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PubMed1989

The molecular basis of chloride channel dysregulation in cystic fibrosis.

The opening and closing of chloride (Cl-) channels in the apical membrane of epithelial cells is regulated by hormones, neurotransmitters and enterotoxins (intestine) acting through a variety of intracellular messengers, including cyclic nucleotides (cAMP, cGMP), calcium (Ca) and diacylglycerol (DAG). The chloride impermeability of epithelial membranes observed in cystic fibrosis (CF) patients does not result from a defect in the Cl- conducting properties of the channel or in channel recruitment but stems either from a defect in a key regulator of the channel, presumably a phosphoprotein, or from the hyperactivation of a channel closing mechanism, presumably a protein phosphatase or a down-regulating protein kinase (i.e. protein kinase C). In vitro phosphorylation of isolated intestinal brush border membranes has revealed the existence of a 25,000 molecular weight proteolipid (p25) acting as cosubstrate for both cGMP- and cAMP-dependent protein kinases and cross-reacting with antibodies directed against the cytoplasmic tail of the band 3 anion exchanger from erythrocytes. The putative role of p25 in Cl- channel regulation and its relationship to an unidentified GTP-binding protein recently implicated in Cl- channel activation is discussed on the basis of a regulatory model indicating potential sites of the CF defect at a molecular level.

Cell Membrane Permeability↗
PubMed1993

Chloride channels.

Cl- channels have various functions such as regulation of cell volume, transepithelial transport, and control of excitability in nerve and muscle. Several different structural classes of Cl- channels have been identified recently by molecular cloning. The importance of these different classes of Cl- channels can be seen from the inherited diseases resulting from mutations in some of the genes encoding them. Mutagenesis studies are beginning to shed light on their structure-function relationships.

Animals↗
PubMed1988

Secretin-regulated chloride channel on the apical plasma membrane of pancreatic duct cells.

Using the patch clamp technique we have identified a small conductance ion channel that typically occurs in clusters on the apical plasma membrane of pancreatic duct cells. The cell-attached current/voltage (I/V) relationship was linear and gave a single channel conductance of about 4 pS. Since the reversal potential was close to the resting membrane potential of the cell, and unaffected by changing from Na+-rich to K+-rich pipette solutions, the channel selects for anions over cations in cell-attached patches. The open state probability was not voltage-dependent. Adding 25 mM-bicarbonate to the bath solution caused a slight outward rectification of the I/V relationship, but otherwise, the characteristics of the channel were unaffected. In excised, inside-out, patches the I/V relationship was linear and gave a single channel conductance of about 4 pS. A threefold chloride concentration gradient across the patch (sulphate replacement) shifted the single channel current reversal potential by -26 mV, indicating that the channel is chloride selective. Stimulation of duct cells with secretin (10 nM), dibutyryl cyclic AMP (1 mM) and forskolin (1 microM) increased channel open state probability and also increased the number of channels, and/or caused disaggregation of channel clusters, in the apical plasma membrane. Coupling of this channel to a chloride/bicarbonate exchanger would provide a mechanism for electrogenic bicarbonate secretion by pancreatic duct cells.

Animals↗
PubMed2006

Flow-activated chloride channels in vascular endothelium. Shear stress sensitivity, desensitization dynamics, and physiological implications.

Although activation of outward rectifying Cl(-) channels is one of the fastest responses of endothelial cells (ECs) to shear stress, little is known about these channels. In this study, we used whole-cell patch clamp recordings to characterize the flow-activated Cl(-) current in bovine aortic ECs (BAECs). Application of shear stress induced rapid development of a Cl(-) current that was effectively blocked by the Cl(-) channel antagonist 5-nitro-2-(3-phenopropylamino)benzoic acid (100 microM). The current initiated at a shear stress as low as 0.3 dyne/cm(2), attained its peak within minutes of flow onset, and saturated above 3.5 dynes/cm(2) approximately 2.5-3.5-fold increase over pre-flow levels). The Cl(-) current desensitized slowly in response to sustained flow, and step increases in shear stress elicited increased current only if the shear stress levels were below the 3.5 dynes/cm(2) saturation level. Oscillatory flow with a physiological oscillation frequency of 1 Hz, as occurs in disturbed flow zones prone to atherosclerosis, failed to elicit the Cl(-) current, whereas lower oscillation frequencies led to partial recovery of the current. Nonreversing pulsatile flow, generally considered protective of atherosclerosis, was as effective in eliciting the current as steady flow. Measurements using fluids of different viscosities indicated that the Cl(-) current is responsive to shear stress rather than shear rate. Blocking the flow-activated Cl(-) current abolished flow-induced Akt phosphorylation in BAECs, whereas blocking flow-sensitive K(+) currents had no effect, suggesting that flow-activated Cl(-) channels play an important role in regulating EC flow signaling.

