natively transport water, molecular dynamics simulations were carried out on human aquaporin 1 (hAQP1) water channels under cell-permeabilizing voltages. Simulations revealed significant changes to water orientation and flux through hAQP1 under PEFs, with voltage-dependent transport rates that diverge from diffusive and osmotic experiments. Surprisingly, hAQP1 was able to transport Cl anions (but not Na cations) when 1-2 volts was applied, despite its inability to do so under physiological conditions. The protein did not affect the construction of discrete lipid electropores around the periphery of the channel, or the timescales of lipid bilayer permeabilization, however a small reduction in the protein's alpha-helicity was detected. hAQP1 Cl conduction was also sensitive to the protonation state of the histidine residue located in the two-stage filter, suggesting that pH may also affect anion conduction. Taken together, these results suggest that PEFs have a direct effect on transmembrane water channels, even before membrane electropores interact with them. Further studies of additional membrane components under PEFs are necessary if optimization of therapeutic electric fields is to be significantly improved.

Peptide inhibitors of chloride channels for treating secretory diarrhea.

Morbidity and mortality associated with diarrheal diseases remain significant burdens on global health. In the developing world, the major sources of secretory diarrhea are infectious, including those caused by bacteria such as enterotoxic Escherichia coli, and viruses such as rotavirus. In many cases of secretory diarrhea, activation of pathways for cyclic nucleotides and/or Ca2+ signaling in the apical membrane of enterocytes increases the conductance of Cl- channels at the enterocyte lumen-facing membrane. Those channels include the cystic fibrosis transmembrane conductance regulator (CFTR) and Ca2+-activated Cl- channel (CaCC). Inhibition of enterocyte Cl- channels is an effective strategy for anti-secretory drug therapy. Small molecules and natural peptides with Cl- channel inhibitory activity have shown efficacy in diarrhea models. Screening of natural peptides via the patch-clamp technique provides evidence that such channel inhibition by an extract of black tea may be responsible for its anti-diarrhea benefits.

Unexpected Connections between Humidity and Ion Transport Discovered Using a Model to Bridge Guard Cell-to-Leaf Scales.

Stomatal movements depend on the transport and metabolism of osmotic solutes that drive reversible changes in guard cell volume and turgor. These processes are defined by a deep knowledge of the identities of the key transporters and of their biophysical and regulatory properties, and have been modeled successfully with quantitative kinetic detail at the cellular level. Transpiration of the leaf and canopy, by contrast, is described by quasilinear, empirical relations for the inputs of atmospheric humidity, CO2, and light, but without connection to guard cell mechanics. Until now, no framework has been available to bridge this gap and provide an understanding of their connections. Here, we introduce OnGuard2, a quantitative systems platform that utilizes the molecular mechanics of ion transport, metabolism, and signaling of the guard cell to define the water relations and transpiration of the leaf. We show that OnGuard2 faithfully reproduces the kinetics of stomatal conductance in Arabidopsis thaliana and its dependence on vapor pressure difference (VPD) and on water feed to the leaf. OnGuard2 also predicted with VPD unexpected alterations in K+ channel activities and changes in stomatal conductance of the slac1 Cl- channel and ost2 H+-ATPase mutants, which we verified experimentally. OnGuard2 thus bridges the micro-macro divide, offering a powerful tool with which to explore the links between guard cell homeostasis, stomatal dynamics, and foliar transpiration.

The effects of ozonated, chlorinated, celestite stone-treated, natural spring and pine resin-treated waters on performance, oxidative stress and carcass parameters in Japanese quail

