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PubMed1998

The murine calcium-sensitive chloride channel (mCaCC) is widely expressed in secretory epithelia and in other select tissues.

The tissue and cellular expression pattern of a recently cloned murine calcium-sensitive chloride channel (mCaCC) was determined. In situ hybridization was performed on formalin-fixed, paraffin-embedded murine tissues using digoxigenin-labeled, single-stranded RNA probes. The data were substantiated with northern blot and reverse transcriptase-polymerase chain reaction analyses. All three assays consistently indicated strong expression in tissues with secretory or ion regulatory functions, such as mammary gland, respiratory and intestinal epithelia, gall bladder, pancreas, kidney, uterus, and epididymis. Additional mCaCC expression was observed in germinal centers of lymphatic tissues, in spermatids, and in keratinocytes of the skin, esophagus, and cornea. The results are in accordance with previous electrophysiological reports on calcium-activated chloride conductances in various murine exocrine secretory epithelia and suggest a role of mCaCC in transepithelial ion transport. However, expression in other than secretory tissues indicates a more complex function.

Animals↗
PubMed1997

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/P) sequence NO > Cl > HCO > 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/P < 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 approximately 5.3 A. 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.

Amino Acid Sequence↗
PubMed1998

Refined localisation of the voltage-gated chloride channel, CLCN3, to 4q33.

Mutations in ion channels have been shown to be responsible for a variety of neurological and muscular diseases. The voltage-gated chloride channel CLCN3 was recently mapped to chromosomal region 4q32. We are analysing a young female patient with Wolf-Hirschhorn syndrome and chorea associated with an inversion-deletion of chromosome 4 [46XX,inv(4)del(4)(qter-->q33::p15.32-->q33]. Considering that chorea in this patient might be due to the disruption of a gene at either of the 4p15.32 or 4q33 breakpoints, CLCN3 was considered as a candidate gene. We showed by FISH analysis with a CLCN3 YAC that the gene was not broken by the inv-del event, and was therefore an unlikely candidate. Using high resolution techniques, we refined the localisation of CLCN3 to 4q33.

Chloride Channels↗
PubMed1996

Asthma prophylaxis agents alter the function of an airway epithelial chloride channel.

A number of recent observations suggest a link between airway Cl-transport and asthma. We have previously described the properties of a voltage- and Ca2+ -dependent chloride channel present in airway epithelium. We now show that agents able to prevent indirectly induced bronchoconstriction (sodium cromoglycate, nedocromil sodium, and furosemide) reduce either the single-channel conductance or the open probability of this channel. The effects of these agents and the Ca2+ dependence of the channel are localized to the same surface, and we show that the channel possesses a specific divalent cation binding site, which responds to concentrations of Ca2+ found on the airway mucosal surface. No alteration of the single-channel properties of this channel were seen in cystic fibrosis epithelium. These data suggest a mechanism by which structurally diverse agents may influence asthma.

Animals↗
PubMed1998

Characterization of calcium-activated chloride channels in patches excised from the dendritic knob of mammalian olfactory receptor neurons.

We investigated the properties of calcium-activated chloride channels in inside-out membrane patches from the dendritic knobs of acutely dissociated rat olfactory receptor neurons. Patches typically contained large calcium-activated currents, with total conductances in the range 30-75 nS. The dose response curve for calcium exhibited an EC50 of about 26 microM. In symmetrical NaCl solutions, the current-voltage relationship reversed at 0 mV and was linear between -80 and +70 mV. When the intracellular NaCl concentration was progressively reduced from 150 to 25 mM, the reversal potential changed in a manner consistent with a chloride-selective conductance. Indeed, modeling these data with the Goldman-Hodgkin-Katz equation revealed a PNa/PCl of 0.034. The halide permeability sequence was PCl > PF > PI > PBr indicating that permeation through the channel was dominated by ion binding sites with a high field strength. The channels were also permeable to the large organic anions, SCN-, acetate-, and gluconate-, with the permeability sequence PCl > PSON > Pacetate > Pgluconate. Significant permeation to gluconate ions suggested that the channel pore had a minimum diameter of at least 5.8 A.