Animals↗
PubMed1991

Immunologically activated chloride channels involved in degranulation of rat mucosal mast cells.

Crosslinking of type I Fc epsilon receptors (Fc epsilon RI) on the surface of basophils or mast cells initiates a cascade of processes leading to the secretion of inflammatory mediators. We report here a correlation between mediator secretion and the activation of Cl- channels in rat mucosal-type mast cells (line RBL-2H3). Stimulation of RBL cells by either IgE and antigen or by a monoclonal antibody specific for the Fc epsilon RI, resulted in the activation of Cl- ion channels as detected by the patch-clamp technique. Channel activation occurred slowly, within minutes after stimulation. The channel has a slope conductance of 32 pS at potentials between 0 and -100 mV, and an increasing open-state probability with increasing depolarization. Activation of apparently the same Cl- channels could be mimicked without stimulation by isolating inside-out membrane patches in tyrode solution. Parallel inhibition of both Cl- channel activity and mediator secretion, as monitored by serotonin release, was observed by two compounds, the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and the anti-allergic drug cromolyn. NPPB inhibited both the antigen-induced Cl- current and the serotonin release, where half-maximal inhibition occurred at similar doses, at 52 microM and 77 microM, respectively. The drug cromolyn, recently found to inhibit immunologically induced mediator secretion from RBL cells upon intracellular application, also blocks Cl- channels (IC50 = 15 microM) when applied to the cytoplasmic side of an inside-out membrane patch. The observed Cl- channel activation upon immunological stimulation and the parallel inhibition of channel current and of serotonin release suggests a functional role for this Cl- channel in mediator secretion from the mast cells studied.

Animals↗
PubMed2000

Dual role of ATP in supporting volume-regulated chloride channels in mouse fibroblasts.

The effects of inhibitors of protein tyrosine kinases (PTKs) on the Cl(-) current (I(Cl(vol))) through volume-regulated anion/chloride (VRAC) channels whilst manipulating cellular ATP have been studied in mouse fibroblasts using the whole-cell patch clamp technique. Removal of ATP from the pipette-filling solution prevented activation of the current during osmotic cell swelling and when the volume of patched cells was increased by the application of positive pressure through the patch pipette to achieve rates exceeding 100%/min. Equimolar substitution of ATP in the pipette solution with its non-hydrolyzable analogs, adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) or adenylyl-(beta,gamma-methylene)-diphosphonate (AMP-PCP), not only supported activation of the current but also maintained its amplitude. The PTK inhibitors, tyrphostins A25, B46, 3-amino-2,4-dicyano-5-(4-hydroxyphenyl)penta-2,4-dienonitrile++ + and genistein (all at 100 microM), inhibited I(Cl(vol)) in a time-dependent manner. Tyrphostin A1, which does not inhibit PTK activity, did not affect the current amplitude. The PTK inhibitors also inhibited I(Cl(vol)) under conditions where ATP in the pipette was substituted with ATPgammaS or AMP-PCP. We conclude that in mouse fibroblasts ATP has a dual role in the regulation of the current: it is required for protein phosphorylation to keep VRAC channels operational and, through non-hydrolytic binding, determines the magnitude of I(Cl(vol)). We also suggest that tyrosine-specific protein kinases and phosphatases exhibit an interdependent involvement in the regulation of VRAC channels.

Adenosine Triphosphate↗
PubMed2004

NAD(P)H oxidase-derived H(2)O(2) signals chloride channel activation in cell volume regulation and cell proliferation.

