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PubMed1996

Role of chloride channels in afferent arteriolar constriction.

The effects of IAA-94, a chloride channel blocker and/or low chloride perfusate on afferent arteriolar (AA) constriction by angiotensin II (Ang II), norepinephrine (NE) and increasing pressure (80 to 160 mm Hg) were assessed using isolated perfused hydronephrotic kidneys. In the first series of experiments, Ang II (0.3 nM) constricted AAs by 33 +/- 3% (N = 5, P < 0.01). Subsequent addition of diltiazem (10 microM) restored the decrements in the AA diameters. In the presence of diltiazem (10 microM), increasing pressure did not constrict AAs. In the second series of experiments. elevation of pressure constricted AAs by 20 +/- 2% (N = 7. P < 0.01). Subsequent addition of IAA-94 (30 microM) failed to alter the basal AA diameter and myogenic responsiveness. However, Ang II-induced AA constriction was abolished by IAA-94. In the third series of experiments, decreasing extracellular chloride exaggerated AA constriction by 0.1 nM of Ang II (from 13 +/- 2 to 20 +/- 3%, N = 6, P < 0.05). Similarly, low chloride perfusate enhanced NE (0.1 microM)-induced AA constriction (from 14 +/- 2 to 19 +/- 2%, N = 6, P < 0.05). In contrast, myogenic responsiveness was not influenced by reducing chloride concentrations. The present data provide evidence that both Ang II and NE induce AA constriction by opening chloride channels and subsequent activation of voltage-dependent calcium channels, and suggest that the myogenic response is mediated by activating voltage-dependent calcium channels independently of chloride channels.

Adrenergic beta-Antagonists↗
PubMed1992

The molecular biology of chloride channels.

The molecular biology of chloride channels is a new field, having begun only 5 years ago with the cloning, on the basis of partial amino acid sequence information, of complementary DNAs for subunits of the neuronal glycine and gamma-aminobutyric acid-receptor channels. The sequences of these subunits that form heterooligomeric channels revealed a general similarity with the structural motif employed by the well-studied and previously cloned nicotinic acetylcholine-receptor subunits. The principal distinguishing feature was the rings of positively charged residues within the loops separating the putative transmembrane helices of the new chloride selective channels. A great diversity of subunit types with specific functional and pharmacologic properties as well as localizations within the central nervous system have been identified and cloned. Chloride channels are known to play an important role in both secretion and reabsorption of salt and fluid across wet epithelia, and the cystic fibrosis transmembrane conductance regulator, cloned by genetic means, has been shown to be centrally involved in both of these processes. It is regulated by a complex mechanism involving both ATP binding and phosphorylation at multiple sites. Expression cloning techniques have yielded a voltage-gated family of channels with a large number of potential membrane spanning segments; one member is responsible for the resting potential of skeletal muscle. An entirely different low molecular weight-voltage-gated channel, possibly regulated by osmolarity changes, is the most recent chloride channel to be cloned. It almost certainly will not be the last.

Animals↗
PubMed2006

Identification and functional characterization of EhClC-A, an Entamoeba histolytica ClC chloride channel located at plasma membrane.

ClC chloride channels perform a wide variety of physiological functions and they had been characterized in animals, yeast, plants and bacteria but not in protozoa. By blast search we found in Entamoeba histolytica, the protozoan responsible for human amoebiasis, two genes (Ehclc-A and Ehclc-B) encoding for putative polypeptides with 25-30% identity to ClC chloride channels of several organisms. Reverse transcription polymerase chain reaction (RT-PCR) experiments showed that both genes are transcribed in trophozoites. Phylogenetic analysis revealed that EhClC-A and EhClC-B polypeptides belong to the eukaryotic branch of plasma membrane ClCs. Specific antibodies against EhClC-A confirmed that it is located at the trophozoite plasma membrane. Xenopus laevis oocytes microinjected with Ehclc-A cRNA elicited anion currents not detected in oocytes microinjected with water. Induced currents were inwardly rectifying and had a permeability sequence of Cl->Br->I->F->>NO3-. The chloride channel blocker 4-acetamido-4'isothiocyanostilbene-2, 2'-disulphonic acid (SITS) strongly inhibited the oocytes anion currents and trophozoites growth. Experiments at diverse pHs suggested that EhClC-A is not a Cl-/H+ exchanger, but it is an ion channel that could be involved in pH regulation. EhClC-A may also participate in cell volume regulation. As far as we know, EhClC-A is the first chloride channel characterized in protozoa.

