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PubMed2006

ClC-3 chloride channel prevents apoptosis induced by thapsigargin in PC12 cells.

Cell volume can be altered by two different ways, swelling and shrinkage. Cell swelling is regulated by volume-regulated Cl- channel (VRC). It is not well understood whether shrinkage is regulated by VRC. We previously found that antisense oligonucleotide specific to ClC-3 (ClC-3 antisense) prevented cell proliferation, which was related to cell swell volume regulation. In the present study, we further studied the role of ClC-3 Cl- channel in cell apoptosis which was related to cell shrinkage volume regulation by using antisense oligonucleotide specific to ClC-3 (ClC-3 antisense) and ClC-3 cDNA transfection techniques. We found that thapsigargin (TG), a specific inhibitor of the endoplasmic reticulum calcium ATPase, evoked apoptotic morphological changes (including cytoplasmic blebbing, condensation of nuclear chromatin, and the formation of apoptotic bodies), DNA laddering, and caspase-3 activation in PC12 cells (Pheochromocytoma-derived cell line). TG increased the cell apoptotic population with a decrease in cell viability. These effects were consistent with the decrease in endogenous ClC-3 protein expression, which was also induced by TG. Overexpression of ClC-3 significantly inhibited TG effect on PC12 cell apoptosis, whereas the ClC-3 antisense produced opposite effects and facilitated apoptosis induced by TG. Our data strongly suggest that ClC-3 channel in PC12 cells mediates TG-induced apoptotic process through inhibitory mechanism. Thus, it appears that ClC-3 Cl- channel mediates both cell proliferation and apoptosis through accelerative and inhibitory fashions, respectively.

Animals↗
PubMed1999

Hyposmotically activated chloride channels in cultured rabbit non-pigmented ciliary epithelial cells.

1. We used whole-cell patch-clamp recording techniques and noise analysis of whole-cell current to investigate the properties of hyposmotic shock (HOS)-activated Cl- channels in SV40-transformed rabbit non-pigmented ciliary epithelial (NPCE) cells. 2. Under conditions designed to isolate Cl- currents, exposure of cells to hyposmotic external solution reversibly increased the whole-cell conductance. 3. The whole-cell current activated with a slow time course (> 15 min), exhibited outward rectification and was Cl- selective. 4. The disulphonic stilbene derivatives 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, 0.5 mM), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS, 0. 5 mM) and 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS, 0.5 mM) produced a voltage-sensitive block of HOS-activated Cl- current at depolarized potentials, whereas niflumic acid produced a voltage-independent block of the current. 5. Under Ca2+-free conditions, HOS stimulation still reversibly activated the Cl- current, but the amplitude of current was reduced and the time course of current activation was slower compared with control (P < 0. 05). 6. The non-specific kinase inhibitor H-7 (100 microM), upregulated HOS-activated Cl- current amplitude in all cells tested (P < 0.05). 7. Noise analysis of whole-cell Cl- current indicated that cell swelling activated a high density of small conductance Cl- channels (< 1 pS). 8. We conclude that HOS primarily activates a high density of volume-sensitive small conductance Cl- channels in rabbit NPCE cells, and that Ca2+ and phosphorylation are involved in channel regulation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

Recording of a large-conductance chloride channel in normal rat lactotrophs.

Membrane current fluctuations resembling channel openings and closings were observed in the whole cell configuration of the patch-clamp technique in normal rat lactotrophs in primary culture. Using high-gain head stage in whole cell configuration, we characterized the nature and pharmacological properties of the ionic channel underlying these fluctuations. This channel, found in small numbers (< 10 per cell), was specific for Cl- because its reversal potential varied with Cl- gradients, according to the Nernst equation, and its unitary amplitude was linearly related to membrane potential from -100 to 0 mV. Slope conductance was close to 100 pS. Analyzing open times, we demonstrated its Ca2+ and potential dependence. Four sublevels were observed. We suggest that this channel, belonging to the background Cl- channel group, takes part in the regulation of intracellular Cl- concentration of normal rat lactotrophs.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1991

Identification and regulation of the cystic fibrosis transmembrane conductance regulator-generated chloride channel.

