Regulation of Chloride Channels in Secretory Epithelia

Fluid and electrolyte secretion from secretory epithelia is a highly regulated process. Chloride channel activity at the apical membrane determines the rate and direction of salt and water secretion. Multiple classes of Cl⁻ channels with distinct gating mechanisms are involved in moving ions and water. Secretory agonists that induce intracellular increases in two second messenger systems, cAMP and [Ca²⁺] _i , are generally associated with secretion. However, changes in cell volume and the membrane potential may also play a role in regulating fluid and electrolyte secretion in some tissues. In this review we discuss the regulation of the different types of Cl⁻ channels found in secretory epithelia. Received: 16 September 1997/Revised: 13 November 1997     

Halide Permeation in Wild-Type and Mutant Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels

Permeation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channels by halide ions was studied in stably transfected Chinese hamster ovary cells by using the patch clamp technique. In cell-attached patches with a high Cl⁻ pipette solution, the CFTR channel displayed outwardly rectifying currents and had a conductance near the membrane potential of 6.0 pS at 22°C or 8.7 pS at 37°C. The current–voltage relationship became linear when patches were excised into symmetrical, N-tris(hydroxymethyl)methyl-2-aminomethane sulfonate (TES)-buffered solutions. Under these conditions, conductance increased from 7.0 pS at 22°C to 10.9 pS at 37°C. The conductance at 22°C was ∼1.0 pS higher when TES and HEPES were omitted from the solution, suggesting weak, voltage-independent block by pH buffers. The relationship between conductance and Cl⁻ activity was hyperbolic and well fitted by a Michaelis-Menten–type function having a K _m of ∼38 mM and maximum conductance of 10 pS at 22°C. Dilution potentials measured with NaCl gradients indicated high anion selectivity (P_Na/P_Cl = 0.003–0.028). Biionic reversal potentials measured immediately after exposure of the cytoplasmic side to various test anions indicated P_I (1.8) > P_Br (1.3) > P_Cl (1.0) > P_F (0.17), consistent with a “weak field strength” selectivity site. The same sequence was obtained for external halides, although inward F⁻ flow was not observed. Iodide currents were protocol dependent and became blocked after 1–2 min. This coincided with a large shift in the (extrapolated) reversal potential to values indicating a greatly reduced I⁻/Cl⁻ permeability ratio (P_I/P_Cl < 0.4). The switch to low I⁻ permeability was enhanced at potentials that favored Cl⁻ entry into the pore and was not observed in the R347D mutant, which is thought to lack an anion binding site involved in multi-ion pore behavior. Interactions between Cl⁻ and I⁻ ions may influence I⁻ permeation and be responsible for the wide range of P_I/P_Cl ratios that have been reported for the CFTR channel. The low P_I/P_Cl ratio usually reported for CFTR only occurred after entry into an altered permeability state and thus may not be comparable with permeability ratios for other anions, which are obtained in the absence of iodide. We propose that CFTR displays a “weak field strength” anion selectivity sequence.     

Decreased Cell Surface Charge in Cystic Fibrosis Epithelia

A colloid titration technique has been used to determine the surface charge of cystic fibrosis (CF) and corresponding non-CF epithelial cells. We have shown that the negative surface charge of CF epithelial cells is significantly reduced in comparison with non-CF cells. This fact may play an important role in CF, where the increased adherence of microorganisms is known to cause chronic lung infection. Neuraminidase treatment removed approximately the same amount of surface charge in both cell lines, indicating no differences in cell surface sialylation. Similar results were obtained by direct measurements of the amount of N-acetylneuraminic acid released by neuraminidase. Therefore, our results indicate that sialic acid residues are not involved in the reduction of the negative surface charge in CF. This conclusion does not support the hypothesis that undersialylation of cell-membrane molecules occurs in cystic fibrosis. © 1997 John Wiley & Sons, Ltd.     