Animals↗
PubMed2002

Effect of an N-terminus deletion on voltage-dependent gating of the ClC-2 chloride channel.

ClC-2, a chloride channel widely expressed in mammalian tissues, is activated by hyperpolarisation and extracellular acidification. Deletion of amino acids 16-61 in rat ClC-2 abolishes voltage and pH dependence in two-electrode voltage-clamp experiments in amphibian oocytes. These results have been interpreted in terms of a ball-and-chain type of mechanism in which the N-terminus would behave as a ball that is removed from an inactivating site upon hyperpolarisation. We now report whole-cell patch-clamp measurements in mammalian cells showing hyperpolarization-activation of rClC-2Delta16-61 differing only in presenting faster opening and closing kinetics than rClC-2. The lack of time and voltage dependence observed previously was reproduced, however, in nystatin-perforated patch experiments. The behaviour of wild-type rClC-2 did not differ between conventional and nystatin-perforated patches. Similar results were obtained with ClC-2 from guinea-pig. One possible explanation of the results is that some diffusible component is able to lock the channel in an open state but does so only to the mutated channel. Alternative explanations involving the osmotic state of the cell and cytoskeleton structure are also considered. Low extracellular pH activates the wild-type channel but not rClC-2Delta16-61 when expressed in oocytes, a result that had been interpreted to suggest that protons affect the ball-and-chain mechanism. In our experiments no difference was seen in the effect of extracellular pH upon rClC-2 and rClC-2Delta16-61 in either recording configuration, suggesting that protons act independently from possible effects of the N-terminus on gating. Our observations of voltage-dependent gating of the N-terminal deleted ClC-2 are an argument against a ball-and-chain mechanism for this channel.

Animals↗
PubMed2000

Stimulatory and Inhibitory Functions of the R Domain on CFTR Chloride Channel.

CFTR is a chloride channel whose gating process involves coordinated interactions among the regulatory (R) domain and the nucleotide-binding folds (NBFs). Protein kinase A phosphorylation of serine residues renders the R domain from inhibitory to stimulatory and enables ATP binding and hydrolysis at the NBFs, which in turn control opening and closing of the chloride channel.

Journal Article↗
PubMed1999

p53 and tumor necrosis factor alpha regulate the expression of a mitochondrial chloride channel protein.

A novel chloride intracellular channel (CLIC) gene, clone mc3s5/mtCLIC, has been identified from differential display analysis of differentiating mouse keratinocytes from p53+/+ and p53-/- mice. The 4.2-kilobase pair cDNA contains an open reading frame of 762 base pairs encoding a 253-amino acid protein with two putative transmembrane domains. mc3s5/mtCLIC protein shares extensive homology with a family of intracellular organelle chloride channels but is the first shown to be differentially regulated. mc3s5/mtCLIC mRNA is expressed to the greatest extent in vivo in heart, lung, liver, kidney, and skin, with reduced levels in some organs from p53-/- mice. mc3s5/mtCLIC mRNA and protein are higher in p53+/+ compared with p53-/- basal keratinocytes in culture, and both increase in differentiating keratinocytes independent of genotype. Overexpression of p53 in keratinocytes induces mc3s5/mtCLIC mRNA and protein. Exogenous human recombinant tumor necrosis factor alpha also up-regulates mc3s5/mtCLIC mRNA and protein in keratinocytes. Subcellular fractionation of keratinocytes indicates that both the green fluorescent protein-mc3s5 fusion protein and the endogenous mc3s5/mtCLIC are localized to the cytoplasm and mitochondria. Similarly, mc3s5/mtCLIC was localized to mitochondria and cytoplasmic fractions of rat liver homogenates. Furthermore, mc3s5/mtCLIC colocalized with cytochrome oxidase in keratinocyte mitochondria by immunofluorescence and was also detected in the cytoplasmic compartment. Sucrose gradient-purified mitochondria from rat liver confirmed this mitochondrial localization. This represents the first report of localization of a CLIC type chloride channel in mitochondria and the first indication that expression of an organellular chloride channel can be regulated by p53 and tumor necrosis factor alpha.

Amino Acid Sequence↗
PubMed2003

Chloride channels as tools for developing selective insecticides.

