Types Of Chloride Channels Research Articles

Comparative Effects of Chloride Channel Inhibitors on LRRC8/VRAC-Mediated Chloride Conductance.

Chloride channels play an essential role in a variety of physiological functions and in human diseases. Historically, the field of chloride channels has long been neglected owing to the lack of powerful selective pharmacological agents that are needed to overcome the technical challenge of characterizing the molecular identities of these channels. Recently, members of the LRRC8 family have been shown to be essential for generating the volume-regulated anion channel (VRAC) current, a chloride conductance that governs the regulatory volume decrease (RVD) process. The inhibitory effects of six commonly used chloride channel inhibitors on VRAC/LRRC8-mediated chloride transport were tested in wild-type HEK-293 cells expressing LRRC8 proteins and devoid of other types of chloride channels (CFTR and ANO1/2). We explored the effectiveness of the inhibitors using the patch-clamp whole-cell approach and fluorescence-based quantification of cellular volume changes during hypotonic challenge. Both DCPIB and NFA inhibited VRAC current in a whole-cell configuration, with IC50 values of 5 ± 1 μM and 55 ± 2 μM, respectively. Surprisingly, GlyH-101 and PPQ-102, two CFTR inhibitors, also inhibited VRAC conductance at concentrations in the range of their current use, with IC50 values of 10 ± 1 μM and 20 ± 1 μM, respectively. T16Ainh-A01, a so-called specific inhibitor of calcium-activated Cl- conductance, blocked the chloride current triggered by hypo-osmotic challenge, with an IC50 of 6 ± 1 μM. Moreover, RVD following hypotonic challenge was dramatically reduced by these inhibitors. CFTRinh-172 was the only inhibitor that had almost no effect on VRAC/LRRC8-mediated chloride conductance. All inhibitors tested except CFTRinh-172 inhibited VRAC/LRRC8-mediated chloride conductance and cellular volume changes during hypotonic challenge. These results shed light on the apparent lack of chloride channel inhibitors specificity and raise the question of how these inhibitors actually block chloride conductances.

Expression of chloride channels in mouse skin incisional wounds

This study aimed to determine the types of chloride channels (CLCs) expressed in mouse skin wounds. Twelve male C57BL/6 mice were randomly divided into the skin wound group (SWG) and normal unwounded group (NUG), with 6 mice in each group. A 5-mm lancet wound was made by cutting into the full-thickness skin in the SWG, and the skin was kept intact in the NUG. The expression of CLCs in both groups was evaluated using real-time PCR (RT-PCR) and western blotting (WB). The results of RT-PCR showed that nine CLCs, including CLC-1, CLC-2, CLC-3, CLC-4, CLC-5, CLC-6, CLC-7, CLC-kα, and CLC-kβ, were expressed in both groups. However, the WB results showed that only CLC-2, CLC-7, CLC-kα, and CLC-kβ were expressed in both groups. The results of this study indicate that CLC-2, CLC-7, CLC-kα, and CLC-kβ were expressed in intact and wounded mouse skin according to both RT-PCR and WB analyses.

Effects of ATP-sensitive potassium channel regulators on chloride channels in the sarcoplasmic reticulum vesicles from rabbit skeletal muscle.

The lipid bilayer technique was used to examine the effects of the ATP-sensitive K+ channel inhibitor (glibenclamide) and openers (diazoxide, minoxidil and cromakalim) and Cl- channel activators (GABA and diazepam) on two types of chloride channels in the sarcoplasmic reticulum (SR) from rabbit skeletal muscle. Neither diazepam at 100 microM nor GABA at 150 microM had any significant effect on the conductance and kinetics of the 75 pS small chloride (SCl) channel. Unlike the 150 pS channel, the SCl channel is sensitive to cytoplasmic glibenclamide with Ki approximately 30 microM. Glibenclamide induced reversible decline in the values of current (maximal current amplitude, Imax and average mean current, I') and kinetic parameters (frequency of opening Fo, probability of the channel being open Po and mean open time, To, of the SCl channel. Glibenclamide increased mean closed time, Tc, and was a more potent blocker from the cytoplasmic side (cis) than from the luminal side (trans) of the channel. Diazoxide increased I', Po, and To in the absence of ATP and Mg2+ but it had no effect on Imax and also failed to activate or remove the glibenclamide- and ATP-induced inhibition of the SCl channel. Minoxidil induced a transient increase in I' followed by an inhibition of Imax, whereas cromakalim reduced Po and I' by increasing channel transitions to the closed state and reducing To without affecting Imax. The presence of diazoxide, minoxidil or cromakalim on the cytoplasmic side of the channel did not prevent [ATP]cis or [glibenclamide]cis from blocking the channel. The data suggest that the action(s) of these drugs are not due to their effects on the phosphorylation of the channel protein. The glibenclamide- and cromakalim-induced effects on the SCl channel are mediated via a "flicker" type block mechanism. Modulation of the SCl channel by [diazoxide]cis and [glibenclamide]cis highlights the therapeutic potential of these drugs in regulating the Ca2+-counter current through this channel.

