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PubMed2003

Regulation of swelling-activated chloride channels in embryonic chick heart cells.

Swelling-activated Cl- currents, I(Cl,swell) were measured during hyposmotic shock in white Leghorn embryonic chick heart cells using the whole-cell recording of patch-clamp technique. Genistein, an inhibitor of protein tyrosine kinase (PTK), suppressed I(Cl,swell). Under isosmotic condition phorbol 12-myristate 13-acetate (PMA), an activator of PKC, elicited the Cl- current similar to that in hyposmotic solution, whereas hyposmotic shock did not elicit I(Cl,swell) in chelerythrine chloride(an inhibitor of PKC)-treated cells. Confocal microscopy experiments using FITC-phalloidin as a fluorescent label of F-actin showed that the actin network was moved from cortical region of the cell to the center after hyposmotic shock as compared with the image under isosmotic condition. When the cells were treated with cytochalasin B (CB) or cytochalasin D (CD) under isosmotic condition the disruption of the F-actin integrity was observed, and I(Cl,swell) was not elicited. With combination treatment of CB with PMA, hyposmotic solution could not elicited I(Cl,swell). The results suggested that the role of PTK, probably receptor tyrosine kinase, for regulation of I(Cl,swell) appeared to be at upstream site related to the role of F-actin. Then PKC signal pathway was activated somehow and finally change in the polymerization state of cytoskeleton led to activate the swelling-activated Cl- channels. These results demonstrate clearly that PTK, PKC and F-actin are important factors for regulation of I(Cl,swell), in embryonic chick heart cells as compared with often controversial results reported in different cell types.

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

Restoration of cyclic adenosine monophosphate-stimulated chloride channel activity in human cystic fibrosis tracheobronchial submucosal gland cells by adenovirus-mediated and cationic lipid-mediated gene transfer.

In human airways, the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is predominantly expressed in serous cells of the tracheobronchial glands. Despite considerable evidence that submucosal glands are important contributors to the pathophysiology of CF lung disease, most attempts at CFTR gene transfer have primarily targeted airway surface epithelial cells. In this study, we systematically evaluated CFTR gene transfer into cultures of immortalized CF human tracheobronchial submucosal gland (6CFSMEO) cells using adenovirus and cationic lipid vectors. We found that the efficiency of adenovirus-mediated gene transfer was comparable in 6CFSMEO and CFT1 cells (a surface airway epithelial cell line isolated from a subject with CF). So was the ranking order of adenovirus vectors containing different enhancers/promoters (CMV >> E1a approximately phosphoglycerokinase), as determined by both X-Gal staining and quantitative measurement of beta-galactosidase activity. Further, we provide the first demonstration that cationic lipids mediate efficient gene transfer into 6CFSMEO cells in vitro. The transfection efficiency at optimal conditions was higher in 6CFSMEO than in CFT1 cells. Finally, either infection with adenoviral vectors or transfection with cationic lipid:plasmid DNA complexes encoding CFTR significantly increased chloride (Cl-) permeability, as assessed using the 6-methoxy-N-(3-sulfopropyl)-quinolinium (SPQ) fluorescence assay, indicating restoration of functional CFTR Cl- channel activity. These data show that although the mechanisms of transfection may be different between the two cell types, 6CFSMEO cells are as susceptible as CFT1 cells to transfection by adenoviral and cationic-lipid gene transfer vectors.

Adenoviridae↗
PubMed1993

Chloride channels activated by osmotic stress in T lymphocytes.

We have used whole-cell and perforated-patch recording techniques to characterize volume-sensitive Cl- channels in T and B lymphocytes. Positive transmembrane osmotic pressure (intracellular osmolality > extracellular osmolality) triggers the slow induction of a Cl- conductance. Membrane stretch caused by cellular swelling may underlie the activation mechanism, as moderate suction applied to the pipette interior can reversibly oppose the induction of Cl- current by an osmotic stimulus. Intracellular ATP is required for sustaining the Cl- current. With ATP-free internal solutions, the inducibility of Cl- current declines within minutes of whole-cell recording, while in whole-cell recordings with ATP or in perforated-patch experiments, the current can be activated for at least 30 min. The channels are anion selective with a permeability sequence of I- > SCN- > NO3-, Br- > Cl- > MeSO3- > acetate, propionate > ascorbate > aspartate and gluconate. GCl does not show voltage- and time-dependent gating behavior at potentials between -100 and +100 mV, but exhibits moderate outward rectification in symmetrical Cl- solutions. Fluctuation analysis indicates a unitary chord conductance of approximately 2 pS at -80 mV in the presence of symmetrical 160 mM Cl-. The relationship of mean current to current variance during the osmotic activation of Cl- current implies that each cell contains on the order of 10(4) activatable Cl- channels, making it the most abundant ion channel in lymphocytes yet described. The current is blocked in a voltage-dependent manner by DIDS and SITS (Ki = 17 and 89 microM, respectively, at +40 mV), the degree of blockade increasing with membrane depolarization. The biophysical and pharmacological properties of this Cl- channel are consistent with a role in triggering volume regulation in lymphocytes exposed to hyposmotic conditions.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1990

