Phosphate stimulates CFTR Cl- channels.

Cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels appear to be regulated by hydrolysis of ATP and are inhibited by a product of hydrolysis, ADP. We assessed the effect of the other product of hydrolysis, inorganic phosphate (P(i)), on CFTR Cl- channel activity using the excised inside-out configuration of the patch-clamp technique. Millimolar concentrations of P(i) caused a dose-dependent stimulation of CFTR Cl- channel activity. Single-channel analysis demonstrated that the increase in macroscopic current was due to an increase in single-channel open-state probability (po) and not single-channel conductance. Kinetic modeling of the effect of P(i) using a linear three-state model indicated that the effect on po was predominantly the result of an increase in the rate at which the channel passed from the long closed state to the bursting state. P(i) also potentiated activity of channels studied in the presence of 10 mM ATP and stimulated Cl- currents in CFTR mutants lacking much of the R domain. Binding studies with a photoactivatable ATP analog indicated that Pi decreased the amount of bound nucleotide. These results suggest that P(i) increased CFTR Cl- channel activity by stimulating a rate-limiting step in channel opening that may occur by an interaction of P(i) at one or both nucleotide-binding domains.     

The interaction of Cl- with a gramicidin-like channel

Molecular dynamics simulations have been used to study the interaction of Cl- with a gramicidin-like channel. The results suggest that there is a high-energy barrier at the entrance of the channel, which would correspond to a permeability 10(-9)-times that of a cation of the same size. This could account for the cationic selectivity of the gramicidin channel and indicates that valence selectivity is kinetically controlled.     

Liquid and solid-state Cl- -sensitive microelectrodes. Characteristics and application to intracellular Cl- activity in Balanus photoreceptor

When intracellular chloride activity (aiCl) was monitored with chloride-sensitive liquid ion exchanges (CLIX) microelectrodes in Balanus photoreceptors, replacement of extracellular chloride (Cl0) by methanesulfonate or glutamate was followed by a rapid but incomplete loss of aiCl. When propionate was used as the extracellular anion substitute, CLIX electrodes detected an apparent gain in aiCl, while a newly designed Ag-AgCl wire-in glass microelectrode showed a loss of aiCl under the same conditions. This discrepancy in Cl- washout when propionate replaced Cl0 is explained by the differences in selectivity of CLIX and Ag-AgCl electrodes for native intracellular anions and for the extracellular anion substitute which also replaces Cli and interferes in the determination of aiCl. Both electrodes indicate that ECl approximately Em when the cells are bathed in normal barnacle saline, and both electrodes showed the rate of Cl washout (tau approximately 5 min) to be independent of Cli when Cl0 was replaced by glutamate. Details of Ag-AgCl microelectrode construction are presented. These electrodes were tested and found to be insensitive to the organic anion substitutes used in this study. Selectivity data of CLIX electrodes for several anions of biological interest are described.     

Potent block of Cl- channels by antiallergic drugs.

Two antiallergic drugs, cromolyn and RU 31156, were examined for their potency to block intermediate-conductance Cl- channels. For this, single Cl- channel activities of mucosal-type mast cells (RBL-2H3) and alternatively, of colonic carcinoma cells (HT29) were monitored employing the patch-clamp technique in the inside-out patch configuration. Here we show that intermediate-conductance Cl- channels of either cell type were blocked by both compounds. Cl- channel inhibition occurred by a slow rather than a flickering block and with a Hill coefficient around 1. RU 31156 was about one order of magnitude more potent (IC50 about 1 microM) than cromolyn. The IC50 values of each compound were not significantly different (p < 0.01) in the two cell types. Our data provide evidence for two novel Cl- channel blockers, which may be of further use in Cl- channel characterization as well as purification.     

Cl- channels in intact human T lymphocytes.