This study was conducted to assess the effects of 5 types of water: Ozonated, Chlorinated, Celestite Stone-treated, Natural Spring Waters and Pin Resin-treated water on performance, oxidative stress and carcass parameters in Japanese quail. A total of 150, 3-day old mixed female-male quails were used into 5 groups of 30 birds for each water group; each group was divided into 3 replicate subgroups of 10 birds. The experiment was continued for 7 weeks and quail were fed one experimental diet, and the different water sources were given ad libitum during in the trial. Statistical differences among water sources were observed on pH and Electrical Conductivity and content of Ca, Mg, Cl and Total Bacteria. Live weight, daily live weight gain, water and feed intake were the highest in the drinkers of Natural Spring Water, while the lowest for drinkers of Resin-treated Water (p<0.001). The lowest level of serum malondialdehyde of was observed in drinkers of Natural Spring Water (1.54 µM/L), and the highest malondialdehyde level in drinkers of Resin-treated Water (4.27 µM/L; P<0.001). The sources of water of trial were determined to have no effects on slaughter weight, carcass weight and carcass yield (p>0.05). As a result, the amount of total dissolved solids of water were the positive effect on live weight, feed consumption, water intake, feed conversion ratio and oxidative stress biomarkers.

Contribution of Resting Conductance, GABAA-Receptor Mediated Miniature Synaptic Currents and Neurosteroid to Chloride Homeostasis in Central Neurons.

Maintenance of a low intraneuronal Cl– concentration, [Cl–]i, is critical for inhibition in the CNS. Here, the contribution of passive, conductive Cl– flux to recovery of [Cl–]i after a high load was analyzed in mature central neurons from rat. A novel method for quantifying the resting Cl– conductance, important for [Cl–]i recovery, was developed and the possible contribution of GABAA and glycine receptors and of ClC-2 channels to this conductance was analyzed. The hypothesis that spontaneous, action potential-independent release of GABA is important for [Cl–]i recovery was tested. [Cl–]i was examined by gramicidin-perforated patch recordings in medial preoptic neurons. Cells were loaded with Cl– by combining GABA or glycine application with a depolarized voltage, and the time course of [Cl–]i was followed by measurements of the Cl– equilibrium potential, as obtained from the current recorded during voltage ramps combined with GABA or glycine application. The results show that passive Cl– flux contributes significantly, in the same order of magnitude as does K+-Cl– cotransporter 2 (KCC2), to [Cl–]i recovery and that Cl– conductance accounts for ∼ 6% of the total resting conductance. A major fraction of this resting Cl– conductance is picrotoxin (PTX)-sensitive and likely due to open GABAA receptors, but ClC-2 channels do not contribute. The results also show that when the decay of GABAA receptor-mediated miniature postsynaptic currents (minis) is slowed by the neurosteroid allopregnanolone, such minis may significantly quicken [Cl–]i recovery, suggesting a possible steroid-regulated role for minis in the control of Cl– homeostasis.

Anoctamin2 (TMEM16B) forms the Ca2+-activated Cl− channel in the retinal pigment epithelium

Chloride channels (Cl channels) play an essential role for the retinal pigment epithelium (RPE). They provide a plasma membrane conductance for Cl− important for transepithelial transport and volume regulation. Ca2+-dependent chloride channels (CaCC) in the RPE were found to adapt Cl− transport to specific needs by increasing intracellular free Ca2+. Although a variety of Cl channels have been identified in the RPE, the molecular identity of the CaCC remains controversial. Sagittal sections of mouse retina were stained against anoctamin2 (Ano2) and analyzed by confocal microscopy. Membrane currents from ARPE-19 cells and primary murine RPE cells were recorded in the whole-cell configuration of the patch-clamp technique. Expression of Ano2 was assessed via immunocytochemistry, PCR and western-blot and down-regulated via siRNA approaches. In the mouse retina, Ano2 was found in the basolateral membrane of the RPE. In primary mouse RPE cells, Ano2 was localized predominantly in the cell membrane. Ano2 mRNA and protein were also detected in rat and primate RPE as well as ARPE-19 cells. Whole-cell currents were elicited by increasing intracellular free Ca2+ via ATP application. These currents were identified as Cl− currents by their reversal potential and blocker sensitivity. Knock-down of Ano2 by siRNA decreased both the Ca2+ dependent chloride conductance and protein expression of Ano2. The biophysical and pharmacological properties of CaCC in ARPE-19 and primary mouse RPE cells resemble those described in previous publications using RPE cells from different species. The siRNA knock-down suggests that Ano2 contributes to Ca2+-dependent chloride conductance in the RPE.