Cystic fibrosis transmembrane conductance regulator (CFTR) generates cAMP-regulated Cl- channels; mutations in CFTR cause defective Cl- channel function in cystic fibrosis epithelia. We used the patch-clamp technique to determine the single channel properties of Cl- channels in cell expressing recombinant CFTR. In cell-attached patches, an increase in cellular cAMP reversibly activated low conductance Cl- channels. cAMP-dependent regulation is due to phosphorylation, because the catalytic subunit of cAMP-dependent protein kinase plus ATP reversibly activated the channel in excised, cell-free patches of membrane. In symmetrical Cl- solutions, the channel had a channel conductance of 10.4 +/- 0.2 (n = 7) pS and a linear current-voltage relation. The channel was more permeable to Cl- than to I- and showed no appreciable time-dependent voltage effects. These biophysical properties are consistent with macroscopic studies of Cl- channels in single cells expressing CFTR and in the apical membrane of secretory epithelia. Identification of the single channel characteristics of CFTR-generated channels allows further studies of their regulation and the mechanism of ion permeation.

Adenosine Triphosphate↗
PubMed1991

Modulation of rat skeletal muscle chloride channels by activators and inhibitors of protein kinase C.

The membrane electrical parameters and component conductances of rat extensor digitorum longus muscle fibres were studied in vitro at 30 degrees C with standard two microelectrode square pulse cable analysis in the presence of protein kinase C (PKC) activators and inhibitors. The PKC activator, 4-beta-phorbol-12,13 dibutyrate (4-beta-PDB), (2-90 nM) blocked up to 67% chloride conductance (GCl) in rat skeletal muscle fibres and induced myotonic hyperexcitability. The concentration necessary to produce a 50% block of the membrane GCl was 23 nM. The "inactive" 4-alpha-phorbol-12,13 dibutyrate had no effect at 2 microM. The blocking effect of 4-beta-PDB on GCl was prevented by preincubation of the preparations with the PKC inhibitors, staurosporine (1-5 microM) and tetrahydropapaverolone (50-100 microM). The blocking effects on membrane GCl of 4-beta-PDB and its antagonism by the inhibitors used support the concept of the involvement of PKC in regulating Cl channels of mammalian skeletal muscle fibres.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1996

ASCT-1 is a neutral amino acid exchanger with chloride channel activity.

The ubiquitous transport activity known as system ASC is characterized by a preference for small neutral amino acids including alanine, serine, and cysteine. ASCT-1 and ASCT-2, recently cloned transporters exhibiting system ASC-like selectivity, are members of a major amino acid transporter family that includes a number of glutamate transporters. Here we show that ASCT1 functions as an electroneutral exchanger that mediates negligible net amino acid flux. The electrical currents previously shown to be associated with ASCT1-mediated transport result from activation of a thermodynamically uncoupled chloride conductance with permeation properties similar to those described for the glutamate transporter subfamily. Like glutamate transporters, ASCT1 activity requires extracellular Na+. However, unlike glutamate transporters, which mediate net flux and complete a transport cycle by countertransport of K+, ASCT-1 mediates only homo- and heteroexchange of amino acids and is insensitive to K+. The properties of ASCT-1 suggest that it may function to equilibrate different pools of neutral amino acids and provide a mechanism to link amino acid concentration gradients.

Alanine↗
PubMed1996

Cytoplasmic loop three of cystic fibrosis transmembrane conductance regulator contributes to regulation of chloride channel activity.

To examine the contribution of the large cytoplasmic loops of the cystic fibrosis transmembrane conductance regulator (CFTR) to channel activity, the three point-mutations (S945L, H949Y, G970R) were characterized that have been detected in the third cytoplasmic loop (CL3, residues 933-990) in patients with cystic fibrosis. Chinese hamster ovary cell lines stably expressing wild-type CFTR or mutant G970R-CFTR yielded polypeptides with apparent masses of 170 kDa as the major products, whereas the major products of mutants S945L-CFTR and H949Y-CFTR had apparent masses of 150 kDa. The 150-kDa forms of CFTR were sensitive to endoglycosidase H digestion, indicating that these mutations interfered with maturation of the protein. Increased levels of mature CFTR (170 kDa) could be obtained for mutant H949Y when cells were grown at a lower temperature (26 degrees C) or incubated in the presence of 10% glycerol. For all mutants, the open probability (P0) of the CFTR channels was significantly altered. S945L-CFTR and G970R-CFTR showed a severe reduction in the P0, whereas the H949Y mutation doubled the P0 relative to wild-type. The changes in P0 predominantly resulted from an alteration of the mean burst durations which suggests that CL3 is involved in obtaining and/or maintaining stability of the open state. In addition, mutants S945L and G970R had current-voltage relationships that were not completely linear over the range +/-80 mV, but showed slight outward rectification. The fact that CL3 mutations can have subtle effects on channel conductance indicates that this region may be physically close to the inner mouth of the pore.