Cellular swelling triggers the activation of Cl(-) channels (volume-sensitive outwardly rectifying (VSOR) Cl(-) channels) in many cell types. Ensuing regulatory volume decrease has been considered the primary function of these channels. However, Cl(-) channels, which share functional properties with volume-sensitive Cl(-) channels, have been shown to be involved in other physiological processes, including cell proliferation and apoptosis, raising the question of their physiological roles and the signal transduction pathways involved in their activation. Here we report that exogenously applied H(2)O(2) elicited VSOR Cl(-) channel activation. Furthermore, activation of these channels was found to be coupled to NAD(P)H oxidase activity. Also, epidermal growth factor, known to increase H(2)O(2) production, activated Cl(-) channels with properties identical to swelling-sensitive Cl(-) channels. It is concluded that NAD(P)H oxidase-derived H(2)O(2) is the common signal transducing molecule that mediates the activation of these ubiquitously expressed anion channels under a variety of physiological conditions.

Cell Division↗
PubMed1995

Volume-activated chloride channels in rat parotid acinar cells.

1. Rat parotid acinar cells undergo a regulatory volume decrease in response to hypotonically induced cell swelling that is sensitive to K+ and Cl- gradients. To investigate the potential mechanisms involved, the whole-cell patch-clamp technique was used to characterize a volume-sensitive Cl- channel in rat parotid acinar cells. 2. Exposure of cells to a hyposmotic gradient induced large Cl- currents that exhibited outward rectification and were not affected by membrane potential or the absence of intracellular Ca2+. Low external pH increased the currents at all potentials without affecting current kinetics. These currents were nearly abolished when the cells were in hypertonic conditions. This decrease in the current amplitude was correlated with a decrease in the cell size. 3. The volume-sensitive currents displayed little or no time dependence, whereas Ca(2+)-activated Cl- channels, present in the same cells, displayed slow activation kinetics and large, time-dependent tail currents upon repolarization to the holding potential. 4. The reversal potential of the osmotically activated channels was close to the predicted chloride equilibrium potential and was sensitive to the physiological extracellular Cl- concentration ([Cl-]o). The relationship between reversal potential and [Cl-]o was fitted to a modified Nernst equation with a slope of 51 mV per decade, consistent with a Cl- selective conductance. 5. The anion permeability sequence of the channel, obtained from the shifts of the reversal potentials of the volume-sensitive Cl- current, was: SCN- > I- > NO-3 > Br- > Cl- > formate > propionate = methanesulphonate = acetate > or = F- > or = butyrate > valerate > gluconate = glucuronate = glutamate. 6. The current through the volume-sensitive channels was inhibited by the Cl- channel blocker SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid) in a voltage-dependent manner. 7. We conclude that rat parotid acinar cells express an outwardly rectifying Cl- current that can be activated by swelling under hypotonic conditions. This Cl- conductance may be an element of the cellular mechanisms of volume regulation in exocrine glands.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1999

Chloride dependence of hyperpolarization-activated chloride channel gates.

1. ClC proteins are a class of voltage-dependent Cl- channels with several members mutated in human diseases. The prototype ClC-0 Torpedo channel is a dimeric protein; each subunit forms a pore that can gate independently from the other one. A common slower gating mechanism acts on both pores simultaneously; slow gating activates ClC-0 at hyperpolarized voltages. The ClC-2 Cl- channel is also activated by hyperpolarization, as are some ClC-1 mutants (e.g. D136G) and wild-type (WT) ClC-1 at certain pH values. 2. We studied the dependence on internal Cl- ([Cl-]i) of the hyperpolarization-activated gates of several ClC channels (WT ClC-0, ClC-0 mutant P522G, ClC-1 mutant D136G and an N-terminal deletion mutant of ClC-2), by patch clamping channels expressed in Xenopus oocytes. 3. With all these channels, reducing [Cl-]i shifted activation to more negative voltages and reduced the maximal activation at most negative voltages. 4. We also investigated the external halide dependence of WT ClC-2 using two-electrode voltage-clamp recording. Reducing external Cl- ([Cl-]o) activated ClC-2 currents. Replacing [Cl-]o by the less permeant Br- reduced channel activity and accelerated deactivation. 5. Gating of the ClC-2 mutant K566Q in normal [Cl-]o resembled that of WT ClC-2 in low [Cl-]o, i.e. channels had a considerable open probability (Po) at resting membrane potential. Substituting external Cl- by Br- or I- led to a decrease in Po. 6. The [Cl-]i dependence of the hyperpolarization-activated gates of various ClC channels suggests a similar gating mechanism, and raises the possibility that the gating charge for the hyperpolarization-activated gate is provided by Cl-. 7. The external halide dependence of hyperpolarization-activated gating of ClC-2 suggests that it is mediated or modulated by anions as in other ClC channels. In contrast to the depolarization-activated fast gates of ClC-0 and ClC-1, the absence of Cl- favours channel opening. Lysine 556 may be important for the relevant binding site.