Permeability of Wild-Type and Mutant Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels to Polyatomic Anions

Permeability of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel to polyatomic anions of known dimensions was studied in stably transfected Chinese hamster ovary cells by using the patch clamp technique. Biionic reversal potentials measured with external polyatomic anions gave the permeability ratio (P_X/P_Cl) sequence NO₃ ⁻ > Cl⁻ > HCO₃ ⁻ > formate > acetate. The same selectivity sequence but somewhat higher permeability ratios were obtained when anions were tested from the cytoplasmic side. Pyruvate, propanoate, methane sulfonate, ethane sulfonate, and gluconate were not measurably permeant (P_X/P_Cl < 0.06) from either side of the membrane. The relationship between permeability ratios from the outside and ionic diameters suggests a minimum functional pore diameter of ∼5.3 Å. Permeability ratios also followed a lyotropic sequence, suggesting that permeability is dependent on ionic hydration energies. Site-directed mutagenesis of two adjacent threonines in TM6 to smaller, less polar alanines led to a significant (24%) increase in single channel conductance and elevated permeability to several large anions, suggesting that these residues do not strongly bind permeating anions, but may contribute to the narrowest part of the pore.     

CFTR Regulation of Intracellular Calcium in Normal and Cystic Fibrosis Human Airway Epithelia

In cystic fibrosis airway epithelia, mutation of the CFTR protein causes a reduced response of Cl⁻ secretion to secretagogues acting via cAMP. Using a Ca²⁺ imaging system, the hypothesis that CFTR activation may permit ATP release and regulate [Ca²⁺] _i via a receptor-mediated mechanism, is tested in this study. Application of external nucleotides produced a significant increase in [Ca²⁺] _i in normal (16HBE14o⁻ cell line and primary lung culture) and in cystic fibrosis (CFTE29o⁻ cell line) human airway epithelia. The potency order of nucleotides on [Ca²⁺] _i variation was UTP ≫ ATP > UDP > ADP > AMP > adenosine in both cell types. The nucleotide [Ca²⁺] _i response could be mimicked by activation of CFTR with forskolin (20 μm) in a temperature-dependent manner. In 16HBE14o⁻ cells, the forskolin-induced [Ca²⁺] _i response increased with increasing temperature. In CFTE29o⁻ cells, forskolin had no effect on [Ca²⁺] _i at body temperature-forskolin-induced [Ca²⁺] _i response in CF cells could only be observed at low experimental temperature (14°C) or when cells were cultured at 26°C instead of 37°C. Pretreatment with CFTR channel blockers glibenclamide (100 μm) and DPC (100 μm), with hexokinase (0.5 U/mg), and with the purinoceptor antagonist suramin (100 μm), inhibited the forskolin [Ca²⁺] _i response. Together, these results demonstrate that once activated, CFTR regulates [Ca²⁺] _i by mediating nucleotide release and activating cell surface purinoceptors in normal and CF human airway epithelia. Received: 3 April 2000/Revised: 30 June 2000     

Characterization of K+ Channels in the Basolateral Membrane of Rat Tracheal Epithelia

To study K⁺ channels in the basolateral membrane of chloride-secreting epithelia, rat tracheal epithelial monolayers were cultured on permeable filters and mounted into an Ussing chamber system. The mucosal membrane was permeabilized with nystatin (180 μg/ml) in the symmetrical high K⁺ (145 mm) Ringer solution. During measurement of the macroscopic K⁺ conductance properties of the basolateral membrane under a transepithelial voltage clamp, we detected at least two types of K⁺ currents: one is an inwardly rectifying K⁺ current and the other is a slowly activating outwardly rectifying K⁺ current. The inwardly rectifying K⁺ current is inhibited by Ba²⁺. The slowly activating K⁺ current was potentiated by cAMP and inhibited by clofilium, phorbol 12-myristae 13-acetate (PMA) and lowering temperature. This is consistent with the biophysical characteristics of I _SK channel. RT-PCR analysis revealed the presence of I _SK cDNA in the rat trachea epithelia. Although 0.1 mm Ba²⁺ only had minimal affect on short-circuit current (I _sc) induced by cAMP in intact epithelia, 0.1 mm clofilium strongly inhibited it. These results indicate that I _SK might be important for maintaining cAMP-induced chloride secretion in the rat trachea epithelia. Received: 1 March 1996/Revised: 5 August 1996     

背景氯离子通道研究进展

综述了目前了解得最为充分的一类电压门控氯通道——背景氯通道,内容涉及选择性、门控和药理学以及通道蛋白的克隆和分子结构.氯通道广泛存在于细胞膜和细胞器膜,作为“总管家”参与细胞pH,体积,静息膜电位和兴奋性等多种细胞过程的调节.由于种种原因,对氯通道的研究起步较晚.目前应用膜片钳和分子生物学技术对氯通道结构功能的研究已经成为一个热点.     