Animals↗
PubMed1996

Heterogeneity of volume-sensitive chloride channels in basolateral membranes of A6 epithelial cells in culture.

A new technique allowing single-channel patch-clamp recordings from basolateral membranes of A6 renal epithelial cells in culture was developed. Using this technique we studied the chloride channels activated in these basolateral membranes during hypo-osmotic stress. Four different types of channel were identified and classified according to their current/voltage (I/V) relationships as observed in the on-cell configuration of the patch-clamp technique. Three of these channels had linear I/V relationships with unitary conductances of 12, 30 and 42 pS. The fourth type had an outwardly rectifying I/V curve with inward and outward conductances of 16 and 57 pS respectively. The kinetic properties of each class of channel were studied and kinetic models developed for two of them: the 42 pS channel and the outward rectifier. These models permitted the study of the evolution of the kinetic parameters during hypo-osmotic shock and revealed two different kinetic schemes of channel activation. The results of experiments made on the basolateral membranes were also compared with those of a set of analogous patch-clamp experiments carried out on isolated A6 cells. In these latter, the frequency of successful observations of active channels in a patch was 13%, whereas it was 31% for basolateral membranes. Also, of the four types of channel observed in basolateral membranes, two were never found in isolated cells, only the 12 pS channel and the outward rectifier were present in these isolated cells.

Animals↗
PubMed1996

Effects of pH on a high conductance Ca-dependent chloride channel: a patch-clamp study in Ascaris suum.

Plasma membrane vesicles prepared from the bag region of the somatic muscle cells of the parasitic nematode Ascaris suum contain high conductance, voltage sensitive, Ca-dependent chloride channels, suggested to be involved in the excretion of carboxylic acids produced by the anaerobic respiration of glucose (Valkanov, Martin & Dixon, 1994). The effect of altered pH on this channel was investigated using the patch-clamp technique and isolated inside-out membrane patches. Changes in pH had little effect on channel conductances and only a small effect on reversal potentials. Under control conditions (symmetrical pH 7.2) the channel had the highest probability of opening at approximately -35 mV (the resting membrane potential of the cell). At positive membrane potentials the probability of opening decreased. The Boltzmann equation was used to describe the relationship between membrane potential and probability of channel opening, and to calculate the effective gating charge. Reduction of external pH produced an increase in the probability of channel opening at hyperpolarized membrane potentials. An increase in internal pH caused a voltage-independent increase in the probability of channel opening and made the effective gating charge less negative. The effect of reducing internal pH was marked: the channel then opened most frequently at positive membrane potentials and the probability of opening at -35 mV was greatly reduced. The decrease in internal pH changed the polarity of the effective gating charge. A simple model was constructed to describe the effects of pH on channel gating.

Animals↗
PubMed2000

Molecular mechanisms of ion conduction in ClC-type chloride channels: lessons from disease-causing mutations.

The muscle Cl- channel, ClC-1, is a member of the ClC family of voltage-gated Cl- channels. Mutations in CLCN1, the gene encoding this channel, cause two forms of inherited human muscle disorders: recessive generalized myotonia congenita (Becker) and dominant myotonia (Thomsen). The functional characterization of these naturally occurring mutations not only allowed a better understanding of the pathophysiology of myotonia, it also provided important insights into the structure and function of the entire ClC channel family. This review describes recent experiments using a combination of cellular electrophysiology, molecular genetics, and recombinant DNA technology to study the molecular basis of ion permeation and selection in ClC-type chloride channels.

Amino Acid Sequence↗
PubMed1999

Identification of three novel mutations in the major human skeletal muscle chloride channel gene (CLCN1), causing myotonia congenita.

