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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.

Animals↗

Intrarenal mRNA expression of the rat MDCK-type chloride channel.

We have reported the isolation of a rat cDNA for MDCK-type chloride channel (RKCL) which is expressed in many tissues including the kidney. In the present study, intrarenal expression distribution of RKCL mRNA and the effect of dehydration on its expression were examined. In situ hybridization showed that RKCL mRNA is more abundantly expressed in the renal medulla and papilla than in the cortex. Reverse transcription and polymerase chain reaction using microdissected nephron segments showed that RKCL mRNA is expressed in several nephron segments, notably the ascending thin limb of Henle's loop and inner medullary collecting duct. Under dehydration, renal RKCL mRNA expression level was not significantly changed for 5 days. In conclusion, RKCL mRNA is expressed in most nephron segments, playing a role in the renal tubular chloride ion homeostasis.

Animals↗

Inositol polyphosphates modify the kinetics of a small chloride channel in skeletal muscle sarcoplasmic reticulum.

The actions of D-myo-inositol 1,4,5-trisphosphate (IP3) and D-myo-inositol 1,3,4,5-tetrakisphospate (IP4) on small chloride (SCl) channels from rabbit skeletal muscle sarcoplasmic reticulum are reported. We find that the inositol polyphosphates (6-40 microM) are potent reversible blockers of SCl channels in lipid bilayers at -40 mV with >10(-5) m cis (cytoplasmic) Ca2+ when added to the cis, but not trans, chamber. IP3 or IP4 at 20 microM reduced the mean open time from 89 +/- 16 msec to 11 +/- 2 msec or to 8.0 +/- 1.0 msec respectively, by abolishing the longest time constant component in the open time distribution. Neither IP3 nor IP4 altered the six single-channel conductance levels. The fraction of low conductance events increased approximately 4-fold and the dwell time at the lower conductance levels increased approximately 3-fold. Channel gating was altered so that most transitions were between the closed level and an open level, in contrast to control channels which remained open for long periods with many transitions between the six open levels. The actions of the inositol polyphosphates were: (1) not prevented by 20 microg/ml cis heparin (an IP3 receptor blocker); (2) mimicked by 10 microM cis synthetic inositol polyphosphates, L-chiro-inositol 1,4,6-trisphosphate and L-chiro-inositol 1,4,6-trisphosphorothionate (which do not bind to IP3 receptors); (3) mimicked by cis additions of the polyanions heparin or hepran (20 microg/ml each) and vanadate (500 microM). The results suggest that an interaction between polyanions and SCl channels would allow the channels to be modulated in vivo by inositol polyphosphates.

Animals↗

Topology of the human skeletal muscle chloride channel hClC-1 probed with hydrophilic epitope insertion.

To investigate the membrane topology of the skeletal muscle chloride channel ClC-1, we inserted the small antigenic flag (DYKDDDDK) and/or HSV (QPELAPEDPED) epitope tags into nine predicted extra- and intracellular loops along the channel protein. Functional integrity of the modified proteins was tested by measuring the chloride currents conducted by these channels expressed in tsA201 cells. Insertion of the tags into the linkers D1D2, D4D5, D6D7, D8D9 or D11D12 did not alter channel function significantly, whereas insertion into D3D4, D5D6, D9D10 and D10D11 led to loss of function. Intra- or extracellular localisation of the tags was determined by immunofluorescent staining of intact and permeabilised tsA201 cells transiently transfected with the functional epitope-inserted constructs. Intact cells stained for the epitope tags inserted into D1D2, D6D7 and D8D9, indicating that these linkers face the extracellular side of the membrane. No conclusions could be drawn for the location of D4D5 and D11D12. Insertion of the flag epitope at position P260 (linker D4D5), a putative pore-lining region, did not change any of the channel function properties markedly, suggesting that the region surrounding P260 cannot directly line the ion conduction pathway of ClC-1.

Cell Line, Transformed↗

Alterations in the gamma-aminobutyric acid-gated chloride channel following transient forebrain ischemia in the gerbil.