Ligand-gated chloride channels underlie inhibition in excitable membranes and are proven target sites for insecticides. The gamma-aminobutyric acid (GABA(1)) receptor/chloride ionophore complex is the primary site of action for a number of currently used insecticides, such as lindane, endosulfan, and fipronil. These compounds act as antagonists by stabilizing nonconducting conformations of the chloride channel. Blockage of the GABA-gated chloride channel reduces neuronal inhibition, which leads to hyperexcitation of the central nervous system, convulsions, and death. We recently investigated the mode of action of the silphinenes, plant-derived natural compounds that structurally resemble picrotoxinin. These materials antagonize the action of GABA on insect neurons and block GABA-mediated chloride uptake into mouse brain synaptoneurosomes in a noncompetitive manner. In mammals, avermectins have a blocking action on the GABA-gated chloride channel consistent with a coarse tremor, whereas at longer times and higher concentrations, activation of the channel suppresses neuronal activity. Invertebrates display ataxia, paralysis, and death as the predominant signs of poisoning, with a glutamate-gated chloride channel playing a major role. Additional target sites for the avermectins or other chloride channel-directed compounds might include receptors gated by histamine, serotonin, or acetylcholine.The voltage-sensitive chloride channels form another large gene family of chloride channels. Voltage-dependent chloride channels are involved in a number of physiological processes including: maintenance of electrical excitability, chloride ion secretion and resorption, intravesicular acidification, and cell volume regulation. A subset of these channels is affected by convulsants and insecticides in mammals, although the role they play in acute lethality in insects is unclear. Given the wide range of functions that they mediate, these channels are also potential targets for insecticide development.

Animals↗
PubMed2000

Distribution of chloride channel-2-immunoreactive neuronal and astrocytic processes in the hippocampus.

The chloride homeostasis of neurons and non-neuronal cells is maintained in part by a voltage-sensitive inwardly rectifying chloride conductance through the chloride channel-2. This channel is activated by hyperpolarization and extracellular hypotonicity. In the present study, hippocampal sections were immunostained for chloride channel-2, and somata and dendrites of both pyramidal and non-pyramidal cells were found to be immunoreactive. In addition, glial processes in the vicinity of small blood vessels were also immunostained, whereas the neuropil of strata pyramidale and lacunosum-moleculare contained chloride channel-2-positive punctate structures. Electron microscopy and double immunostaining using antibodies against chloride channel-2 and glial fibrillary acidic protein confirmed that the dense network of chloride channel-2-positive processes corresponds to the end feet of astrocytes. The distribution of chloride channel-2-immunoreactive astrocytes was inhomogeneous throughout the hippocampus: strata oriens, pyramidale and lacunosum-moleculare of CA1-CA3 and the outer molecular layer of the dentate gyrus contained the majority of immunoreactive end feet, whereas the other layers showed sparse labeling. Subcellular studies demonstrated that, in addition to astrocytes, chloride channel-2 was localized in the membrane of dendrites, dendritic spines, cell bodies and axon initial segments of neurons, frequently close to, or within active zones of, symmetrical synapses.Thus, chloride channel-2 appears to be involved in transmembrane chloride movements associated with GABAergic synaptic transmission. The specific laminar distribution of chloride channel-2-positive astroglial processes coinciding with that of GABAergic axon terminals suggests that the network of astrocytes may be able to siphon and deliver Cl(-) ions to layers with intense GABAergic transmission, thereby increasing the efficacy of GABA(A) receptor-mediated inhibition.

Animals↗
PubMed1993

Molecular cloning and expression of a rat cDNA encoding MDCK-type chloride channel.

A cDNA encoding MDCK-type chloride channel was cloned from the rat kidney cDNA library using the homology screening approach. The deduced amino acid sequence of cloned cDNA has a 92.4% homology with the MDCK chloride channel, and electrophysiological analysis using Xenopus oocyte expression system showed the functional character of chloride channel. The rat kidney chloride channel, abbreviated RKCL, is not specific to the kidney, but widely expressed in various tissues. It is suggested that RKCL is important for most cells.

Animals↗
PubMed2006

Large movement in the C terminus of CLC-0 chloride channel during slow gating.