Myotonia congenita (MC) is a genetic disease characterized by mutations in the CLCN1 gene (OMIM*118425) encoding the skeletal muscle voltage-gated chloride channel (ClC-1). Autosomal dominant and recessive forms are observed, characterized by impaired muscle relaxation after forceful contraction (myotonia), which is more pronounced after inactivity and improves with exercise. We report three novel and one known mutations of the CLCN1 gene in four unrelated MC families. In two families the mutations were missense: 803C>T (T268M) and 1272C>G (I424M) in exons 7 and 12, respectively. The third was a splice mutation in intron 5 (696+2T>A), which induced a frame shift with a stop codon in exon 6 (fs213X). In the fourth family the previously-reported missense mutation 689G>A (G230E) was found. We also report two known polymorphisms: 261C>T (T87T) and 2154T>C (D718D) in exons 2 and 17 of two MC families; also found in 14 (33%) and 28 (67%) of 42 healthy controls, respectively. These findings expand our knowledge of mutations responsible for myotonia congenita, reducing the proportion of MC patients in whom genetic alterations have not been found.

Chloride Channels↗
PubMed1996

Flickery block of single CFTR chloride channels by intracellular anions and osmolytes.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation- and nucleotide-dependent chloride channel. Single CFTR currents recorded on cell show slight outward rectification, which has previously been suggested to be due to an asymmetrical chloride ion gradient or to a specific interaction between permeant intracellular anions and the channel. Using a single-channel recording from Chinese hamster ovary cells stably expressing CFTR, we have found that both the sparingly permeant anion glutamate and the impermeant anion gluconate cause a rapid, voltage-dependent block of CFTR channels when applied to the intracellular, but not the extracellular, face of excised patches. Both the affinity and the voltage dependence of block were affected by the extracellular chloride concentration in a manner consistent with chloride ions being able to repel these blocking ions from the pore. These results are discussed in terms of previous models of CFTR current outward rectification, and it is suggested that this rectification may result from a combination of asymmetrical chloride concentrations and voltage-dependent block of the channel by large cytoplasmic anions. In addition, we find that CFTR conductance is decreased by high concentrations of intracellular sucrose, sorbitol, and urea in a manner consistent with a rapid block of the channel by these molecules.

Animals↗
PubMed2006

Consequences of point mutations in trout anion exchanger 1 (tAE1) transmembrane domains: evidence that tAE1 can behave as a chloride channel.

In this study, we have shown that, when expressed in Xenopus oocytes, trout anion exchanger 1 (tAE1) was able to act as a bifunctional protein, either an anion exchanger or a chloride conductance. Point mutations of tAE1 were carried out and their effect on Cl- conductance and Cl- unidirectional flux were studied. We have shown that mutations made in transmembrane domain 7 had dramatic effects on tAE1 function. Indeed, when these residues were mutated, either individually or together (mutants E632K, D633G, and ED/KG), Cl- conductance was reduced to 28-44% that of wild-type tAE1. Moreover, ion substitution experiments showed that anion selectivity was altered. However, the exchanger function was unchanged, as evidenced by the fact that Cl- influx and K(m) were identical for each of these mutants and similar to the wild-type protein parameters. By contrast, mutations made in the C-terminal domains of the protein (R819M, Q829K) affected both transport functions. Cl- conductance was increased by approximately 200% with respect to tAE1 and anion selectivity was impaired. Likewise, Cl- influx was increased by approximately 260% and was no longer saturable. These and other mutations carried out in transmembrane domains 7, 8, 12-14 of tAE1 allow us to demonstrate without doubt that, in addition to its anion exchanger activity, tAE1 can also function as a chloride channel. Above all, this work led us to identify amino acids involved in this double function organization.

Amino Acid Sequence↗
PubMed2004

Ginkgolide B preferentially blocks chloride channels formed by heteromeric glycine receptors in hippocampal pyramidal neurons of rat.