The role of inhibitory neurotransmission in selective neuronal degeneration after transient forebrain ischemia was studied by binding of t-[35S]butylbicyclophosphorothionate ([35S]TBPS) to the gamma-aminobutyric acid (GABA)-gated chloride channel and measurement of GABAA receptor function in Mongolian gerbil brain. [35S]TBPS binding to the hippocampus, striatum, and cortex quantified by autoradiography and muscimol-stimulated 36Cl- uptake in synaptoneurosomes of the same regions were examined 1, 4, and 29 days after a 5-min bilateral carotid occlusion. [35S]TBPS binding was decreased in the pyramidal cell dendritic layers, stratum oriens, and stratum lacunosum-moleculare of the CA1 hippocampus, 4 and 29 days after occlusion, and in the stratum radiatum 29 days after occlusion. [35S]TBPS binding sites in the lateral striatum decreased 47% 4 days after occlusion. At the same time, there was a corresponding decrease in muscimol-stimulated 36Cl- uptake in the striatal synaptoneurosomes. Muscimol-stimulated 36Cl- uptake in the hippocampus decreased slightly 4 days after occlusion and more so after 29 days, although these decreases were not significant. No changes were observed in somatosensory cortex at any time point. These data suggest that a portion of GABAA receptors in areas sensitive to ischemic insult are associated with degenerating neurons, whereas other GABAA) receptors are spared.

Animals↗

Effect of a chloride channel blocker on bullfrog corneal epithelium.

The effect of the chloride channel blocker, diphenylamine-2-carboxylate (DPC), was investigated on the bullfrog cornea epithelium using microelectrodes. Perfusion of 2 X 10(-4) M DPC on the tear side resulted in a 44% reduction of the corneal transepithelial potential difference (TEP) and a 9% reduction in corneal conductance. The cornea epithelial cells hyperpolarized 7.2 +/- 1.5 mV, and the apical fractional resistance increased 35% (n = 8 tissues). The basolateral potential difference depolarized 5.5 +/- 1.8 mV. The relative apical conductance decreased 29%, whereas relative changes of basolateral membranes increased 56% with prolonged DPC exposure, indicating that the DPC inhibition was acting at the apical cell membrane. DPC-treated corneas could be stimulated with isoproterenol (10(-6) M), indicating that the DPC inhibition could be reversed with the stimulation of chloride conductance. The DPC inhibitory effect was dependent on basolateral chloride. Intracellular chloride activity increases with DPC, indicating that the integrity of the basolateral chloride entry mechanism was not altered. The DPC inhibitory action was due to a reduction in apical chloride conductance.

Animals↗

Cloning and functional expression of rat CLC-5, a chloride channel related to kidney disease.

We have cloned a novel member of the CLC chloride channel family from rat brain, rCLC-5. The cDNA predicts a 83-kDa protein belonging to the branch including CLC-3 and CLC-4, with which it shares approximately 80% identity. Expression of rCLC-5 in Xenopus oocytes elicits novel anion currents. They are strongly outwardly rectifying and have a conductivity sequence of NO3- > Cl- > Br- > I- >> glutamate-. Although CLC-5 has consensus sites for phosphorylation by protein kinase A, raising the intracellular cAMP concentration had no effect on these currents. Currents were also unchanged when rCLC-5 was coexpressed with rCLC-3 and rCLC-4, either singly or in combination. rCLC-5 is expressed predominantly in kidney and also in brain, lung, and liver. Along the nephron, rCLC-5 message is detectable in all tubule segments investigated, but expression in the glomerulus and the S2 segment of the proximal tubule is low.

Animals↗

Developmental aspects of benzodiazepine receptors and GABA-gated chloride channels in primary cultures of spinal cord neurons.

The developmental aspects of gamma-aminobutyric acid (GABA)-benzodiazepine receptor ionophore complex in mouse cultured spinal cord neurons was investigated in vitro. We examined benzodiazepine binding, GABA enhancement of benzodiazepine binding and GABA activation of chloride channels to intact cultured neurons during the development (days 1-14). Specific benzodiazepine binding increased with time from day 3 (Bmax = 546 fmol/mg protein) to day 14 (Bmax = 1746 fmol/mg protein). There was no significant difference in the affinity (Kd) of [3H]flunitrazepam for its receptor sites from day 3 to 14. Specific binding of [3H]flunitrazepam to the intact cells was also increased in the presence of GABA during all stages of development. The EC50 value of the GABA concentration (14-16 microM) required to enhance [3H]flunitrazepam binding did not change from day 3 to 14. GABA receptor-activated chloride channels, studied by 36Cl-influx was also observed at all ages in culture, and indicated an early development (as early as day 3) of these GABA-gated channels. These studies indicate that GABA-benzodiazepine-ionophore complex develop early in cultured spinal cord neurons, and these neurons provide an ideal system to study GABAergic events.

Animals↗

Loss of the chloride channel ClC-7 leads to lysosomal storage disease and neurodegeneration.