Chloride channels and transporters of the CLC gene family are expressed in virtually all cell types and are crucial in the regulation of membrane potential, chloride homeostasis and intravesicular pH. There are two gating processes that open CLC channels-fast and slow. The fast gating process in CLC channels has recently been linked to a small movement of a glutamate side chain. However, the molecular mechanism underlying the slow gating process is still elusive. Using spectroscopic microscopy, we observed a large backbone movement in the C terminus of the CLC-0 chloride channel that was functionally linked to slow gating. We further showed that the C-terminal movement had a time course similar to slow gating. In addition, a mutation known to lock the slow gate open prevented movement of the C terminus. When combined with recent structural information on the CLC C terminus, our findings provide a structural model for understanding the conformational changes linked to slow gating in CLC transport proteins.

Cell Line↗
PubMed2000

The role of voltage-gated chloride channels in type II pyrethroid insecticide poisoning.

Pyrethroids act on mammalian sodium channels, but we have previously shown that low concentrations of the type II pyrethroid deltamethrin also decrease the open channel probability (P(o)) of voltage-gated chloride channels. This effect would be expected to amplify the sodium channel-mediated signs of poisoning produced by pyrethroids. In the present study we evaluated potential chloride channel agonists in vitro, and then tested the most effective of these on pyrethroid-poisoned rats to determine the practical significance of chloride channel effects in vivo. Patch clamp experiments showed that, for voltage-gated maxi chloride channels in excised, inside-out patches from mouse N1E 115 neuroblastoma cells, ivermectin (10(-7) M) and pentobarbitone (10(-6) M) significantly increased open channel probability (p </= 0.01 and p </= 0.02, respectively), whereas phenobarbitone, hexobarbitone, mephobarbitone, thiopentone, and barbituric acid did not. This suggested that, if chloride channels were important in vivo, ivermectin and pentobarbitone should antagonize type II pyrethroid poisoning and phenobarbitone should not. Male F344 rats were then pretreated with ivermectin (4 mg/kg iv), equisedative doses of either pentobarbitone (15 mg/kg ip) or phenobarbitone (45 mg/kg ip), or solvent controls. This was followed by deltamethrin (1.5 or 2 mg/kg iv) or the type I pyrethroid cismethrin (4 mg/kg iv). Ivermectin produced a marked fall in deltamethrin-induced salivation (p </= 0.05) and also (in anesthetized rats) in repetitive electromyogram discharge and muscle twitch (p </= 0.01 and p </= 0.05, respectively). Pentobarbitone significantly reduced the motor signs score due to deltamethrin (p </= 0.01). Ivermectin therefore protected against the peripheral signs of deltamethrin poisoning and pentobarbitone protected against the central signs. As expected phenobarbitone had no protective effects. The motor signs produced by the type I pyrethroid cismethrin (which does not act on chloride channels) were not diminished by either barbiturate. The peripheral benzodiazepine receptor blocker PK11195 did not diminish the protective action of ivermectin on the muscle twitch (p </= 0.05), although it partially reversed the block of salivation (p </= 0.05). These results support the hypothesis that the voltage-dependent chloride channel is a toxicologically significant additional site of action for deltamethrin and that the use of chloride channel agonists can provide a rationale for a novel and effective therapy against type II pyrethroid poisoning.

Animals↗
PubMed2000

Quaternary structure of the chloride channel ClC-2.

The chloride channel ClC-2 is thought to be essential for chloride homeostasis in neurons and critical for chloride secretion by the developing respiratory tract. In the present work, we investigated the quaternary structure of ClC-2 required to mediate chloride conduction. We found using chemical cross-linking and a novel PAGE system that tagged ClC-2 expressed in Sf9 cells exists as oligomers. Fusion of membranes from Sf9 cells expressing this protein confers double-barreled channel activity, with each pore exhibiting a unitary conductance of 32 pS. Polyhistidine-tagged ClC-2 from Sf9 cells can be purified as monomers, dimers, and tetramers. Purified, reconstituted ClC-2 monomers do not possess channel function whereas both purified ClC-2 dimers and tetramers do mediate chloride flux. In planar bilayers, reconstitution of dimeric ClC-2 leads to the appearance of a single, anion selective 32 pS pore, and tetrameric ClC-2 confers double-barreled channel activity similar to that observed in Sf9 membranes. These reconstitution studies suggest that a ClC-2 dimer is the minimum functional structure and that ClC-2 tetramers likely mediate double-barreled channel function.

Animals↗
PubMed2005

How Bartter's and Gitelman's syndromes, and Dent's disease have provided important insights into the function of three renal chloride channels: ClC-Ka/b and ClC-5.