Purification of polluted water with spent mushroom (Agaricus bisporus) substrate: from agricultural waste to biosorbent of phenanthrene, Cd and Pb

ABSTRACTThe present research was aimed to (i) report the recycling of spent A. bisporus substrate (SAS) to remove heavy metals (Cd and Pb) and phenanthrene (Phe) from polluted water and (ii) assess the possibility to use the treated water for irrigation. Batch experiments were carried out to assess, firstly, the effect of interaction time between pollutants with SAS and, secondly, the pH of the polluted water. Then a biofilter was designed by using pressurized glass columns. Chemical parameters such as pH, electrical conductivity and content of Pb, Cd, Phe, nutrients (NPK) and Cl− were determined. Equilibrium for contaminants was quickly reached (1–2 h). The pH of the polluted water was the key factor for pollutants’ adsorption. The polluted water’s pH was increased after biofilter interaction. Phe was not detected in any fraction. Pb and Cd sorption rates were higher than 99%. The pollutant concentrations were within the permitted range to be used for agriculture purposes. Purified water showed significant concentrations of NPK, indicating its potential use as fertilizer. The SAS shows potential to be used as Phe, Pb and Cd biosorbent and the resulting treated water can be used for irrigation according to pollutant contents and agronomical evaluation.

Geothermal solute flux monitoring and the source and fate of solutes in the Snake River, Yellowstone National Park, WY

The combined geothermal discharge from over 10,000 features in Yellowstone National Park (YNP) can be can be estimated from the Cl flux in the Madison, Yellowstone, Falls, and Snake Rivers. Over the last 30 years, the Cl flux in YNP Rivers has been calculated using discharge measurements and Cl concentrations determined in discrete water samples and it has been determined that approximately 12% of the Cl flux exiting YNP is from the Snake River. The relationship between electrical conductivity and concentrations of Cl and other geothermal solutes was quantified at a monitoring site located downstream from the thermal inputs in the Snake River. Beginning in 2012, continuous (15 min) electrical conductivity measurements have been made at the monitoring site. Combining continuous electrical conductivity and discharge data, the Cl and other geothermal solute fluxes were determined. The 2013–2015 Cl fluxes (5.3–5.8 kt/yr) determined using electrical conductivity are comparable to historical data. In addition, synoptic water samples and discharge data were obtained from sites along the Snake River under low-flow conditions of September 2014. The synoptic water study extended 17 km upstream from the monitoring site. Surface inflows were sampled to identify sources and to quantify solute loading. The Lewis River was the primary source of Cl, Na, K, Cl, SiO2, Rb, and As loads (50–80%) in the Snake River. The largest source of SO4 was from the upper Snake River (50%). Most of the Ca and Mg (50–55%) originate from the Snake Hot Springs. Chloride, Ca, Mg, Na, K, SiO2, F, HCO3, SO4, B, Li, Rb, and As behave conservatively in the Snake River, and therefore correlate well with conductivity (R2 ≥ 0.97).

Revealing the activation pathway for TMEM16A chloride channels from macroscopic currents and kinetic models.

TMEM16A (ANO1), the pore-forming subunit of calcium-activated chloride channels, regulates several physiological and pathophysiological processes such as smooth muscle contraction, cardiac and neuronal excitability, salivary secretion, tumour growth and cancer progression. Gating of TMEM16A is complex because it involves the interplay between increases in intracellular calcium concentration ([Ca(2+)]i), membrane depolarization, extracellular Cl(-) or permeant anions and intracellular protons. Our goal here was to understand how these variables regulate TMEM16A gating and to explain four observations. (a) TMEM16A is activated by voltage in the absence of intracellular Ca(2+). (b) The Cl(-) conductance is decreased after reducing extracellular Cl(-) concentration ([Cl(-)]o). (c) ICl is regulated by physiological concentrations of [Cl(-)]o. (d) In cells dialyzed with 0.2 μM [Ca(2+)]i, Cl(-) has a bimodal effect: at [Cl(-)]o <30mM TMEM16A current activates with a monoexponential time course, but above 30mM, [Cl(-)]o ICl activation displays fast and slow kinetics. To explain the contribution of Vm, Ca(2+) and Cl(-) to gating, we developed a 12-state Markov chain model. This model explains TMEM16A activation as a sequential, direct, and Vm-dependent binding of two Ca(2+) ions coupled to a Vm-dependent binding of an external Cl(-) ion, with Vm-dependent transitions between states. Our model predicts that extracellular Cl(-) does not alter the apparent Ca(2+) affinity of TMEM16A, which we corroborated experimentally. Rather, extracellular Cl(-) acts by stabilizing the open configuration induced by Ca(2+) and by contributing to the Vm dependence of activation.