Voltage dependence and stability of the gating kinetics of the fast chloride channel from rat skeletal muscle.

1. The voltage dependence and stability of the gating kinetics of the fast Cl-channel in excised patches of membrane from cultured rat skeletal muscle were studied with the patch clamp technique. Up to 10(6) open and shut intervals were analysed from each of five different patches containing a single fast Cl-channel. 2. To test for kinetic stability, plots of the mean durations of consecutive groups of 5-500 open and shut intervals were examined at each voltage. After excluding infrequent entries into both an apparent subconductance state and a long-lived (inactive) shut state, there were no abrupt and sustained changes in the moving means, indicating the absence of obvious shifts to other kinetic modes. The moving means did, however, fluctuate about the overall mean. 3. A comparison of experimental and simulated data indicated that most, but not all, of the fluctuation in the moving means was due to the stochastic variation inherent in the gating process. The fluctuation not accounted for by stochastic variation was mainly expressed as a slow, low-amplitude, component of drift about the mean. This slow component was unlikely to have arisen from measurement errors. 4. To examine whether the slow drift reflected detectable changes in kinetic modes, the data were divided into consecutive groups of 50,000 intervals. The exponential components describing the distributions were remarkably similar among the different groups, with stochastic variation accounting for most of the observed differences. This finding implies a single kinetic mode throughout the experiment. Thus, any changes in channel activity associated with the slow drift would have little effect on the analysis of gating kinetics presented here. 5. Depolarization increased channel open probability, Popen, for all five channels. This increase had a voltage sensitivity of 17 +/- 4 mV per e-fold change (effective gating charge of 1.6 +/- 0.32 electronic charges at 23 degrees C). Popen was 0.5 at -31 +/- 4 mV. 6. The depolarization-induced increase in Popen typically arose from a decrease in the mean shut time (19 +/- 4 mV per e-fold change; effective gating charge of 1.3 +/- 0.3 at 23 degrees C) and an increase in the mean open time (109 +/- 61 mV per e-fold change; effective gating charge of -0.24 +/- 0.13). 7. Neither plots of Popen versus voltage nor plots of the mean open and mean shut time versus voltage were completely described by a single Boltzmann distribution, suggesting multiple voltage-sensitive steps in channel gating.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1997

Calcium-activated chloride channels in bovine pulmonary artery endothelial cells.

1. We characterized Ca(2+)-activated Cl- currents in calf pulmonary artery endothelial (CPAE) cells by using a combined patch clamp and fura-2 microfluorescence technique to simultaneously measure ionic currents and the intracellular Ca2+ concentration, [Ca2+]i. 2. Various procedures that increased [Ca2+]i, such as stimulation with ATP or ionomycin, or loading the cells with Ca2+ via the patch pipette, activated a strongly outwardly rectifying current with a reversal potential close to the Cl- equilibrium potential. Changing the extracellular Cl- concentration shifted this reversal potential as predicted for a Cl- current. Buffering Ca2+ rises with BAPTA prevented ATP from activating the current. 3. Ca(2+)-activated Cl- currents could be distinguished from volume-activated Cl- currents, which were sometimes coactivated in the same cell. The latter showed much less outward rectification, their activation was voltage independent, and they could be inhibited by exposing the cells to hypertonic solutions. 4. The permeability ratio for the Ca(2+)-activated conductance of the anions iodide:chloride: gluconate was 1.71 +/- 0.06:1:0.39 +/- 0.03 (n = 12). 5. This Ca(2+)-activated Cl- current, ICl, Ca, inactivated rapidly at negative potentials and activated slowly at positive potentials. Outward tail currents were slowly decaying, while inward tail currents decayed much faster. 6. 4,4'-Diisothiocyanatostilbene-2,2'-disulphonic-acid (DIDS) and niflumic acid inhibited Icl,Ca in a voltage-dependent manner, i.e. they exerted a more potent block at positive potentials. The block by N-phenylanthracilic acid (NPA), 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and tamoxifen was voltage independent. Niflumic acid and tamoxifen were the most potent blockers. 7. The single-channel conductance was 7.9 +/- 0.7 pS (n = 15) at 300 mM extracellular Cl-. The channel open probability was high at positive potentials, but very small at negative potentials. 8. It is concluded that [Ca2+]i activates small-conductance Cl- channels in endothelial cells, which coexist with the volume-activated Cl- channels described previously.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1995