Animals↗
PubMed1984

Inhibition of epithelial chloride channels by a semi-purified fraction of Crotalus atrox venom.

Rattlesnake venom has a strong effect on the ionic permeability of plasma membranes. Rattlesnake venom and one of its semipurified fractions (CAV-C2) has been implicated in affecting both sodium and chloride transfer across epithelia. The mechanism of CAV-C2 action on epithelia, however, is still open to question. Aplysia californica intestine has been shown to contain both chloride conductive channels and sodium dependent chloride uptake mechanisms in the mucosal membrane of its enterocytes which makes it an ideal model to differentiate the CAV-C2 action on either of these membrane transport processes. In the absence of sodium in the bathing medium, chloride is the only ion actively transported. CAV-C2 and piretanide have similar types of inhibitory effects on net chloride transport while furosemide had no effect on chloride movement across the intestine. It was concluded that CAV-C2 action is on chloride conductive channels located in the mucosal membrane of the enterocytes.

Animals↗
PubMed2003

Beta-adrenergic receptors couple to CFTR chloride channels of intercalated mitochondria-rich cells in the heterocellular toad skin epithelium.

In the heterocellular toad skin epithelium the beta-adrenergic receptor agonist isoproterenol activates cyclic AMP-dependent Cl(-) channels that are not located in the principal cells. With four experimental approaches, in the present study, we tested the hypothesis that the signalling pathway targets apical CFTR-chloride channels of mitochondria-rich cells. (i) Serosal application of isoproterenol (log(10)EC50=-7.1+/0.2; Hill coefficient=1.1+/0.2), as well as nor-adrenaline, activated an anion pathway with an apical selectivity sequence, G(Cl)>G(Br)> or =G(NO(3))>G(I), comparable to the published selectivity sequence of cloned human CFTR expressed in Xenopus oocytes. (ii) Known modulators of human CFTR, glibenclamide (200 micromol/l) and genistein (50 micromol/l), depressed and activated, respectively, the receptor-stimulated G(Cl). Genistein did not modify the anion selectivity. (iii) Transcellular voltage clamp studies of single isolated mitochondria-rich cells revealed functional beta-adrenergic receptors on the basolateral membrane. With approximately 60,000 mitochondria-rich cells per cm(2), the saturating activation of 11.9+/-1.6 ns/cell accounted for the measured isoproterenol-activated transepithelial conductance of 600-900 micros/cm(2). In forskolin-stimulated cells, glibenclamide (200 micromol/l) reversibly inhibited the transcellular conductance by 9.6+/1.6 ns/cell. (iv) A nucleotide sequence of one third of the Bufo bufo CFTR gene corresponding to the R-domain and part of the first nucleotide binding domain (NBD1) including its Walker motif was amplified from gallbladder epithelium. Somewhat smaller sequences of the BbCFTR were cloned from lung and isolated skin epithelium. The above new results taken together with our previously identified small-conductance CFTR-like Cl(-) channel in the apical membrane of isolated mitochondria-rich cells provide compelling evidence that the toad's CFTR gene codes for a functional Cl(-) channel in the apical plasma membrane of this minority cell type.

Amino Acid Sequence↗
PubMed1999

Chloride channels activated by hypotonicity in N2A neuroblastoma cell line.

By using the patch-clamp technique we have shown that, in hypotonic extracellular solutions, the mouse neuroblastoma cells Neuro2A (N2A) develop ionic currents mediated by a chloride-selective channel which is also permeable to other anions in accordance with the permeability sequence: I->Br->Cl->gluconate->glutamate-. The currents persist for several hours when Mg-ATP is present in the recording pipette but occur only transiently in the absence of Mg-ATP. Typical blockers of anions channels such as La3+ and Zn2+ do not affect the hypotonicity-activated channel; conversely, the stilbene sulfonate-derivatives, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), reversibly inhibit the channel in a voltage-dependent manner. Also intact cells exposed to hyposmotic solutions activate volume-regulation mechanisms which decrease the transient volume increase that develops immediately after the application of the hyposmotic challenge. Since N2A neurons have been used as an expression system of exogenous channels, the presence of osmolarity-regulated channels in these cells is an important aspect that deserves the attention of researchers who may wish to express and study the properties of transport proteins in this cell line.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1989

Histamine directly gates a chloride channel in lobster olfactory receptor neurons.