It has been found recently that the platelet activating factor antagonist ginkgolide B is a selective use-dependent blocker of glycine-gated chloride channels. GABAA receptor antagonist picrotoxin is known to block alpha homomeric glycine (Gly) receptors, being less effective for heteromeric alpha1/beta glycine receptors. Studying pyramidal hippocampal neurons of rat, we have confirmed that the effect of picrotoxin depends on the age of the animals. Its blocking ability was characterised by IC50=140+/-12 microM and IC50=354+/-43 microM for 7 and 14 days old rats, respectively, indicating at a possibly increased contribution of heteromeric receptors with animals age. We have revealed that the blocking action of ginkgolide B is subjected to a more drastic change in the same range of ages: the IC50 value is decreased from 1.6+/-0.2 microM for 7 days old rats to 0.27+/-0.01 microM for 14 days old rats. When measured on the background of ginkgolide B (1 microM), IC50 for picrotoxin was 92+/-16 microM. Taken together, these findings indicate that ginkgolide B has higher affinity to heteromeric Gly receptor-gated channels than to the homomeric ones.

Age Factors↗
PubMed2005

Sulfamoyl-4-oxoquinoline-3-carboxamides: novel potentiators of defective DeltaF508-cystic fibrosis transmembrane conductance regulator chloride channel gating.

The synthesis of a small collection of sulfamoyl-4-oxoquinoline-3-carboxamides is described for use as correctors of defective gating of the DeltaF508-cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Several compounds with submicromolar potency were obtained. N-Ethyl 6-(ethylphenylsulfamoyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (7b) was found to be the most effective sulfonamide corrector of defective DeltaF508-CFTR gating.

Chloride Channels↗
PubMed2003

Mutations in CLCN2 encoding a voltage-gated chloride channel are associated with idiopathic generalized epilepsies.

Idiopathic generalized epilepsy (IGE) is an inherited neurological disorder affecting about 0.4% of the world's population. Mutations in ten genes causing distinct forms of idiopathic epilepsy have been identified so far, but the genetic basis of many IGE subtypes is still unknown. Here we report a gene associated with the four most common IGE subtypes: childhood and juvenile absence epilepsy (CAE and JAE), juvenile myoclonic epilepsy (JME), and epilepsy with grand mal seizures on awakening (EGMA; ref. 8). We identified three different heterozygous mutations in the chloride-channel gene CLCN2 in three unrelated families with IGE. These mutations result in (i) a premature stop codon (M200fsX231), (ii) an atypical splicing (del74-117) and (iii) a single amino-acid substitution (G715E). All mutations produce functional alterations that provide distinct explanations for their pathogenic phenotypes. M200fsX231 and del74-117 cause a loss of function of ClC-2 channels and are expected to lower the transmembrane chloride gradient essential for GABAergic inhibition. G715E alters voltage-dependent gating, which may cause membrane depolarization and hyperexcitability.

Adolescent↗
PubMed1994

Novel plasma membrane action of estrogen and antiestrogens revealed by their regulation of a large conductance chloride channel.

Antiestrogens antagonize many genomic effects of estrogen through binding to the nuclear estrogen receptor. We report here that NIH3T3 fibroblasts grown in the presence of colchicine acquire the activation of a large conductance chloride channel upon exposure to extracellular but not intracellular antiestrogens. This effect can be prevented by extracellular 17 beta-estradiol, but not intracellular 17 beta-estradiol or extracellular 17 alpha-estradiol. This is the first demonstration of a regulatory role for antiestrogens and estrogens in the regulation of ionic channels occurring through an interaction of these compounds with a plasma membrane binding site distinct from the classical estrogen receptor and subsequent activation of intracellular second messenger pathway (or pathways).

3T3 Cells↗
PubMed2005

Propofol suppresses synaptic responsiveness of somatosensory relay neurons to excitatory input by potentiating GABA(A) receptor chloride channels.