Animals↗
PubMed1993

Endothelin and vasopressin activate low conductance chloride channels in aortic smooth muscle cells.

The non-contractile aortic smooth muscle cell line A7r5 was used to study the membrane events involved in the effect of vasoconstrictor peptides. Whole-cell voltage-clamp and membrane potential recording techniques were used to demonstrate the contribution of an increased Cl- conductance to the late depolarization induced by endothelin-1 and vasopressin. During cell-attached patch recording with N-methyl-D-glucamine in the pipette, bath application of endothelin or vasopressin induced single-channel inward currents in the following minutes. The current/potential (I/V) curve of the most frequently observed channel type--a small conductance Cl- (SCl) channel--reversed near the cell membrane potential and showed a single-channel conductance of 1.8 pS for inward currents. After patch excision in an extracellular solution containing CaCl2 (2 mM), the frequency of SCl channel openings increased. Patch excision in the absence of peptide stimulation also produced this channel activity. Replacement of CaCl2 by a Ca2+ chelator on the intracellular face of a patch reversibly inhibited the channel activity, indicating that these SCl channels are Ca(2+)-activated Cl- channels. The single-channel I/V characteristic showed outward rectification above +50 mV. An analysis of the gating kinetics of the SCl channel is given. Another channel type was recorded less frequently after peptide stimulation. It had a lower conductance (1.0-1.3 pS) and slower kinetics and was designated a very small conductance Cl- channel. It is concluded that activation of two types of Cl- channels (at least one of which is Ca2+ dependent) is involved in the late depolarization produced by vasoconstrictor peptides in vascular smooth muscle cells of the aortic cell line A7r5.

Adenosine Triphosphate↗
PubMed2003

A chloride channel blocker reduces acetylcholine uptake into synaptic vesicles at the frog neuromuscular junction.

A key mechanism for loading acetylcholine (ACh(+)) into synaptic vesicles uses energy to transport H(+) into the vesicle interior and then exchanges H(+) for ACh(+). This mechanism requires anions to follow the H(+) into the vesicles to prevent the building up of an overwhelming electrical gradient across the vesicle membrane. Frog nerve-muscle preparations were treated with hypertonic solution in which sodium gluconate was the major constituent, which substantially increases the sizes of the quanta by increasing their ACh(+) content. The Cl(-) channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) antagonized the increases in quantal size, so it seems likely that Cl(-) follows H(+) to prevent the buildup of a potential gradient across the vesicular membrane.

Acetylcholine↗
PubMed2003

Effects of chloride channel blockers on rat renal vascular responses to angiotensin II and norepinephrine.

The aim of the present study was to investigate the role of Ca2+-activated Cl- channels in the renal vasoconstriction elicited by angiotensin II (ANG II) and norepinephrine (NE). Renal blood flow (RBF) was measured in vivo using electromagnetic flowmetry. Ratiometric photometry of fura 2 fluorescence was used to estimate intracellular free Ca2+ concentration ([Ca2+]i) in isolated preglomerular vessels from rat kidneys. Renal arterial injection of ANG II (2-4 ng) and NE (20-40 ng) produced a transient decrease in RBF. Administration of ANG II (10-7 M) and NE (5 x 10-6 M) to the isolated preglomerular vessels caused a prompt increase in [Ca2+]i. Renal preinfusion of DIDS (0.6 and 1.25 micromol/min) attenuated the ANG II-induced vasoconstriction to approximately 35% of the control response, whereas the effects of NE were unaltered. Niflumic acid (0.14 and 0.28 micromol/min) and 2-[(2-cyclopentenyl-6,7-dichloro-2,3-dihydro-2-methyl-1-oxo-1H-inden-5-yl)oxy]acetic acid (IAA-94; 0.045 and 0.09 micromol/min) did not affect the vasoconstrictive responses of these compounds. Pretreatment with niflumic acid (50 microM) or IAA-94 (30 microM) for 2 min decreased baseline [Ca2+]i but did not change the magnitude of the [Ca2+]i response to ANG II and NE in the isolated vessels. The present results do not support the hypothesis that Ca2+-activated Cl- channels play a crucial role in the hemodynamic effects of ANG II and NE in rat renal vasculature.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1997

CFTR-like chloride channels in non-ciliated bronchiolar epithelial (Clara) cells.