We recently described a large, multiple-conductance Cl- channel in excised patches from normal T lymphocytes. The properties of this channel in excised patches are similar to maxi-Cl- channels found in a number of cell types. The voltage dependence in excised patches permitted opening only at nonphysiological voltages, and channel activity was rarely seen in cell-attached patches. In the present study, we show that Cl- channels can be activated in intact cells at physiological temperatures and voltages and that channel properties change after patch excision. Maxi-Cl- channels were reversibly activated in 69% of cell-attached patches when the temperature was above 32 degrees C, whereas fewer than 2% of patches showed activity at room temperature. Upon excision, the same patches displayed large, multiple-conductance Cl- channels with characteristics like those we previously reported for excised patches. After patch excision, warm temperatures were not essential to allow channel activity; 37% (114/308) of inside-out patches had active channels at room temperature. The voltage dependence of the channels was markedly different in cell-attached recordings compared with excised patches. In cell-attached patches, Cl- channels could be open at cell resting potentials in the normal range. Channel activation was not related to changes in intracellular Ca2+ since neither ionomycin nor mitogens activated the channels in cell-attached patches, Ca2+ did not rise in response to warming and the Cl- channel was independent of Ca2+ in inside-out patches. Single-channel currents were blocked by internal or external Zn2+ (100-200 microM), 4-acetamido-4' isothiocyanostilbene-2,2'-disulfonate (SITS, 100-500 microM) and 4,4'-diisothiocyanostilbene 2,2'-disulfonate (DIDS, 100 microM). NPPB (5-nitro-2-(3-phenylpropylamino)-benzoate) reversibly blocked the channels in inside-out patches.     

Regulation of Cl- channels by multifunctional CaM kinase.

cAMP kinase has been shown to mediate the cAMP pathway for regulation of Cl- channels in lymphocytes, but the mediator of an alternative, Ca2+ pathway has not been identified. We show here that Ca2+ ionophore activates Cl- currents in cell-attached and whole-cell patch-clamp recordings of Jurkat T lymphocytes, but this activation is not direct. The effect of Ca2+ ionophore on whole-cell Cl- currents is inhibited by a specific peptide inhibitor of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase). Furthermore, Cl- channels are activated in excised patches by purified CaM kinase in a fashion that mimics the effect of Ca2+ ionophore in cell-attached recordings. These results suggest that CaM kinase mediates the Ca2+ pathway of Cl- channel activation.     

Cl- regulates the structure of the fibrin clot.

The differences between coarse and fine fibrin clots first reported by Ferry have been interpreted in terms of nonspecific ionic strength effects for nearly 50 years and have fostered the notion that fibrin polymerization is largely controlled by electrostatic forces. Here we report spectroscopic and electron microscopy studies carried out in the presence of different salts that demonstrate that this long-held interpretation needs to be modified. In fact, the differences are due entirely to the specific binding of Cl- to fibrin fibers and not to generic ionic strength or electrostatic effects. Binding of Cl- opposes the lateral aggregation of protofibrils and results in thinner fibers that are also more curved than those grown in the presence of inert anions such as F-. The effect of Cl- is pH dependent and increases at pH > 8.0, whereas fibers grown in the presence of F- remain thick over the entire pH range from 6.5 to 9.0. From the pH dependence of the Cl- effect it is suggested that the anion exerts its role by increasing the pKa of a basic group ionizing around pH 9.2. The important role of Cl- in structuring the fibrin clot also clarifies the role played by the release of fibrinopeptide B, which leads to slightly thicker fibers in the presence of Cl- but actually reduces the size of the fibers in the presence of F-. This effect becomes more evident at high, close to physiological concentrations of fibrinogen. We conclude that Cl- is a basic physiological modulator of fibrin polymerization and acts to prevent the growth of thicker, stiffer, and straighter fibers by increasing the pKa of a basic group. This discovery opens new possibilities for the design of molecules that can specifically modify the clot structure by targeting the structural domains responsible for Cl- binding to fibrin.     

The development of an increased rate of Cl- uptake in the ascomycete Neocosmospora vasinfecta.

Freshly harvested mycelium of the filamentous ascomycete Neocosmospora vasinfecta accumulated C1- against a concentration gradient by a process probably requiring the expenditure of metabolic energy. When mycelium, washed free of growth medium, was incubated in deionized water or tris (hydroxymethyl) aminomethane sulfate at pH 7.5 for 4 h and then transferred to K36C1 solutions, the C1- uptake rate was, on the average, 3.77 +/- 0.26 (+/-SE, N = 20) times the uptake rate exhibited by freshly harvested mycelium. This development of an increased rate of Cl- uptake could be blocked by the presence of an inhibitor of ribonucleic acid synthesis (azaguanine) or of protein synthesis (cycloheximide, fluorophenylalanine, or puromycin). The combined presence of glucose and a potassium salt in the preincubation solution virtually arrested the development of enhanced Cl- uptake. The rate of Cl- uptake by freshly harvested mycelium did not vary greatly with the age of the culture on harvest but the ability to develop an increased rate declined with age. The fact that it is possible to obtain mycelium possessing widely different capacities for Cl- uptake should assist in biochemical characterization of the Cl- uptake system.     

Na+-K+-2Cl- cotransporters and Cl- channels regulate citric acid cough in guinea pigs.