Propofol is a widely used intravenous general anesthetic. Propofol-induced unconsciousness in humans is associated with inhibition of thalamic activity evoked by somatosensory stimuli. However, the cellular mechanisms underlying the effects of propofol in thalamic circuits are largely unknown. We investigated the influence of propofol on synaptic responsiveness of thalamocortical relay neurons in the ventrobasal complex (VB) to excitatory input in mouse brain slices, using both current- and voltage-clamp recording techniques. Excitatory responses including EPSP temporal summation and action potential firing were evoked in VB neurons by electrical stimulation of corticothalamic fibers or pharmacological activation of glutamate receptors. Propofol (0.6 - 3 microM) suppressed temporal summation and spike firing in a concentration-dependent manner. The thalamocortical suppression was accompanied by a marked decrease in both EPSP amplitude and input resistance, indicating that a shunting mechanism was involved. The propofol-mediated thalamocortical suppression could be blocked by a GABAA receptor antagonist or chloride channel blocker, suggesting that postsynaptic GABAA receptors in VB neurons were involved in the shunting inhibition. GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) were evoked in VB neurons by electrical stimulation of the reticular thalamic nucleus. Propofol markedly increased amplitude, decay time, and charge transfer of GABAA IPSCs. The results demonstrated that shunting inhibition of thalamic somatosensory relay neurons by propofol at clinically relevant concentrations is primarily mediated through the potentiation of the GABAA receptor chloride channel-mediated conductance, and such inhibition may contribute to the impaired thalamic responses to sensory stimuli seen during propofol-induced anesthesia.

Anesthetics, Intravenous↗
PubMed1998

Multiple mechanisms of block by the anesthetic isoflurane of a gamma-aminobutyric acid activated chloride channel in crayfish.

Outside-out patches were excised from the membrane of the deep extensor abdominal muscle (DEAM), containing gamma-aminobutyric acid (GABA)-activated chloride channels, in the crayfish Astacus astacus. GABA and isoflurane (iso) were applied in pulses by a liquid filament switch, and their effects on the GABA-elicited chloride currents were investigated. Application of iso alone elicited no current responses and pre-application of iso prior to GABA had no effects on the GABA-elicited current. Co-application of GABA and iso resulted in a reduction of the initial chloride current and subsequent decline of the current to a steady state, indicating that iso binds to the receptor after GABA has bound. Recovery currents at the end of the co-application pulse, their amplitudes decreasing with pulse duration, confirmed this suggestion. Open-time distributions of the blocked channel showed a shift of the long open-time towards a new time constant, indicating a second block mechanism via the long open state A5Os of the channel. Removal of GABA and iso after reaching the equilibrium state of the block resulted in recovery currents containing exclusively openings from the long open state A5Os, confirming the suggestion of an open channel block only at one of the open states.

Anesthetics↗
PubMed1993

Protein kinase A (PKA) still activates CFTR chloride channel after mutagenesis of all 10 PKA consensus phosphorylation sites.

The cystic fibrosis transmembrane conductance regulator (CFTR) plays a central role in transepithelial ion transport by acting as a tightly regulated apical chloride channel. Regulation is achieved by the concerted action of ATP at conserved nucleotide binding folds and serine phosphorylation at multiple sites by protein kinases A (PKA) and C (PKC). A previous investigation concluded that activation by PKA is critically dependent on phosphorylation at four of the nine predicted PKA sites in the R domain (S660A, S737A, S795A, S813A), because a "Quad" mutant lacking these sites could not be activated. We show in the present work that not only can this mutant be phosphorylated and activated, but a mutant in which all 10 predicted PKA sites have been altered still retains significant PKA-activated function. Potentiation of the PKA response by PKC is also preserved in this mutant. Thus CFTR may be regulated by cryptic PKA sites which also mediate interactions between different kinases. Such hierarchical phosphorylation of CFTR by obvious and cryptic PKA sites could provide a metered response to secretagogues.

Amino Acid Sequence↗