CFTR-independent ATP release from epithelial cells triggered by mechanical stimuli.

Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated ATP efflux has been proposed as an autocrine mechanism for regulating chloride secretion through other types of chloride channels. Although we found in previous studies that wild-type CFTR channels bathed with high-ATP solutions do not conduct ATP at rates that can be measured with the patch-clamp technique, those experiments would not have detected very small or electroneutral ATP fluxes through CFTR or ATP efflux through other pathways that might be regulated by CFTR. To examine these possibilities, we have now used a sensitive luciferase luminometric assay to measure ATP efflux from epithelial and nonepithelial cell lines. Adenosine 3',5'-cyclic monophosphate (cAMP) stimulation did not raise external ATP concentration above the background noise in any of the cell lines tested [T84, Calu-3, 9HTEo- and sigma CFTE29o- (colonic and airway human epithelial cells, respectively), NIH/3T3 fibroblasts, and Chinese hamster ovary cells], and variations in ATP release were not correlated with CFTR expression. The rate of ATP release was unaffected by cAMP but was exquisitely sensitive to mechanical perturbations in both CFTR expressing and nonexpressing cells. Mechanically induced, CFTR-independent ATP release may be a physiologically relevant mechanism of epithelial regulation, which has not previously been fully appreciated.

Multiple types of chloride channels in bovine pulmonary artery endothelial cells.

We have characterized two different types of Cl- currents in calf pulmonary artery endothelial (CPAE) cells by using a combined patch-clamp and Fura-2 microfluorescence technique to measure simultaneously ionic currents and the intracellular Ca2+ concentration, [Ca2+]i. Exposure of CPAE cells to 28% hypotonic solution induces cell swelling without a change in membrane capacitance and [Ca2+]i, and concomitantly activates a current. This current, I(Cl, vol), is closely correlated with the changes in cell volume and shows a modest outward rectification. It slowly inactivates at potentials more positive than +60 mV but is time- and voltage-independent at other potentials. Increase in [Ca2+]i by different maneuvers, such as application of vasoactive agonists (ATP), ionomycin, or loading of the cells directly with Ca2+ also activates a Cl- current, I(Cl, Ca). This current slowly activates at positive potentials, inactivates quickly at negative potentials and shows strong outward rectification. A time-independent component of the current activated by elevation of [Ca2+]i alone can be inhibited by cell shrinking by exposing the cells to hypertonic solution, indicating that an increase in [Ca2+]i also co-activates I(Cl, vol). Forskolin or cAMP never activated a current in CPAE cells, which indicates the lack of cAMP-activated channels in these cells. There is also no evidence for the existence of voltage-gated Cl- channels in resting, nonstimulated cells. Challenging a cell with elevated [Ca2+]i and hypotonic solutions activated I(Cl, vol) on top of I(Cl, Ca), suggesting that I(Cl, Ca) and I(Cl, vol) are different channels. We conclude that CPAE cells do not express voltage-gated (ClC-type) or cAMP-gated (CFTR-type) Cl- channels, but activate large Cl- currents after volume (mechanical?) or chemical (Ca2+) stimulation.

CAMP-induced chloride transport in NCL-SG3 sweat gland cells.

cAMP-induced ion transport in a human sweat gland cell line, NCL-SG3, was investigated by X-ray microanalysis and patch-clamp technique. Stimulation with cAMP caused a decrease in the cellular Cl and K. cAMP had no significant effect on the intracellular Na and Ca. Chloride channel blockers (9-AC, DPC and NPPB) inhibited the cAMP-induced chloride efflux. In patch-clamp experiments the inward current increased over a period of 5-15 min on addition of membrane-permeable cAMP in 66% of the attempts when the cell was held at 0 mV and pulsed to negative membrane potentials. The inward current was completely blocked by chloride channel blockers. Washout reversed the effect of cAMP. The inward current was not diminished by substitution of impermeable cations for Na in the bath and was insensitive to TEA (tetraethylammoniumchloride). It is concluded that the inward current is mainly a chloride current. Cystic fibrosis transmembrane regulator (CFTR) could not be demonstrated in the NCL-SG3 cells. It is therefore possible that the chloride efflux is mediated by other types of chloride channels.