Subunit Stoichiometry of Human Muscle Chloride Channels

Voltage-gated Cl⁻ channels belonging to the ClC family appear to function as homomultimers, but the number of subunits needed to form a functional channel is controversial. To determine subunit stoichiometry, we constructed dimeric human skeletal muscle Cl⁻ channels in which one subunit was tagged by a mutation (D136G) that causes profound changes in voltage-dependent gating. Sucrose-density gradient centrifugation experiments indicate that both monomeric and dimeric hClC-1 channels in their native configurations exhibit similar sedimentation properties consistent with a multimeric complex having a molecular mass of a dimer. Expression of the heterodimeric channel in a mammalian cell line results in a homogenous population of Cl⁻ channels exhibiting novel gating properties that are best explained by the formation of heteromultimeric channels with an even number of subunits. Heteromultimeric channels were not evident in cells cotransfected with homodimeric WT-WT and D136G-D136G constructs excluding the possibility that functional hClC-1 channels are assembled from more than two subunits. These results demonstrate that the functional hClC-1 unit consists of two subunits.     

Mechanism of Ion Permeation in Skeletal Muscle Chloride Channels

Voltage-gated Cl⁻ channels belonging to the ClC family exhibit unique properties of ion permeation and gating. We functionally probed the conduction pathway of a recombinant human skeletal muscle Cl⁻ channel (hClC-1) expressed both in Xenopus oocytes and in a mammalian cell line by investigating block by extracellular or intracellular I⁻ and related anions. Extracellular and intracellular I⁻ exert blocking actions on hClC-1 currents that are both concentration and voltage dependent. Similar actions were observed for a variety of other halide (Br⁻) and polyatomic (SCN⁻, NO₃ ⁻, CH₃SO₃ ⁻) anions. In addition, I⁻ block is accompanied by gating alterations that differ depending on which side of the membrane the blocker is applied. External I⁻ causes a shift in the voltage-dependent probability that channels exist in three definable kinetic states (fast deactivating, slow deactivating, nondeactivating), while internal I⁻ slows deactivation. These different effects on gating properties can be used to distinguish two functional ion binding sites within the hClC-1 pore. We determined K _D values for I⁻ block in three distinct kinetic states and found that binding of I⁻ to hClC-1 is modulated by the gating state of the channel. Furthermore, estimates of electrical distance for I⁻ binding suggest that conformational changes affecting the two ion binding sites occur during gating transitions. These results have implications for understanding mechanisms of ion selectivity in hClC-1, and for defining the intimate relationship between gating and permeation in ClC channels.     

Downregulation of Chloride Channel ClC-2 by Janus Kinase 3

Janus kinase-3 (JAK3) fosters proliferation and counteracts apoptosis of lymphocytes and tumor cells. The gain of function mutation ^A572VJAK3 has been discovered in acute megakaryoplastic leukemia. JAK3 is inactivated by replacement of lysine by alanine in the catalytic subunit (^K855AJAK3). Regulation of cell proliferation and apoptosis involves altered activity of Cl^? channels. The present study, thus, explored whether JAK3 modifies the function of the small conductance Cl^? channel ClC-2. To this end, ClC-2 was expressed in Xenopus oocytes with or without wild-type JAK3, ^A568VJAK3 or ^K851AJAK3, and the Cl^? channel activity determined by dual-electrode voltage clamp. Channel protein abundance in the cell membrane was determined utilizing chemiluminescence. As a result, expression of ClC-2 was followed by a marked increase of cell membrane conductance. The conductance was significantly decreased following coexpression of JAK3 or ^A568VJAK3, but not by coexpression of ^K851AJAK3. Exposure of the oocytes expressing ClC-2 together with ^A568VJAK3 to the JAK3 inhibitor WHI-P154 (4-[(3’-bromo-4’-hydroxyphenyl)amino]-6,7-dimethoxyquinazoline, 22 μM) increased the conductance. Coexpression of ^A568VJAK3 decreased the ClC-2 protein abundance in the cell membrane of ClC-2 expressing oocytes. The decline of conductance in ClC-2 and ^A568VJAK3 coexpressing oocytes following inhibition of channel protein insertion by brefeldin A (5 μM) was similar in oocytes expressing ClC-2 with ^A568VJAK3 and oocytes expressing ClC-2 alone, indicating that ^A568VJAK3 might slow channel protein insertion into rather than accelerating channel protein retrieval from the cell membrane. In conclusion, JAK3 downregulates ClC-2 activity and thus counteracts Cl^? exit—an effect possibly influencing cell proliferation and apoptosis.     