ClC-7 is a chloride channel of late endosomes and lysosomes. In osteoclasts, it may cooperate with H(+)-ATPases in acidifying the resorption lacuna. In mice and man, loss of ClC-7 or the H(+)-ATPase a3 subunit causes osteopetrosis, a disease characterized by defective bone resorption. We show that ClC-7 knockout mice additionally display neurodegeneration and severe lysosomal storage disease despite unchanged lysosomal pH in cultured neurons. Rescuing their bone phenotype by transgenic expression of ClC-7 in osteoclasts moderately increased their lifespan and revealed a further progression of the central nervous system pathology. Histological analysis demonstrated an accumulation of electron-dense material in neurons, autofluorescent structures, microglial activation and astrogliosis. Like in human neuronal ceroid lipofuscinosis, there was a strong accumulation of subunit c of the mitochondrial ATP synthase and increased amounts of lysosomal enzymes. Such alterations were minor or absent in ClC-3 knockout mice, despite a massive neurodegeneration. Osteopetrotic oc/oc mice, lacking a functional H(+)-ATPase a3 subunit, showed no comparable retinal or neuronal degeneration. There are important medical implications as defects in the H(+)-ATPase and ClC-7 can underlie human osteopetrosis.

Animals↗

Molecular cloning and characterization of an intracellular chloride channel in the proximal tubule cell line, LLC-PK1.

CLC5 is an intracellular chloride channel of unknown function, expressed in the renal proximal tubule. The subcellular localization and function of CLC5 were investigated in the LLC-PK1 porcine proximal tubule cell line. We cloned a cDNA for the porcine CLC5 ortholog (pCLC5) that is predicted to encode an 83-kDa protein with 97% amino acid sequence identity to rat and human CLC5. By immunofluorescence, pCLC5 was localized to early endosomes of the apical membrane fluid-phase endocytotic pathway and to the Golgi complex. Xenopus oocytes injected with pCLC5 cRNA exhibited outwardly rectifying whole cell currents with a relative conductance profile (nitrate Cl(-) approximately Br(-) > I(-) > acetate > gluconate) different from that of control oocytes. Acidification of the extracellular medium reversibly inhibited this outward current with a pK(a) of 6.0 and a Hill coefficient of 1. Overexpression of CLC5 in LLC-PK1 cells resulted in morphological changes, including loss of cell-cell contacts and the appearance of multiple prominent vesicles. These findings are consistent with a potential role for CLC5 in the acidification of membrane compartments of both the endocytic and the exocytic pathway and suggest that its function may be important for normal intercellular adhesion and vesicular trafficking.

Amino Acid Sequence↗

Chloride channels in cultured glomus cells of the rat carotid body.

As part of our investigations on the chemosensory mechanisms in the rat carotid body, we are studying the physiology of the parenchymal glomus cells by the patch-clamp technique. Here we characterize a large-conductance chloride channel (approximately 296 pS) with random open and closed kinetics in inside-out patches of cultured glomus cells. The open-state probability (Po; mean = 0.61) was hardly affected by membrane potential (-50 to +50 mV) and cytoplasmic calcium (0-1 mM). Similarly, the channel did not appear to be regulated by cytoplasmic nucleotides (1 mM) or pH (6.5-8). Ion-substitution experiments yielded the following selectivity sequence: chloride greater than bicarbonate greater than sulfate greater than glutamate approximately sodium. Single-channel currents were reversibly reduced or blocked by anthracene-9-carboxylic acid (5-10 mM) but were unaffected by stilbene derivatives (0.5-1 mM), by furosemide (1 mM), and by 5-nitro-2-(3-phenyl-propylamino)benzoic acid (0.01 mM). Because these cultured glomus cells have been shown to express carbonic anhydrase, it is inferred that the chloride channels may play an important role in the physiology of glomus cells by aiding in the regulation of pHi and the resting potential via bicarbonate and chloride permeability.

Animals↗

Regulation of calcium-activated chloride channels in smooth muscle cells: a complex picture is emerging.

Calcium-activated chloride channels (ClCa) are ligand-gated anion channels as they have been shown to be activated by a rise in intracellular Ca2+ concentration in various cell types including cardiac, skeletal and vascular smooth muscle cells, endothelial and epithelial cells, as well as neurons. Because ClCa channels are normally closed at resting, free intracellular Ca2+ concentration (approximately 100 nmol/L) in most cell types, they have generally been considered excitatory in nature, providing a triggering mechanism during signal transduction for membrane excitability, osmotic balance, transepithelial chloride movements, or fluid secretion. Unfortunately, the genes responsible for encoding this class of ion channels is still unknown. This review centers primarily on recent findings on the properties of these channels in smooth muscle cells. The first section discusses the functional significance and biophysical and pharmacological properties of ClCa channels in smooth muscle cells, and ends with a description of 2 candidate gene families (i.e., CLCA and Bestrophin) that are postulated to encode for these channels in various cell types. The second section provides a summary of recent findings demonstrating the regulation of native ClCa channels in vascular smooth muscle cells by calmodulin-dependent protein kinase II and calcineurin and how their fine tuning by these enzymes may influence vascular tone.