Distal airways are believed to be a major site of disease in cystic fibrosis. The product of the CF gene, CFTR, is expressed in non-ciliated bronchiolar epithelial (Clara) cells. Clara cells in primary culture are capable of Cl- secretion which can be stimulated by cAMP and extracellular nucleotides. We used the patch clamp technique to look for Cl- channels in the apical membrane of rabbit Clara cells. In cell-attached patches, we recorded Cl- channels with a conductance for outward currents of 7.5 +/- 0.4 pS (n = 10) and for inward currents of 3.2 +/- 0.5 pS (n = 10); these channels typically exhibited slow kinetics and were not inhibited by the Cl- channel blockers NPPB and DIDS. Channel activity was not noticeably dependent on pipette potential. Addition of chlorophenylthio-cyclic AMP (cpt-cAMP) to the bath increased the percentage of cell-attached patches with active channels (33.8% vs 56.7%; p< 0.05) and the channel open probability (0.49 +/- 0.03 vs 0.84 +/- 0.02; p< 0.05). Extracellular ATP increased the percentage of cell-attached patches with active channels (28.7% to 50.0%; p< 0.05) but had no significant effect on the channel open probability (0.62 +/- 0.07 vs 0.60 +/- 0.06). In conclusion, rabbit non-ciliated bronchiolar epithelial cells express low-conductance Cl- channels that share many similarities with the CFTR-related Cl- channel and are regulated by cAMP and extracellular ATP.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2004

General anesthetic octanol and related compounds activate wild-type and delF508 cystic fibrosis chloride channels.

1. Cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is defective during cystic fibrosis (CF). Activators of the CFTR Cl(-) channel may be useful for therapy of CF. Here, we demonstrate that a range of general anesthetics like normal-alkanols (n-alkanols) and related compounds can stimulate the Cl(-) channel activity of wild-type CFTR and delF508-CFTR mutant. 2. The effects of n-alkanols like octanol on CFTR activity were measured by iodide ((125)I) efflux and patch-clamp techniques on three distinct cellular models: (1). CFTR-expressing Chinese hamster ovary cells, (2). human airway Calu-3 epithelial cells and (3). human airway JME/CF15 epithelial cells which express the delF508-CFTR mutant. 3. Our data show for the first time that n-alkanols activate both wild-type CFTR and delF508-CFTR mutant. Octanol stimulated (125)I efflux in a dose-dependent manner in CFTR-expressing cells (wild-type and delF508) but not in cell lines lacking CFTR. (125)I efflux and Cl(-) currents induced by octanol were blocked by glibenclamide but insensitive to 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, as expected for a CFTR Cl(-) current. 4. CFTR activation by octanol was neither due to cell-to-cell uncoupling properties of octanol nor to an intracellular cAMP increase. CFTR activation by octanol requires phosphorylation by protein kinase-A (PKA) since it was prevented by H-89, a PKA inhibitor. 5. n-Alkanols chain length was an important determinant for channel activation, with rank order of potencies: 1-heptanol<1-octanol<2-octanol<1-decanol. Our findings may be of valuable interest for developing novel therapeutic strategies for CF.

Animals↗
PubMed1997

Novel activation stimulus of chloride channels by potassium in human osteoblasts and human leukaemic T lymphocytes.

1. The whole-cell recording mode of the patch-clamp technique was used to study the effect of extracellular K+ and Rb+ on membrane currents in human osteoblasts, in a human osteoblast-like cell line, and in the Jurkat human leukaemic T cell line. 2. Increasing the extracellular concentration of K+ increased the membrane conductance of the cells in a concentration-dependent manner. This increase in membrane conductance was due to the activation of a Cl- conductance. Rb+ also induced this conductance, but conductance was less than half that seen in K+. 3. The Cl- channel blockers 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 4-acetamido-4'-isothiocyanatostilbene 2,2'-disulphonic acid (SITS) blocked the K(+)-induced Cl- current in a voltage-dependent manner. The degree of blockade increased with membrane depolarization to a maximum level at 40 mV. At potentials above this value the block appeared to decrease. 4. Both tonicity and K+ were required for maximal activation of the Cl- conductance since the K(+)-induced Cl- conductance could be inhibited by hypertonic solutions and the activation of a volume-sensitive Cl- conductance by hypotonic solutions could be enhanced by extracellular K+. 5. We conclude that an outwardly rectifying Cl- conductance can be activated either upon osmotic swelling or by an increase in extracellular K+. Both activation pathways may be involved in cell volume regulation and seem to apply to volume-sensitive Cl- channels in general since we observe this phenomenon in two different cell types, in human osteoblasts as well as in human leukaemic T lymphocytes.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1999

Gating of cystic fibrosis transmembrane conductance regulator chloride channels by adenosine triphosphate hydrolysis. Quantitative analysis of a cyclic gating scheme.