Permeant Anions Control Gating of Calcium-dependent Chloride Channels

The effects of external anions (SCN⁻, NO₃⁻, I⁻, Br⁻, F⁻, glutamate, and aspartate) on gating of Ca²⁺-dependent Cl⁻ channels from rat parotid acinar cells were studied using the whole-cell configuration of the patch-clamp technique. Shifts in the reversal potential of the current induced by replacement of external Cl⁻ with foreign anions, gave the following selectivity sequence based on permeability ratios (P_x/P_Cl): SCN⁻>I⁻>NO₃⁻>Br⁻>Cl⁻>F⁻>aspartate>glutamate. Using a continuum electrostatic model we calculated that this lyotropic sequence resulted from the interaction between anions and a polarizable tunnel with an effective dielectric constant of ∼23. Our data revealed that anions with P_x/P_Cl > 1 accelerated activation kinetics in a voltage-independent manner and slowed deactivation kinetics. Moreover, permeant anions enhanced whole-cell conductance (g, an index of the apparent open probability) in a voltage-dependent manner, and shifted leftward the membrane potential-g curves. All of these effects were produced by the anions with an effectiveness that followed the selectivity sequence. To explain the effects of permeant anions on activation kinetics and g_Cl we propose that there are 2 different anion-binding sites in the channel. One site is located outside the electrical field and controls channel activation kinetics, while a second site is located within the pore and controls whole-cell conductance. Thus, interactions of permeant anions with these two sites hinder the closing mechanism and stabilize the channel in the open state.This revised version was published online in August 2005 with a corrected cover date.     

Role of Potassium Ions in Regulation of Calcium-Activated Chloride Channels

Doklady Biochemistry and Biophysics - Using the patch-clamp method in the whole cell configuration, it was shown that external potassium ions play an important role in the regulation of...     

Chloride Channels and Transporters of the CLC Family in Plants

Russian Journal of Plant Physiology - Proteins of the chloride channel family CLC (ChLoride Channel) are found in prokaryotes and eukaryotes. The family includes anion channels and anion/proton...     

Evolutionary Relationship of the Ligand-Gated Ion Channels and the Avermectin-Sensitive,Glutamate-Gated Chloride Channels

Two cDNAs, GluClα and GluClβ, encoding glutamate-gated chloride channel subunits that represent targets of the avermectin class of antiparasitic compounds, have recently been cloned from Caenorhabditis elegans (Cully et al., Nature, 371, 707–711, 1994). Expression studies in Xenopus oocytes showed that GluClα and GluClβ have pharmacological profiles distinct from the glutamate-gated cation channels as well as the γ-aminobutyric acid (GABA)- and glycine-gated chloride channels. Establishing the evolutionary relationship of related proteins can clarify properties and lead to predictions about their structure and function. We have cloned and determined the nucleotide sequence of the GluClα and GluClβ genes. In an attempt to understand the evolutionary relationship of these channels with the members of the ligand-gated ion channel superfamily, we have performed gene structure comparisons and phylogenetic analyses of their nucleotide and predicted amino acid sequences. Gene structure comparisons reveal the presence of several intron positions that are not found in the ligand-gated ion channel superfamily, outlining their distinct evolutionary position. Phylogenetic analyses indicate that GluClα and GluClβ form a monophyletic subbranch in the ligand-gated ion channel superfamily and are related to vertebrate glycine channels/receptors. Glutamate-gated chloride channels, with electrophysiological properties similar to GluClα and GluClβ, have been described in insects and crustaceans, suggesting that the glutamate-gated chloride channel family may be conserved in other invertebrate species. The gene structure and phylogenetic analyses in combination with the distinct pharmacological properties demonstrate that GluClα and GluClβ belong to a discrete ligand-gated ion channel family that may represent genes orthologous to the vertebrate glycine channels. Received: 30 September 1996 / Accepted: 15 November 1996     