Cloning of an epithelial chloride channel from bovine trachea.

We have isolated and cloned a novel epithelial Cl- channel protein from a bovine tracheal cDNA expression library using an antibody probe. The antibody (alpha p38) was raised against a 38-kDa component of a homopolymeric protein that behaves as a Ca2+/calmodulin kinase II-, DIDS-, and dithiothreitol (DTT)-sensitive, anion-selective channel when incorporated into planar lipid bilayers. The full-length cDNA is 3001 base pairs long and codes for a 903-amino acid protein. The clone does not show any significant homology to any other previously reported Cl- channel sequence. Northern analysis of bovine tracheal mRNA with a cDNA probe corresponding to the cloned sequence revealed a band at 3.1 kilobases, suggesting that close to the full-length sequence has been cloned. The full-length open reading frame (2712 base pairs) has been expressed in Xenopus oocytes and in mammalian COS-7 cells. In oocytes, expression of the clone was associated with the appearance of a novel DIDS-, and DTT-sensitive, anion-selective conductance that was outwardly rectified and exhibited a reversal potential close to 0 mV. Whole-cell patch clamp studies in COS-7 cells transfected with the clone identified an ionomycin-, and DTT-sensitive chloride conductance that was not apparent in mock-transfected or control cells. In vitro translation studies have shown that the primary transcript codes for a protein migrating at 140 kDa under reduced conditions, significantly larger than the polypeptide recognized by alpha p38. We therefore suggest that either the 140-kDa translated product is a prepro form of the 38-kDa subunit of the previously identified bovine tracheal anion channel and that the primary transcript is post-translationally cleaved to yield the final product, or that the cloned channel and the previously identified bovine tracheal anion channel protein share an epitope that is recognized by the alpha p38 antibody.

Amino Acid Sequence↗
PubMed1982

Isolated-patch recording from liposomes containing functionally reconstituted chloride channels from Torpedo electroplax.

Small unilamellar vesicles formed from purified phospholids by detergent/dialysis methods may be enlarged to 30-microns diameter by freezing and thawing. Very-high-resistance seals were formed by applying a glass micropipette to the surface of these large liposomes, and single bilayer "patches" of membrane were isolated from the liposome surface while remaining sealed to the micropipette. The exogenous channel-forming peptides gramicidin and alamethicin induced characteristic single-channel fluctuation behavior in these excised patches held under voltage-clamp conditions. Large liposomes were formed from the small unilamellar vesicles made from cholate extracts of Torpedo electroplax plasma membrane vesicles. Isolated patches formed from these reconstituted membranes displayed current fluctuations due to single voltage-gated Cl- channels from non-innervated-face membranes; the properties of these Cl- channels are identical to those observed in planar bilayer membranes after direct insertion from native membranes. This liposome-patch method combines the advantages of membrane protein incorporation into liposomes with high-resolution electrical recording methods and may provide a generally applicable approach to the study of integral membrane channel proteins after solubilization and reconstitution.

Animals↗
PubMed1987

The "small" conductance chloride channel in the luminal membrane of the rectal gland of the dogfish (Squalus acanthias).