Gating of the cystic fibrosis transmembrane conductance regulator (CFTR) involves a coordinated action of ATP on two nucleotide binding domains (NBD1 and NBD2). Previous studies using nonhydrolyzable ATP analogues and NBD mutant CFTR have suggested that nucleotide hydrolysis at NBD1 is required for opening of the channel, while hydrolysis of nucleotides at NBD2 controls channel closing. We studied ATP-dependent gating of CFTR in excised inside-out patches from stably transfected NIH3T3 cells. Single channel kinetics of CFTR gating at different [ATP] were analyzed. The closed time constant (tauc) decreased with increasing [ATP] to a minimum value of approximately 0.43 s at [ATP] >1.00 mM. The open time constant (tauo) increased with increasing [ATP] with a minimal tauo of approximately 260 ms. Kinetic analysis of K1250A-CFTR, a mutant that abolishes ATP hydrolysis at NBD2, reveals the presence of two open states. A short open state with a time constant of approximately 250 ms is dominant at low ATP concentrations (10 microM) and a much longer open state with a time constant of approximately 3 min is present at millimolar ATP. These data suggest that nucleotide binding and hydrolysis at NBD1 is coupled to channel opening and that the channel can close without nucleotide interaction with NBD2. A quantitative cyclic gating scheme with microscopic irreversibility was constructed based on the kinetic parameters derived from single-channel analysis. The estimated values of the kinetic parameters suggest that NBD1 and NBD2 are neither functionally nor biochemically equivalent.

3T3 Cells↗
PubMed2006

Volume-sensitive chloride channels involved in apoptotic volume decrease and cell death.

Apoptosis is an essential process in organ development, tissue homeostasis, somatic cell turnover, and the pathogenesis of degenerative diseases. Apoptotic cell death occurs in response to a variety of stimuli in physiological and pathological circumstances. Efflux of K(+) and Cl(-) leads to apoptotic volume decrease (AVD) of the cell. Both mitochondrion-mediated intrinsic, and death receptor-mediated extrinsic, apoptotic stimuli have been reported to rapidly activate Cl(-) conductances in a large variety of cell types. In epithelial cells and cardiomyocytes, the AVD-inducing anion channel was recently determined to be the volume-sensitive outwardly rectifying (VSOR) Cl(-) channel which is usually activated by swelling under non-apoptotic conditions. Blocking the VSOR Cl(-) channel prevented cell death in not only epithelial and cardiac cells, but also other cell types, by inhibiting the induction of AVD and subsequent apoptotic events. Ischemia-reperfusion-induced apoptotic death in cardiomyocytes and brain neurons was also prevented by Cl(-) channel blockers. Furthermore, cancer cell apoptosis induced by the anti-cancer drug cisplatin was recently found to be associated with augmented activity of the VSOR Cl(-) channel and to be inhibited by a Cl(-) channel blocker. The apoptosis-inducing VSOR Cl(-) channel is distinct from ClC-3 and its molecular identity remains to be determined.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2004

Molecular determinants of differential pore blocking of kidney CLC-K chloride channels.

The highly homologous Cl(-) channels CLC-Ka and CLC-Kb are important for water and salt conservation in the kidney and for the production of endolymph in the inner ear. Mutations in CLC-Kb lead to Bartter's syndrome and mutations in the small CLC-K subunit barttin lead to Bartter's syndrome and deafness. Here we show that CLC-Ka is blocked by the recently identified blocker 2-(p-chlorophenoxy)-3-phenylpropionic acid of the rat channel CLC-K1 with an apparent K(D) approximately 80 microM. We also found that DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid), a generic Cl(-) channel blocker, inhibits CLC-Ka (K(D) approximately 90 microM). Surprisingly, the highly homologous channel CLC-Kb is fivefold to sixfold less sensitive to both compounds. Guided by the crystal structure of bacterial CLC proteins, we identify two amino acids, N68/D68 and G72/E72, in CLC-Ka and CLC-Kb, respectively, that are responsible for the differential drug sensitivity. Both residues expose their side chains in the extracellular pore mouth, delineating the probable drug binding site. These novel CLC-K channel blockers are promising lead compounds for the development of new diuretic drugs.

2-Methyl-4-chlorophenoxyacetic Acid↗