Properties of lipid electropores II: Comparison of continuum-level modeling of pore conductance to molecular dynamics simulations

Electrical conductance of an aqueous pore in the lipid bilayer has an important role in the process of membrane electroporation, i.e., formation of pores induced by electric pulses. In our present study we compare the pore conductance as predicted by a theoretical model based on the continuum Poisson–Nernst–Planck theory to the pore conductance obtained with molecular dynamics simulations (Casciola et al., Bioelectrochemistry 109:108–116, 2016). Our analysis demonstrates that the Poisson–Nernst–Planck model is able to quantitatively predict the dependence of the pore conductance on the pore radius when considering the toroidal shape of the pore. In order to correctly describe the difference in the pore conductance for Cl and Na ions (the pore selectivity), however, it is necessary to take into account the electric double layer next to the lipid–water interface and the electroosmotic flow through the pore. We further show that simplified analytical descriptions of pore conductance can lead to incorrect predictions of the pore size extracted from experimental measurements, and can affect the predictions of electroporation models. Overall, this study demonstrates that continuum modeling can be efficiently used as complementary method to molecular scale models for investigating lipid pores.

Highly conductive side chain block copolymer anion exchange membranes.

Block copolymers based on poly(styrene) having pendent trimethyl styrenylbutyl ammonium (with four carbon ring-ionic group alkyl linkers) or benzyltrimethyl ammonium groups with a methylene bridge between the ring and ionic group were synthesized by reversible addition-fragmentation radical (RAFT) polymerization as anion exchange membranes (AEMs). The C4 side chain polymer showed a 17% increase in Cl(-) conductivity of 33.7 mS cm(-1) compared to the benzyltrimethyl ammonium sample (28.9 mS cm(-1)) under the same conditions (IEC = 3.20 meq. g(-1), hydration number, λ = ∼7.0, cast from DMF/1-propanol (v/v = 3 : 1), relative humidity = 95%). As confirmed by small angle X-ray scattering (SAXS), the side chain block copolymers with tethered ammonium cations showed well-defined lamellar morphologies and a significant reduction in interdomain spacing compared to benzyltrimethyl ammonium containing block copolymers. The chemical stabilities of the block copolymers were evaluated under severe, accelerated conditions, and degradation was observed by (1)H NMR. The block copolymer with C4 side chain trimethyl styrenylbutyl ammonium motifs displayed slightly improved stability compared to that of a benzyltrimethyl ammonium-based AEM at 80 °C in 1 M NaOD aqueous solution for 30 days.

Transport proteins NHA1 and NHA2 are essential for survival, but have distinct transport modalities