Loop diuretics have been shown to inhibit cough and other airway defensive reflexes via poorly defined mechanisms. We test the hypothesis that the furosemide-sensitive Na+-K+-2Cl- cotransporter (NKCC1) is expressed by sensory nerve fibers innervating the airways where it plays an important role in regulating sensory neural activity. NKCC1 immunoreactivity was present on the cell membranes of most nodose and jugular ganglia neurons projecting to the trachea, and it was present on the peripheral terminals of putative mechanosensory nerve fibers in the airways. In urethane-anesthetized, spontaneously breathing guinea pigs, bolus application of citric acid (1 mM to 2 M) to an isolated and perfused segment of the tracheal mucosa evoked coughing and respiratory slowing. Removal of Cl- from the tracheal perfusate evoked spontaneous coughing and significantly potentiated cough and respiratory slowing reflexes evoked by citric acid. The NKCC1 inhibitor furosemide (10-100 microM) significantly reduced both the number of coughs evoked by citric acid and the degree of acid-evoked respiratory slowing (P < 0.05). Localized tracheal pretreatment with the Cl- channel inhibitors DIDS or niflumic acid (100 microM) also significantly reduced cough, whereas the GABAA receptor agonist muscimol potentiated acid-evoked responses. These data suggest that vagal sensory neurons may accumulate Cl- due to the expression of the furosemide-sensitive Cl- transporter, NKCC1. Efflux of intracellular Cl-, in part through calcium-activated Cl- channels, may play an important role in regulating airway afferent neuron activity.     

Molecular Physiology of an Extra-renal Cl- Uptake Mechanism for Body Fluid Cl- Homeostasis

The development of an ion regulatory mechanism for body fluid homeostasis was an important trait for vertebrates during the evolution from aquatic to terrestrial life. The homeostatic mechanism of Cl^- in aquatic fish appears to be similar to that of terrestrial vertebrates; however, the mechanism in non-mammalian vertebrates is poorly understood. Unlike in mammals, in which the kidney plays a central role, in most fish species, the gill is responsible for the maintenance of Cl^- homeostasis via Cl^- transport uptake mechanisms. Previous studies in zebrafish identified Na⁺-Cl^- cotransporter (NCC) 2b-expressing cells in the gills and skin as the major ionocytes responsible for Cl^- uptake, similar to distal convoluted tubular cells in mammalian kidney. However, the mechanism by which basolateral ions exit from NCC cells is still unclear.Of the in situ hybridization signals of twelve members of the clc Cl^- channel family, only that of clc-2c exhibited an ionocyte pattern in the gill and embryonic skin. Double in situ hybridization/immunocytochemistry confirmed colocalization of apical NCC2b with basolateral CLC-2c. Acclimation to a low Cl^- environment increased mRNA expression of both clc-2c and ncc2b, and also the protein expression of CLC-2c in embryos and adult gills. Loss-of-function of clc-2c resulted in a significant decrease in whole body Cl^- content in zebrafish embryos, a phenotype similar to that of ncc2b mutants; this finding suggests a role for CLC-2c in Cl^- uptake. Translational knockdown of clc-2c stimulated ncc2b mRNA expression and vice versa, revealing cooperation between these two transporters in the context of zebrafish Cl^- homeostasis. Further comparative genomic and phylogenetic analyses revealed that zebrafish CLC-2c is a fish-specific isoform that diverged from a kidney-predominant homologue, in the same manner as NCC2b and its counterparts (NCCs).Several lines of molecular and cellular physiological evidences demonstrated the cofunctional role of apical NCC2b and basolateral CLC-2c in the gill/skin Cl^- uptake pathway. Taking the phylogenetic evidence into consideration, fish-specific NCC2b and CLC-2c may have coevolved to perform extra-renal Cl^- uptake during the evolution of vertebrates in an aquatic environment.     

The branchial chloride pump in the goldfish Carassius auratus: relationship between Cl-/HCO3- and Cl-/Cl-- exchanges and the effect of thiocyanate.

1. The effect of thiocyanate on chloride and sodium fluxes across the gill was studied in the goldfish Carassius auratus. At low external chloride concentrations, addition of SCN- to the bathing solution markedly inhibited chloride influx and efflux, the net flux being reversed, SCN- injection was without effect. SCN- had no effect on sodium fluxes when injected or added to the external medium. 2. The inhibition of chloride influx by SCN- was of a mixed type involving simultaneous modifications of the affinity constant of the carrier for Cl- and of the maximal Cl- influx. The affinity constant of the carrier for SCN- was 10 times lower than that for Cl-. 3. The gill of the goldfish was found to be practically impermeable to SCN-. 4. In the presence of external SCN-, the Cl-/HCO3- exchange was reversed: Cl- was lost against HCO3- which is absorbed. This suggests an obligatory exchange. 5. Exchange diffusion for chloride was also demonstrated. 6. A kinetic model is proposed to explain chloride and bicarbonate transport across the gill of Carassius auratus.     