Animals↗

Determinants of slow gating in ClC-0, the voltage-gated chloride channel of Torpedo marmorata.

Membrane hyperpolarization normally activates the slow gate of the Torpedo voltage-gated chloride channel (ClC-0). To elucidate the structural basis of this process, carboxy terminus truncation mutants and chimeras were constructed, expressed in Xenopus oocytes, and evaluated using a two-microelectrode voltage clamp. Introduction of stop codons at several positions between transmembrane domains 12 and 13 (D12 and D13) showed no expression, whereas a truncation just after D13 yielded wild-type currents. A chimera (022) entailing the substitution of the carboxy-terminal cytoplasmic tail after Lys-520 with the corresponding region of ClC-2 lacked slow gating, whereas a more conservative construct (chimera 002), in which D13 was replaced with its ClC-2 analog, retained its capacity to slow gate. These findings suggest that important structures reside within the interdomain stretch (IDS) between D12 and D13. Unlike ClC-2, in which transplantation of "ball" structures could restore gating to constitutively open mutants, transplantation of the ClC-0 IDS to the amino terminus of chimera 022 did not restore gating. Surprisingly, replacement of the IDS by the analogous regions of either ClC-1 or ClC-2 showed slow voltage-activated gating, although the gating was altered. Our findings lead us to conclude that both the functional expression and the slow voltage gating of ClC-0 rely on structures at the carboxy terminus of the channel.

Animals↗

Mutations in CLCN5 chloride channel in Japanese patients with low molecular weight proteinuria.

Mutations in the CLCN5 gene have been demonstrated in three disorders of hypercalciuric nephrolithiasis, i.e., Dent's disease, X-linked recessive nephrolithiasis, and X-linked recessive hypophosphatemic rickets. Recently, a number of Japanese children with low molecular weight proteinuria (LMWP) showing symptoms similar to those shown by patients with Dent's disease in British families have also been reported to have mutations in the CLCN5 gene. The present study examines five unrelated Japanese families with LMWP, two of which lacked any signs other than LMWP, and three of which had several signs other than LMWP, i.e., hypercalciuria, aminoaciduria, hypophosphatemia, and rickets. One nonsense (E118X) and one missense (W22G) mutation were found in three patients in the two families having only LMWP. One genomic deletion including exons 5 to 8 in the CLCN5 gene was found in a patient with hypophosphatemic rickets, and a nonsense mutation (R347X) was found in one patient with LMWP and slight hypercalciuria. No mutations of the exons and exon-intron boundaries in the CLCN5 gene were found in one patient with LMWP, aminoaciduria, and hypokalemia. In addition to the predicted loss of chloride channel function in these nonsense and deletion mutations, the loss of function in the missense mutation W22G was confirmed in the Xenopus oocyte expression system. These results clarified four novel mutations in the CLCN5 genes, and additionally suggested that the loss-of-function mutation of the CLCN5 does not necessarily lead to hypercalciuria and nephrocalcinosis in the early stage of the disease, and that LMWP is an early and essential manifestation of disorders of the CLC-5 chloride channel.

Adult↗

G-protein regulation of outwardly rectified epithelial chloride channels incorporated into planar bilayer membranes.

Experiments were designed to test if immunopurified outwardly rectified chloride channels (ORCCs) and the cystic fibrosis transmembrane conductance regulator (CFTR) incorporated into planar lipid bilayers are regulated by G-proteins. pertussis toxin (PTX) (100 ng/ml) + NAD (1 mM) + ATP (1 mM) treatment of ORCC and CFTR in bilayers resulted in a 2-fold increase in single channel open probability (Po) of ORCC but not of CFTR. Neither PTX, NAD, nor ATP alone affected the biophysical properties of either channel. Further, PTX conferred a linearity to the ORCC current-voltage curve, with a slope conductance of 80 +/- 3 picosiemens (pS) in the +/- 100 mV range of holding potentials. PKA-mediated phosphorylation of these PTX + NAD-treated channels further increased the Po of the linear 80-pS channels from 0.66 +/- 0.05 to >0.9, and revealed the presence of a small (16 +/- 2 pS) linear channel in the membrane. PTX treatment of a CFTR-immunodepleted protein preparation incorporated into bilayer membranes resulted in a similar increase in the Po of the larger conductance channel and restored PKA-sensitivity that was lost after CFTR immunodepletion. The addition of guanosine 5'-3-O-(thio)triphosphate (100 mum) to the cytoplasmic bathing solutions decreased the activity of the ORCC and increased its rectification at both negative and positive voltages. ADP-ribosylation of immunopurified material revealed the presence of a 41-kDa protein. These results demonstrate copurification of a channel-associated G-protein that is involved in the regulation of ORCC function.