The cation/proton antiporter (CPA) family includes the well-known sodium/proton exchanger (NHE; SLC9A) family of Na(+)/H(+) exchangers, and the more recently discovered and less well understood CPA2s (SLC9B), found widely in living organisms. In Drosophila, as in humans, they are represented by two genes, Nha1 (Slc9b1) and Nha2 (Slc9b2), which are enriched and functionally significant in renal tubules. The importance of their role in organismal survival has not been investigated in animals, however. Here we show that single RNAi knockdowns of either Nha1 or Nha2 reduce survival and in combination are lethal. Knockdown of either gene alone results in up-regulation of the other, suggesting functional complementation of the two genes. Under salt stress, knockdown of either gene decreases survival, demonstrating a key role for the CPA2 family in ion homeostasis. This is specific to Na(+) stress; survival on K(+) intoxication is not affected by sodium/hydrogen antiporter (NHA) knockdown. A direct functional assay in Xenopus oocytes shows that Nha2 acts as a Na(+)/H(+) exchanger. In contrast, Nha1 expressed in Xenopus oocytes shows strong Cl(-) conductance and acts as a H(+)-Cl(-) cotransporter. The activity of Nha1 is inhibited by chloride-binding competitors 4,4'-diiso-thiocyano-2,2'-disulfonic acid stilbene and 4,4'-dibenzamido-2,2'-stilbenedisulphonate. Salt stress induces a massive up-regulation of NHA gene expression not in the major osmoregulatory tissues of the alimentary canal, but in the crop, cuticle, and associated tissues. Thus, it is necessary to revise the classical view of the coordination of different tissues in the coordination of the response to osmoregulatory stress.

ENaC inhibition stimulates HCl secretion in the mouse cortical collecting duct. II. Bafilomycin-sensitive H+ secretion.

Epithelial Na(+) channel (ENaC) blockade stimulates stilbene-sensitive conductive Cl(-) secretion in the mouse cortical collecting duct (CCD). This study's purpose was to determine the co-ion that accompanies benzamil- and DIDS-sensitive Cl(-) flux. Thus transepithelial voltage, VT, as well as total CO2 (tCO2) and Cl(-) flux were measured in CCDs from aldosterone-treated mice consuming a NaCl-replete diet. We reasoned that if stilbene inhibitors (DIDS) reduce conductive anion secretion they should reduce the lumen-negative VT. However, during ENaC blockade (benzamil, 3 μM), DIDS (100 μM) application to the perfusate reduced net H(+) secretion, which increased the lumen-negative VT. Conversely, ENaC blockade alone stimulated H(+) secretion, which reduced the lumen-negative VT. Application of an ENaC inhibitor to the perfusate reduced the lumen-negative VT, increased intercalated cell intracellular pH, and reduced net tCO2 secretion. However, benzamil did not change tCO2 flux during apical H(+)-ATPase blockade (bafilomycin, 5 nM). The increment in H(+) secretion observed with benzamil application contributes to the fall in VT observed with application of this diuretic. As such, ENaC blockade reduces the lumen-negative VT by inhibiting conductive Na(+) absorption and by stimulating H(+) secretion by type A intercalated cells. In conclusion, 1) in CCDs from aldosterone-treated mice, benzamil application stimulates HCl secretion mediated by the apical H(+)-ATPase and a yet to be identified conductive Cl(-) transport pathway; 2) benzamil-induced HCl secretion is reversed with the application of stilbene inhibitors or H(+)-ATPase inhibitors to the perfusate; and 3) benzamil reduces VT not only by inhibiting conductive Na(+) absorption, but also by stimulating H(+) secretion.

The ClC-7 Chloride Channel Is Downregulated by Hypoosmotic Stress in Human Chondrocytes.

Articular chondrocytes in osteoarthritis (OA) patients are exposed to hypoosmotic stress because the osmolality of this synovial fluid is significantly decreased. Hypoosmotic stress can cause an efflux of Cl(-) and an associated decrease of cell volume. We have previously reported that a Cl(-) conductance contributes to the regulation of resting membrane potential and thus can alter intracellular Ca(2+) concentration ([Ca(2+)]i) in human chondrocytes. The molecular identity and pathologic function of these Cl(-) channels, however, remained to be determined. Here, we show that the ClC-7 Cl(-) channel is strongly expressed in a human chondrocyte cell line (OUMS-27) and that it is responsible for Cl(-) currents that are activated by extracellular acidification (pH 5.0). These acid-sensitive currents are inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; IC50 = 13 μM) and are markedly reduced by small-interfering RNA-induced knockdown of ClC-7. DIDS hyperpolarized these chondrocytes, and this was followed by an increase in [Ca(2+)]i. ClC-7 knockdown caused a similar hyperpolarization of the membrane potential. Short-term culture (48 hours) in hypoosmotic medium (270 mOsm) reduced the expression of ClC-7 and decreased the acid-sensitive currents. Interestingly, these hypoosmotic culture conditions, or ClC-7 knockdown, resulted in enhanced cell death. Taken together, our results show that the significant hyperpolarization due to ClC-7 impairment in chondrocytes can significantly increase [Ca(2+)]i and cell death. Thus, downregulation of ClC-7 channels during the hypoosmotic stress that accompanies OA progression is one important concept of the complex etiology of OA. These findings suggest novel targets for therapeutic intervention(s) and drug development for OA.