Cl- Fluxes and Cl- Content of Dunaliella acidophila--An Alga with a Positive Membrane Potential

The Cl^– fluxes across the plasma membrane and the Cl^–content of the acid–resistant alga Dunaliella acidophila(optimal growthat pH 1.0, positive membrane potential) werestudied in the presence of 0.01–300 mM Cl^–. Up to40 mM Cl^– in the medium, theinternal Cl^– concentrationis higher than that predicted by the electrochemical equilibrium,whereas at higher external Cl^– concentrations internalCl^– levels are lower than expected for the electrochemicalequilibrium. Growth in the absence of Cl^– is significantlylower than in the standard growth medium (2.2 mM Cl^–)and this reduction cannot be overcome by the addition ofothermonovalent anions such as Br^– or NO^–₃ The latterimplies a specific Cl^– requirement in addition to therole of Cl^– as apermeant anion during ion translocations.Growth and photosynthesis tolerate an excess of Cl^– upto 300 mM (without stepwiseadaptation to increasing salinity).The uptake of Cl^– (measured by tracer techniques) exhibitsMichaelis–Menten kinetics (K_M = 0.75 mM Cl^–) andis stimulated by light and high H⁺ concentrations. Internalacidification by acetic acid causes an inhibition of Cl^–uptake. The uptake of Cl^– is inhibited by the monovalentanions Br^–, I^–, and NO^–₃ with K¹, values notvery much different from the K_M. value for Cl^–. The aniontransport inhibitors SITS and DIDS do not affect photosynthesis,but strongly suppressthe uptake of Cl^–. The Cl^–channel blockers A–9–C and NPPB cause inhibitionsof Cl^– uptake as well as of photosynthesis andthe ATPpool. FCCP strongly depresses the internal ATP–pool withouta marked effect on Cl^– uptake. Cl^– efflux was inhibitedbyDIDS and SITS, but stimulated or inhibited by FCCP, dependingon the external Cl^– concentration. Results are in agreementwiththe hypothesis that Cl^– uptake into D. acidophila is dueto catalysed diffusion and is primarily independent of the hydrolysisofATP. Cl^– efflux is assumed to be coupled to an activepump. Data suggest tight co–operativity between the systemsresponsiblefor Cl^– uptake and Cl^– efflux, with thecytoplasmic pH and the membrane potential being important mediators. Key words: Acid resistance, chloride carrier, chloride channels, Dunaliella acidophila, membrane potential, plasma membrane     

Cl- transport by gastric mucosa: cellular Cl- activity and membrane permeability

The mechanism of Cl- secretion in the isolated, resting (i.e. cimetidine-treated) gastric mucosa of Necturus has been investigated with radioisotopic and electrophysiological techniques. Measurement of transepithelial 36Cl- fluxes (mucosal to serosal (M leads to S), Jms Cl-; S leads to M, Jsm Cl-) during control conditions show that at open circuit, when the transepithelial potential difference psi ms = 20 mV (S ground), Jms Cl- = Jsm Cl-, i.e. Jnet Cl- = 0, but during short-circuit current conditions Jnet Cl- = I sc = 2 mu equiv cm-2 h. Experiments with low [Cl-] solutions indicate that Cl- exchange diffusion does not contribute significantly to either Jms Cl- or Jsm Cl-. Double-barrelled, Cl- -selective microelectrodes showed that in open circuit, the cellular (C) chemical potential for Cl-, psi c Cl- = 31 mV (apparent [Cl-] = 29 mM), the electrical potential across the M membrane, psi m = -34 mV (mucosa ground) while that across the S membrane, psi s = -52 mV (serosa ground). During short-circuit current conditions, psi m = psi s = -49 mV and [Cl-]c = 30 mM. The permeability of the M membrane to Cl- (Pm Cl-) was calculated both from the tracer experiments and the electrode measurements by using the constant-field equation. Short-term (45 s) uptake of 36Cl- at [Cl]m = 96 mM during short circuit conditions gave Pm Cl- = 2.6 x 10(-5) cm s-1. Measurement of [Cl-]c by means of the electrodes when [Cl-]m was changed from 96 to 2 mM or from 2 to 96 mM gave Pm Cl- = 2.9-5.7 x 10(-5) cm s-1. Our results indicate that during open circuit conditions Cl- is accumulated across the S membrane into gastric cells in an energy-requiring step, but since Jnet Cl- = 0, Cl- must leak back into the S solution at a rate equal to the entry rate. When the tissue is short-circuited, Cl- secretion occurs (Jnet Cl- = Isc) owing to the same energy-requiring accumulation of Cl- by the cells and a passive (apparently electrodiffusive) movement across the mucosal membrane.     