Adenosine Diphosphate Ribose↗

Cell volume regulation and swelling-activated chloride channels.

Maintenance of a constant volume is essential for normal cell function. Following cell swelling, as a consequence of reduction of extracellular osmolarity or increase of intracellular content of osmolytes, animal cells are able to restore their original volume by activation of potassium and chloride conductances. The loss of these ions, followed passively by water, is responsible for the homeostatic response called regulatory volume decrease (RVD). Activation of a chloride conductance upon cell swelling is a key step in RVD. Several proteins have been proposed as candidates for this chloride conductance. The status of the field is reviewed, with particular emphasis on ClC-3, a member of the ClC family which has been recently proposed as the chloride channel involved in cell volume regulation.

Animals↗

Low-conductance chloride channel from crayfish skeletal muscle incorporated into planar lipid bilayers.

Low-conductance chloride channel from skeletal muscle SR vesicles of the crayfish Astacus fluviatilis was incorporated into planar lipid bilayers and its basic characteristics were investigated. The channel has a relatively low unitary conductance of 26 pS in symmetrical 160 mmol/l choline-chloride. The dependence of the channel conductance on Cl- concentration shows saturating behavior with a maximum conductance of 37 pS and an ionic activity for half-maximum conductance Km = 75 mmol/l. The channel exhibits a complex kinetics with several modes of activity. Open state probability slightly decreases with the increasing absolute value of voltage. The channel activity does not appear to be dependent on the presence of Ca2+ ions. The channel is effectively inhibited by DIDS, a stilbene derivative. The permeability properties of the channel are similar to the specific behavior of the "double-barrelled" channel from Torpedo electroplax described by Miller and White (1984).

Animals↗

Chloride channel 7 (ClCN7) gene mutations and autosomal dominant osteopetrosis, type II.

UNLABELLED: ADO2 is an uncommon sclerosing bone disorder with incomplete penetrance and variable expressivity. Positional candidate studies were performed to identify the gene responsible for ADO2. In 11 of 12 kindreds, five different missense mutations were identified in the ClCN7 gene, indicating the genetic basis and possible dominant negative mechanism for ADO2. INTRODUCTION: Autosomal dominant osteopetrosis, type II (ADO2) is an uncommon sclerosing bone disorder with a distinct radiographic appearance and unique clinical characteristics. We present the results from our genetic studies designed to identify the ADO2 gene through a positional candidate approach. METHODS: Having identified 12 families with ADO2, we initially performed linkage studies in our seven largest kindreds and observed a summed maximum LOD score of 15.91 at marker D16S521 on chromosome 16p13.3. Critical meiotic recombination events further narrowed the putative gene region to a 7.6-cM area, which contains the candidate genes ATP6L and chloride channel 7 (ClCN7). We screened affected individuals from each ADO2 family for mutations in these genes using direct sequencing. Identified mutations were subsequently confirmed through direct sequencing or restriction fragment length polymorphism analysis. We then calculated the overall disease penetrance rate after all available at-risk family members were assessed for ClCN7 gene mutations. RESULTS: No ATP6L mutations were identified in affected subjects. Subsequently, as CICN7 gene mutations were being reported, we identified two novel (L213F, R762L) and three known (G215R, R286W, R767W) missense mutations in 11 kindreds. In our large sample, disease penetrance was 66% (62 clinically affected individuals/94 subjects with the gene mutation). To date, nine different mutations have been discovered in the ClCN7 gene in 22 of 23 ADO2 families studied. CONCLUSIONS: We conclude that mutations in the CICN7 gene are responsible for ADO2 and that genetic heterogeneity is unlikely to exist in this disorder. Based on the preponderance of missense mutations and the knowledge that chloride channels probably function as dimers, it seems that heterozygous ClCN7 gene mutations may cause ADO2 through a dominant negative mechanism.

Chloride Channels↗