Chloride channels are expressed in almost all cell membranes and are potentially involved in a wide variety of functions. The kidney expresses 8 of the 9 chloride channels of the ClC family that have been cloned so far to date in mammals. This review focuses on the pathophysiology of two renal disorders that have contributed recently to our understanding of the physiological role of chloride channels belonging to the ClC family. First are the related syndromes of Bartter's and Gitelman's, in which inactivating mutations of the genes encoding either of the ClC-Ks, or their regulatory beta-subunit barttin, have shown the important contribution of these chloride channels to renal tubular sodium and chloride (NaCl) transport along the loop of Henle and distal tubule. Second is the renal Fanconi syndrome known as Dent's disease, in which ClC-5 disruption has revealed the key role of this endosomal chloride channel in the megalin-mediated endocytotic pathway in the proximal tubule. The underlying pathophysiology of this inherited disorder demonstrates how ClC-5 is directly or indirectly required for the reabsorption of filtered low-molecular-weight proteins and bioactive peptides, also expression of membrane transporters, and clearance of calcium-based stone-forming crystals.

Animals↗
PubMed1992

Chloride channels of intracellular organelles and their potential role in cystic fibrosis.

Chloride channels were previously purified from bovine kidney cortex membranes using a drug affinity column. Reconstitution of the purified proteins into artificial liposomes and planar bilayers yielded chloride channels. A 64 x 10(3) M(r) protein, p64, identified as a component of this chloride channel, was used to generate antibodies which depleted solubilized kidney membranes of all chloride channel activity. This antibody has now been used to identify a clone, H2B, from a kidney cDNA library. Antibodies, affinity-purified against the fusion protein of H2B, from a kidney cDNA library. Antibodies, affinity-purified against the fusion protein of H2B, also depleted solubilized kidney cortex from all chloride channel activity. The predicted amino acid sequence of p64 shows that it contains two and possibly four putative transmembrane domains and potential phosphorylation sites by protein kinases A and C. There was no significant homology to other protein (or DNA) sequences in the data base including other anion channels or the cystic fibrosis transmembrane conductance regulator. The protein is expressed in all cells tested and probably represents the chloride channel of intracellular organelles. Cystic fibrosis (CF) is associated with a defect in a cyclic-AMP-activated chloride channel in secretory epithelia which leads to decreased fluid secretion. In addition, many mucus glycoproteins show decreased sialylation but increased sulfation. We have recently shown that the pH of intracellular organelles is more alkaline in CF cells, an abnormality that is due to defective chloride conductance in the vesicle membranes. We postulate that the defect in the intracellular chloride channel, and hence the alkalization, could explain the glycosylation abnormalities since the pH optimum of Golgi sialyltransferase is acid while that of focusyl- and sulfotransferases is alkaline. Defects in sialyation of glycolipids might also generate receptors for Pseudomonas, which is known to colonize the respiratory tract of CF patients.

Adenosine Triphosphatases↗
PubMed1996

Cystic fibrosis gene encodes a cAMP-dependent chloride channel in heart.

cAMP-dependent chloride channels in heart contribute to autonomic regulation of action potential duration and membrane potential and have been inferred to be due to cardiac expression of the epithelial cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. In this report, a cDNA from rabbit ventricle was isolated and sequenced, which encodes an exon 5 splice variant (exon 5-) of CFTR, with >90% identity to human CFTR cDNA present in epithelial cells. Expression of this cDNA in Xenopus oocytes gave rise to robust cAMP-activated chloride currents that were absent in control water-injected oocytes. Antisense oligodeoxynucleotides directed against CFTR significantly reduced the density of cAMP-dependent chloride currents in acutely cultured myocytes, thereby establishing a direct functional link between cardiac expression of CFTR protein and an endogenous chloride channel in native cardiac myocytes.

Alternative Splicing↗
PubMed1994

Reconstitution, identification, and purification of the Torpedo californica electroplax chloride channel complex.