Biogenic amines mediate many types of intercellular communication in multicellular organisms. Heretofore, little direct evidence has indicated that biogenic amines produce intracellular responses other than by triggering the enzymatic production of second messengers. Our electrophysiological studies of lobster olfactory receptor neurons now reveal that one biogenic amine, histamine, can directly gate an ion channel. The channel responds to histamine concentrations of 1 microM or more, is permeable primarily to Cl-, is more active at depolarized potentials, and has a conductance of 44 pS in the American lobster and 66 pS in the Caribbean spiny lobster. The expression of this ligand-gated channel in olfactory receptor neurons implies that these neurons are targets of a regulatory or feedback process.

Animals↗
PubMed1993

Effects of high salt concentration on the open probability of the background chloride channel.

We report here single channel data showing that high concentration of NaCl or CsCl at the cytoplasmic side of the membrane increases the open probability of the background Cl channel from hippocampal neurons. The open probability vs. voltage curve was shifted by 36 mV towards more negative voltages, when salt concentration was raised from 300 to 1000 mM. The steepness of the curve remained unchanged. Possible explanations for this effect are discussed.

Animals↗
PubMed1995

A voltage-gated chloride channel in ascidian embryos modulated by both the cell cycle clock and cell volume.

1. Eggs of the ascidian Boltenia villosa have an inwardly rectifying Cl- current whose amplitude varies by more than 10-fold during each cell cycle, the largest amplitude being at exit from M-phase. We examined whether this current was also sensitive to changes in cell volume. 2. Cell swelling, produced by direct inflation through a whole-cell recording pipette, greatly increased the amplitude of the Cl- current at all stages of the cell cycle in activated eggs. Swelling was much less effective in unfertilized eggs. 3. The increase in Cl- current amplitude continued for 10-20 min after an increase in diameter that was complete in 10 s, suggesting the involvement of a second messenger system in the response. 4. Treatment of unfertilized eggs with 6-dimethylaminopurine (DMAP), an inhibitor of cell cycle-dependent protein kinases, increased the amplitude of the Cl- current and its sensitivity to swelling to levels characteristic of fertilized eggs. 5. Osmotically produced swelling also increased Cl- current amplitude in unfertilized eggs. 6. We propose that dephosphorylation renders the Cl- channel functional, and that swelling or activation of the egg increases the sensitivity of the channel to dephosphorylation, perhaps by disrupting its links to the cytoskeleton.

Adenine↗
PubMed1992

Two types of chloride channel in the apical membrane of rat choroid plexus epithelial cells.

The patch clamp technique has been used to study ion channel activity in the apical (ventricular) membrane of epithelial cells from the rat choroid plexus. Two different classes of Cl(-)-selective channel were identified. A low conductance (26 pS) channel which was the predominant feature in cell-attached and inside-out patches. The occurrence of this channel appeared to increase in tissue bathed in forskolin. It was activated in inside-out patches by increasing the Ca2+ concentration at the intracellular face of the membrane and by depolarising potentials. The second class of channel was observed infrequently (2% of patches) and appeared to be similar to 'maxi'-Cl- channels which have been described in many other cell types. It had a conductance of 320 pS, opened to sub-conductance levels and displayed a marked voltage dependence in inside-out patches. The possible contribution of these channels to Cl- transport during the production of cerebrospinal fluid (CSF) is discussed.

Animals↗
PubMed2001

Chloride channels in the loop of Henle.

Cl- transport in the loop of Henle is responsible for reclamation of 25-40% of the filtered NaCl load and for the formation of dilute urine. Our understanding of the physiologic and molecular mechanisms responsible for Cl- reabsorption in both the thin ascending limb and thick ascending limb of Henle's loop has increased greatly over the last decade. Plasma membrane Cl- channels are known to play an integral role in transcellular Cl- transport in both the thin and thick ascending limbs. This review focuses on the functional characteristics and molecular identities of these Cl- channels, as well as the role of these channels in the pathophysiology of disease.

Amino Acid Sequence↗