Expression and regulation of chloride channels in neonatal rat cardiomyocytes

Using an 125I- efflux assay, we have studied the expression of various types of chloride channels in isolated neonatal rat cardiomyocytes. Three different classes of anion conductances were distinguished: (1) a Ca(2+)-sensitive Cl- conductance, triggered upon stimulation of the cells with endothelin-1 or Ca(2+)-ionophore; (2) a cAMP/protein kinase A-operated Cl- conductance, activated by addition of forskolin. This anion channel could be identified as the Cystic Fibrosis Transmembrane conductance Regulator (CFTR-CI- channel) by Western blotting as well as by its enhanced activity in cultures pretreated with the tyrosine kinase inhibitor genistein; (3) a distinct class of cell volume-regulated Cl- channels, potentiated in the presence of endothelin-1 or the phosphotyrosine phosphatase inhibitor pervanadate. The potential role of each class of Cl- channels in the generation and/or modulation of action potentials as well as in maintaining cell volume is discussed.

Sarcolemmal Chloride and Potassium Channels from Normal and Myotonic Mouse Muscle Studied in Lipid-Supplemented Vesicles

By patch-clamp analysis of lipid supplemented vesicles prepared from the sarcolemma of mouse wildtype skeletal muscle, we could identify two known types of potassium channels, the inward rectifier and Ca2+ activated BK channels, and five types of chloride channels designated CIC-a, CIC-b, CIC-c, CIC-d, and CIC-e. CIC-b corresponds to a known chloride channel, whereas CICa, -c, -d and -e have not been described previously. The diversity may be due to a heterooligomeric composition of different subunits. None of these chloride channels nor the potassium channels were found in the sarcoplasmic reticulum fraction. In vesicles from myotonic mice, Clc-d and -e were not found.

Resolving three types of chloride channels in demyelinated Xenopus axons.

Axons from Xenopus sciatic nerve were demyelinated by intraneural injection of lysolecithin rendering the entire internodal axolema accessible to a patch electrode. In this region, three types of anion selective pores were found and characterized at the single-channel level. These included outwardly rectifying, inwardly rectifying, and maxi Cl- channels. The outwardly rectifying Cl- channels (24 pS) are activated by depolarization with a weak voltage dependence of 42 mV per e-fold change in open probability. The inwardly rectifying Cl- channels (27 pS) are insensitive to voltage, but can be blocked by internal application of 100 microM SITS or DIDS. The I-V curves of rectifying channels are S-shaped and can be fitted by a kinetic model with a single free energy barrier. The rectification may be related to the location of this barrier. The maxi Cl- channel (335 pS) is often open at the resting potential, but is inactivated by a large depolarization. The rectification, voltage dependence, and inactivation of these channels may contribute to the regulation of axonal Cl- balance and resting potential.

Insight into the Structure-Function Relation of Chloride Channels

Chloride channels are highly selective transport proteins ubiquitously expressed in eukaryotic cells. Biophysical methods allow discrimination between several different types of chloride channels with

Chemical probes for anion transporters of mammalian cell membranes.

Mammalian cell membranes harbor several types of chloride channels, chloride-cation symporters/cotransporters, and several classes of anion exchangers/antiporters. These transport systems subserve different cellular or organismic functions, depending on the nature of the cell, the spatial organization of transporters, and their functional interplay. Chemical probing has played a central role in the structural and functional delineation of the various anion transport systems. The design of specific probes or their selection from existing sources coupled with their judicious application to the most appropriate biological system had led to the identification of specific anion transporters and to the elucidation of the underlying molecular transport mechanism. In many instances, chemical probing has remained the major or exclusive analytical tool for the functional definition or identification of a given transport system, particularly for discerning among the various anion transporters which operate in highly heterogeneous cell membrane systems. This work critically reviews the present state of the chemical armamentarium available for the most common anion transporters found in mammalian cell membranes. It encompasses the description of the most useful or commonly used probes in terms of their chemical, biochemical, physiological, and pharmacological properties. The review deals primarily with what chemical probes tell about anion transporters and, most importantly, with the limitations inherent in the use of probes in transport studies.