A voltage-gated chloride channel was identified in the electric organ of the marine ray Torpedo californica by White and Miller (J. Biol. Chem. 254, 10161-10166 (1979)). The experiments reported here concern the purification and identification of this channel which was accomplished by solubilization of electric organ plasma membranes and reconstitution of the channel into vesicles made of phosphatidylethanolamine, phosphatidylserine, and cholesterol. Channel activity was measured in these vesicles by assaying 36Cl- uptake against an outwardly directed chloride chemical gradient as described by Garty et al. (J. Biol. Chem. 258, 13094-13099 (1983)). Maximal uptake occurred by 15 s. Addition of valinomycin after 10 min released intravesicular 36Cl- suggesting that chloride is moving through a channel. Channel activity was inhibited by DIDS (K0.5 of 56 mM) and NBD chloride (K0.5 of 176 mM). In a 40 lipid/1 protein (w/w) reconstitution, approx. 30% of the vesicles contained a functional chloride channel, based upon uptake done in the presence of chlorotriphenyltin (an anion ionophore), indicating that the Torpedo electric organ is an enriched source as shown by White and Miller (Biophys. J. 35, 455-462 (1981)). The chloride channel was purified approx. 40-fold by sedimentation velocity. In this purified preparation, four polypeptides (210, 95, 55, and 40 kDa) were visible by silver-staining after nonreducing SDS-PAGE. Of the four polypeptides, the largest (210 kDa) is not sufficient for Cl- channel activity by itself, but it is labeled by DIDS, an inhibitor of channel activity. Channel activity was approx. 20-fold greater in material that bound to concanavalin A compared to the concanavalin A flow-through; all four polypeptides were present in the bound materia. It is possible that some of these polypeptides are subunits of the chloride channel.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMedDate not supplied

Large chloride channel from pre-eclamptic human placenta.

Chloride transport involving conductive pathways participates in numerous epithelial functions, such as membrane voltage maintenance, solute transport and cell volume regulation. Evidence points to involvement of transepithelial chloride transport in such functions in placental syncytiotrophoblast. A molecular candidate for physiologic conductive chloride transport in apical syncytiotrophoblast membrane is a Maxi-chloride channel with distinct biophysical properties: conductance over 200 pS, multiple substates, voltage dependent open probability, and permeation to anionic amino acids. Pre-eclampsia, a high incidence pathology of pregnancy, exerts great impact on fetal morbi-mortality. This relies, among others, on intrauterine growth restriction (IUGR), thought to be mediated by diminished blood flow to the placenta, with growing knowledge regarding contribution of other factors. The Maxi-chloride channel's properties suggest it could be altered in this pathology. We have characterized the apical chloride channels from pre-eclamptic placentae, reconstituted in giant liposomes suitable for patch-clamp electrophysiological studies. In n=33 experiments from n=6 pre-eclamptic placentae we observed a chloride-permeable channel with similar biophysical properties to the channel from normal tissue (n=29 experiments from n=15 placentae). However, the main conductance state showed diminished magnitude (<150 pS), and the open probability versus voltage relationship exhibited a flattened curve instead of the bell-shaped curve of normal placentae. These results are the first evidence of a functionally altered ionic channel from placental syncytiotrophoblast in pre-eclampsia. Considering the abundance of chloride-conducting channel activity in human apical membrane and their relevance in epithelial function in general, these alterations could greatly disturb numerous placental functions that rely on syncytiotrophoblast integrity.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2000

Blocking chloride channels in the rat lens: localized changes in tissue hydration support the existence of a circulating chloride flux.

PURPOSE: To investigate the effects of inhibitors of chloride channels on lens volume and tissue architecture under isotonic conditions. METHODS: Rat lenses were maintained in organ culture under isotonic conditions in the presence of various putative chloride channel inhibitors. The effect of an inhibitor on lens wet mass and tissue morphology was determined by weighing and histologic examination, respectively. RESULTS: Exposure to 100 microM of either 5-nitro-2- (3-phenylpropylamino) benzoic acid (NPPB) or 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) caused an increase in wet mass and severe tissue disruption in the lens equatorial region. Two distinctly different zones of tissue damage were evident: a peripheral zone of fiber cell swelling and an inner zone of extensive tissue breakdown. Extracellular space dilations caused the extensive tissue damage in the inner zone and preceded the peripheral fiber cell swellings. That the observed effects were a consequence of the inhibition of chloride channels was supported by (1) the effectiveness of NPPB at the lower dose of 10 microM, (2) the absence of any NPPB effect in chloride-free medium, and (3) an identical effect after exposure to tamoxifen, an inhibitor of the chloride channel regulator p-glycoprotein. CONCLUSIONS: Study results indicate that chloride channels are active in the lens under isotonic conditions. The spatial and temporal pattern of morphologic changes that was observed is consistent with a steady state efflux of chloride ions and water from peripheral fiber cells and a corresponding influx into fiber cells deeper in the lens. These observations may therefore represent the first visualization of the chloride flux postulated by others to be a component of the lens internal circulation system.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