Control of Plasma Membrane Cl- Fluxes in Chara corallina by External Cl- and Light

The efflux of Cl^– at the plasma membrane of Chara wasstudied in relation to two treatments known to affect the flux:that of removal of external Cl^– and of light. It is shownthat although removal of external Cl^– results in a rapidreduction in Cl^– efflux (consistent with a direct effectof external Cl^– on the transport system) the magnitudeof this reduction in the dark is greater than the measured darkinflux. Therefore, in the dark at least, it is proposed that1:1 exchange diffusion cannot account for the trans-stimulationof efflux by external Cl^–. Light induces an inhibition of efflux and a concomitant stimulationof Cl^– influx at 20 °C, but at 10 °C the responsesto light of the two fluxes can be separated temporally. It istherefore suggested that the fluxes are not reciprocally dependenton the same factor which mediates in the light response. Furtherconsiderations show that it is unlikely that the cytoplasmicCl^– concentration mediates in the light response of eitherflux, but that changes in cytoplasmic pH may do so.     

The first extracellular domain of claudin-7 affects paracellular Cl- permeability

Tight junctions (TJ) constitute paracellular diffusion channels regulating the passage of ions and solutes across epithelia. We recently demonstrated that overexpression of the TJ membrane protein claudin-7 in LLC-PK1 cells decreases paracellular permeability to Cl(-) and increases paracellular permeability to Na(+). To investigate the effect of charged amino acid residues in extracellular domains (ED) of claudin-7 on paracellular charge selectivity, we created claudin-7 mutants by replacing negatively charged amino acids on ED with positively charged amino acids. Immunofluorescence light microscopy showed that these mutant proteins were correctly targeted to the cell junction. Ultrastructure examination of TJ morphology did not reveal any difference between cells expressing wildtype (WT) and mutant claudin-7. However, electrophysiological studies showed increased Cl(-) permeability in cells expressing first extracellular domain (ED1) mutants, but not second extracellular domain (ED2) mutants, compared to that of WT claudin-7. Our results demonstrate that negatively charged amino acids in ED1 of claudin-7 are involved in modulating paracellular Cl(-) permeability.     

In vivo airway surface liquid Cl- analysis with solid-state electrodes.

The pathogenesis of cystic fibrosis (CF) airways disease remains controversial. Hypotheses that link mutations in CFTR and defects in ion transport to CF lung disease predict that alterations in airway surface liquid (ASL) isotonic volume, or ion composition, are critically important. ASL [Cl-] is pivotal in discriminating between these hypotheses, but there is no consensus on this value given the difficulty in measuring [Cl-] in the "thin" ASL (approximately 30 microm) in vivo. Consequently, a miniaturized solid-state electrode with a shallow depth of immersion was constructed to measure ASL [Cl-] in vivo. In initial experiments, the electrode measured [Cl-] in physiologic salt solutions, small volume (7.6 microl) test solutions, and in in vitro cell culture models, with > or =93% accuracy. Based on discrepancies in reported values and/or absence of data, ASL Cl- measurements were made in the following airway regions and species. First, ASL [Cl-] was measured in normal human nasal cavity and averaged 117.3 +/- 11.2 mM (n = 6). Second, ASL [Cl-] measured in large airway (tracheobronchial) regions were as follows: rabbit trachea and bronchus = 114.3 +/- 1.8 mM; (n = 6) and 126.9 +/- 1.7 mM; (n = 3), respectively; mouse trachea = 112.8 +/- 4.2 mM (n = 13); and monkey bronchus = 112.3 +/- 10.9 mM (n = 3). Third, Cl- measurements were made in small (1-2 mm) diameter airways of the rabbit (108.3 +/- 7.1 mM, n = 5) and monkey (128.5 +/- 6.8 mM, n = 3). The measured [Cl-], in excess of 100 mM throughout all airway regions tested in multiple species, is consistent with the isotonic volume hypothesis to describe ASL physiology.