Noise analysis and single-channel observations of 4 pS chloride channels in human airway epithelia

Apical membranes of human airway epithelial cells have significant chloride permeability, which is reduced in cystic fibrosis (CF), causing abnormal electrochemistry and impaired mucociliary clearance. At least four types of chloride channels have been identified in these cells, but their relative roles in total permeability and CF are unclear. Noise analysis was used to measure the conductance of chloride channels in human nasal epithelial cells. The data indicate that channels with a mean conductance of 4.5 pS carry most of the chloride current, and that the mean number of such channels per cell is approximately 4,000. Chloride channels in this conductance range were also seen in single-channel recordings.

Two types of chloride channels in hen colon epithelial cells identified by patch-clamp experiments

Freshly isolated epithelial cells from hen colon were investigated using the patch-clamp technique. The aim of this investigation was to characterise the cellular conducting site for Cl- secretion. In cell-attached mode two types of Cl(-)-channels were found. Both showed distinct outward rectification. The channel types differed in single channel conductances and the marked voltage dependence of the open probabilities. A low conductance Cl(-)-channel was observed with a mean conductance at negative holding potentials of g- = 9 pS, and of g+ = 34 pS at positive potentials. This channel was predominantly open at negative potentials, corresponding to cell hyperpolarization. The second channel type observed had conductances of g- = 35 pS and g+ = 77 pS, and showed increasing open probabilities with increasing holding potentials (cell depolarisation). Both channel types were blockable by the Cl(-)-channel blocker NPPB. These data in combination with previously published transepithelial transport data on hen colon indicate that these channels are the Cl- secretory sites in colon epithelium.

Purification and Reconstitution of Chloride Channels from Kidney and Trachea

Chloride channels mediate absorption and secretion of fluid in epithelia, and the regulation of these channels is now known to be defective in cystic fibrosis. Indanyl-oxyacetic acid 94 (IAA-94) is a high-affinity ligand for the chloride channel, and an affinity resin based on that structure was developed. Solubilized proteins from kidney and trachea membranes were applied to the affinity matrix, and four proteins with apparent molecular masses of 97, 64, 40, and 27 kilodaltons were eluted from the column by excess IAA-94. A potential-dependent 36Cl- uptake was observed after reconstituting these proteins into liposomes. Three types of chloride channels with single-channel conductances of 26, 100, and 400 picosiemens were observed after fusion of these liposomes with planar lipid bilayers. Similar types of chloride channels have been observed in epithelia.

Reconstitution and Phosphorylation of Chloride Channels from Airway Epithelium Membranes

Airway epithelial chloride secretion is controlled by the apical-membrane chloride permeability. Purified apical-membrane vesicles from bovine tracheal epithelium have now been shown to contain functional chloride channels by using the planar-bilayer technique. Three types of chloride channels were observed; a voltage-dependent, calcium-independent, 71-picoSiemen (in 150 mM NaCl) channel accounted for more than 80 percent of the vesicular chloride conductance and was under strict control of phosphorylation. The channel underwent a fast rundown in less than 2 to 3 minutes of recording, and reactivation required in situ exposure to a phosphorylating "cocktail" containing the catalytic subunit of the adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase. Mean open time and open probability were increased after phosphorylation, whereas slope conductance remained unchanged. Thus, metabolic control of tracheal chloride single channels can now be studied in vitro.

Chloride channels in the luminal membrane of the rectal gland of the dogfish (Squalus acanthias)

The rectal gland of the dogfish (Squalus acanthias) secretes chloride via a chloride channel present in the apical cell membrane. Using the patch clamp technique in isolated perfused rectal gland tubules, two types of chloride channels are demonstrable in the apical membrane of cyclic AMP treated tubule segments. A small channel of about 11 pS and another channel of 40-50 pS are present. The small channel is described in the succeeding report. With NaCl on both sides (excised patches) the current amplitude of the larger channel is an almost linear function of the voltage (+/- 50 mV). However, the open probability of this channel is grossly reduced at negative clamp potentials (corresponding to cell hyperpolarization). Therefore, the macroscopic Cl- current through this channel is reduced with hyperpolarization on the cytosolic side. An analysis of time constants of this channel reveals that at depolarized voltages two open and two closed time constants of about 1 ms and of about 10 ms, respectively, are demonstrable. With hyperpolarized voltages the larger open state time constant is reduced significantly. This type of chloride channel is blocked reversibly by diphenylamine-2-carboxylate (10(-4) mol/l) and by 5-nitro-2-(3-phenylpropylamino)-benzoate (10(-5) mol/l). The channel is selective for Cl- over Na+ and K+ as well as over Br-. It is, however, permeable for NO3-. Since this channel is very rare or absent in nonstimulated rectal gland tubules, it is very likely that this type of channel is responsible for hormone and cAMP dependent chloride secretion in this organ.