ENaC inhibition stimulates HCl secretion in the mouse cortical collecting duct. I. Stilbene-sensitive Cl- secretion.

Inhibition of the epithelial Na(+) channel (ENaC) reduces Cl(-) absorption in cortical collecting ducts (CCDs) from aldosterone-treated rats and mice. Since ENaC does not transport Cl(-), the purpose of the present study was to explore how ENaC modulates Cl(-) absorption in mouse CCDs perfused in vitro. Therefore, we measured transepithelial Cl(-) flux and transepithelial voltage in CCDs perfused in vitro taken from mice that consumed a NaCl-replete diet alone or the diet with aldosterone administered by minipump. We observed that application of an ENaC inhibitor [benzamil (3 μM)] to the luminal fluid unmasks conductive Cl(-) secretion. During ENaC blockade, this Cl(-) secretion fell with the application of a nonselective Cl(-) channel blocker [DIDS (100 μM)] to the perfusate. While single channel recordings of intercalated cell apical membranes in split-open CCDs demonstrated a Cl(-) channel with properties that resemble the ClC family of Cl(-) channels, ClC-5 is not the primary pathway for benzamil-sensitive Cl(-) flux. In conclusion, first, in CCDs from aldosterone-treated mice, most Cl(-) absorption is benzamil sensitive, and, second, benzamil application stimulates stilbene-sensitive conductive Cl(-) secretion, which occurs through a ClC-5-independent pathway.

Etiology of distinct membrane excitability in pre- and posthearing auditory neurons relies on activity of Cl− channel TMEM16A

The developmental rehearsal for the debut of hearing is marked by massive changes in the membrane properties of hair cells (HCs) and spiral ganglion neurons (SGNs). Whereas the underlying mechanisms for the developing HC transition to mature stage are understood in detail, the maturation of SGNs from hyperexcitable prehearing to quiescent posthearing neurons with broad dynamic range is unknown. Here, we demonstrated using pharmacological approaches, caged-Ca(2+) photolysis, and gramicidin patch recordings that the prehearing SGN uses Ca(2+)-activated Cl(-) conductance to depolarize the resting membrane potential and to prime the neurons in a hyperexcitable state. Immunostaining of the cochlea preparation revealed the identity and expression of the Ca(2+)-activated Cl(-) channel transmembrane member 16A (TMEM16A) in SGNs. Moreover, null deletion of TMEM16A reduced the Ca(2+)-activated Cl(-) currents and action potential firing in SGNs. To determine whether Cl(-) ions and TMEM16A are involved in the transition between pre- and posthearing features of SGNs we measured the intracellular Cl(-) concentration [Cl(-)]i in SGNs. Surprisingly, [Cl(-)]i in SGNs from prehearing mice was ∼90 mM, which was significantly higher than posthearing neurons, ∼20 mM, demonstrating discernible altered roles of Cl(-) channels in the developing neuron. The switch in [Cl(-)]i stems from delayed expression of the development of intracellular Cl(-) regulating mechanisms. Because the Cl(-) channel is the only active ion-selective conductance with a reversal potential that lies within the dynamic range of SGN action potentials, developmental alteration of [Cl(-)]i, and hence the equilibrium potential for Cl(-) (ECl), transforms pre- to posthearing phenotype.