钙激活氯通道电生理特性及调节机制

钙激活氯通道(Calcium-activated Chloride Channels,CaCC)在许多生理过程中起着重要作用,如分泌蛋白及盐的跨上皮转运、神经元兴奋、平滑肌生理特性的维持、卵母细胞受精和心脏动作电位的复极化等.CaCC电生理特性较复杂,其全细胞电流随胞内钙离子浓度和刺激电压的不同而表现出不同整流特性和时间电压依赖性,不同组织的单通道电导有很大差异,大小介于1～70pS.CaCC可被NFA、9-AC、DPC、DIDS所阻断.目前CaCC通道蛋白的分子结构还不清楚,已克隆的两种蛋白家族CLCA和Bestrophin可作为其候选分子.本文综述了CaCC的电生理特性、调节机制、分子结构和生理功能.     

Activation and Inhibition of TMEM16A Calcium-Activated Chloride Channels

Calcium-activated chloride channels (CaCC) encoded by family members of transmembrane proteins of unknown function 16 (TMEM16) have recently been intensely studied for functional properties as well as their physiological roles as chloride channels in various tissues. One technical hurdle in studying these channels is the well-known channel rundown that frequently impairs the precision of electrophysiological measurements for the channels. Using experimental protocols that employ fast-solution exchange, we circumvented the problem of channel rundown by normalizing the Ca²⁺-induced current to the maximally-activated current obtained within a time period in which the channel rundown was negligible. We characterized the activation of the TMEM16A-encoded CaCC (also called ANO1) by Ca²⁺, Sr²⁺, and Ba²⁺, and discovered that Mg²⁺ competes with Ca²⁺ in binding to the divalent-cation binding site without activating the channel. We also studied the permeability of the ANO1 pore for various anions and found that the anion occupancy in the pore–as revealed by the permeability ratios of these anions–appeared to be inversely correlated with the apparent affinity of the ANO1 inhibition by niflumic acid (NFA). On the other hand, the NFA inhibition was neither affected by the degree of the channel activation nor influenced by the types of divalent cations used for the channel activation. These results suggest that the NFA inhibition of ANO1 is likely mediated by altering the pore function but not through changing the channel gating. Our study provides a precise characterization of ANO1 and documents factors that can affect divalent cation activation and NFA inhibition of ANO1.     

High-Efficiency,Selection-free Gene Repair in Airway Stem Cells from Cystic Fibrosis Patients Rescues CFTR Function in Differentiated Epithelia

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Activity-Dependent Dendritic Spine Shrinkage and Growth Involve Downregulation of Cofilin via Distinct Mechanisms

A current model posits that cofilin-dependent actin severing negatively impacts dendritic spine volume. Studies suggested that increased cofilin activity underlies activity-dependent spine shrinkage, and that reduced cofilin activity induces activity-dependent spine growth. We suggest instead that both types of structural plasticity correlate with decreased cofilin activity. However, the mechanism of inhibition determines the outcome for spine morphology. RNAi in rat hippocampal cultures demonstrates that cofilin is essential for normal spine maintenance. Cofilin-F-actin binding and filament barbed-end production decrease during the early phase of activity-dependent spine shrinkage; cofilin concentration also decreases. Inhibition of the cathepsin B/L family of proteases prevents both cofilin loss and spine shrinkage. Conversely, during activity-dependent spine growth, LIM kinase stimulates cofilin phosphorylation, which activates phospholipase D-1 to promote actin polymerization. These results implicate novel molecular mechanisms and prompt a revision of the current model for how cofilin functions in activity-dependent structural plasticity.