Besides the "larger" Cl- channel, with a single channel conductance of about 45 pS, a "small" channel was observed in the luminal membrane of the dogfish rectal gland. In cell excised (inside out) patches with NaCl solution on both sides, the latter channel had a single channel conductance of 11 +/- 1 pS (n = 21), and its current-voltage relationship was linear in the voltage range +90 to -90 mV. The open state probability increased moderately with negative clamp potentials. Ionic replacement studies revealed a high selectivity of Cl- over gluconate, sulfate, and iodide, whereas bromide was permeable to some extent. Also the channel is impermeable for Na+. The Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate did not affect this "small" conductance Cl- channel. It can be concluded that the luminal membrane of stimulated rectal gland cells possesses two types of Cl- channels, which differ markedly in their characteristics.

Animals↗
PubMed1989

Macrolide compounds, ivermectin and milbemycin D, stimulate chloride channels sensitive to GABAergic drugs in cultured chick spinal neurons.

1. The effects of ivermectin (IVM) and milbemycin D (MMD) on cultured embryonic spinal neurons were investigated, using the whole-cell recording technique. 2. IVM and MMD caused Cl- current under voltage-clamp conditions. 3. The reversal potential of the MMD-evoked Cl- current was shifted in a positive direction when half of the external Cl- was substituted by isethionate. 4. The MMD-evoked current was inhibited by picrotoxinin and bicuculline, whereas it was enhanced by diazepam. 5. Different characteristics of desensitization were observed between the MMD- (IVM-) and muscimol-induced effects. 6. These results suggest that IVM and MMD directly activate the Cl- channel through their specific sites modulated by GABAergic drugs.

Animals↗
PubMedDate not supplied

Activation of multiple-conductance state chloride channels in spinal neurones by glycine and GABA.

In the mammalian central nervous system, glycine and gamma-aminobutyric acid (GABA) bind to specific and distinct receptors and cause an increase in membrane conductance to CI- (refs 5-7). Neurones in various regions of the nervous system show differential sensitivity to glycine and GABA; thus GABA and glycine receptors are spatially distinct from one another. However, on the basis of desensitization experiments on spinal cord neurones, it was suggested that the receptors for glycine and GABA may share the same CI- channel. We now report that in small membrane patches, isolated from the soma of spinal neurones, both receptor channels display several (multiple) conductance states. Two of the states are common to both receptor channels. However, the most frequently observed 'main conductance states' of the GABA and glycine receptor channels are different. Both channels display the same anion selectivity. We propose that one class of multistate CI- channel is coupled to either GABA or glycine receptors. The main conductance state adopted by this channel is determined by the receptor to which it is coupled.

Animals↗
PubMed1986

Interaction of t-butylbicyclophosphorothionate with gamma-aminobutyric acid-gated chloride channels in cultured cerebral neurons.

The role of t-butylbicyclophosphorothionate (TBPS) as an antagonist of gamma-aminobutyric acid (GABA) was studied with primary cultures of neurons from the chick embryo cerebrum. The addition of GABA stimulated the uptake of 36Cl- by neurons and the dose dependence of this effect followed hyperbolic kinetics with a K0.5 = 1.3 microM for GABA. TBPS proved to be a potent inhibitor of GABA-dependent Cl- uptake (IC50 = 0.30 microM). Analysis of the kinetics of this process revealed that TBPS is a noncompetitive inhibitor (Ki = 0.15 microM) with respect to GABA. Scatchard analysis of direct binding of [35S]TBPS to membranes isolated from neuronal cultures gave curvilinear plots. These could be resolved by nonlinear regression methods into two components with KD values of 3.1 nM and 270 nM. The TBPS binding constant for this lower affinity site agreed well with the IC50 and Ki values for inhibition of Cl- flux, suggesting that this site is physiologically relevant to GABA antagonism. GABA was a noncompetitive displacer of [35S]TBPS binding to the lower affinity site. The Ki value for this displacement by GABA (1.7 microM) was comparable to the value for GABA enhancement of Cl- flux. The binding of [35S]TBPS to its low-affinity site on neuronal membranes was ninefold higher in the presence of Cl- than with gluconate, an impermeant anion. The rank order for anion stimulation of [35S]TBPS binding was Br- greater than or equal to SCN- greater than Cl- greater than or equal to NO3- greater than I- greater than F- greater than gluconate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Quantitative description of three modes of activity of fast chloride channels from rat skeletal muscle.

The steady-state kinetic properties of single Cl- channels with fast kinetics active at resting membrane potentials in cultured rat skeletal muscle were studied using the patch-clamp technique. Membrane patches containing single active Cl- channels were often observed, and binomial analysis of the percentage open time in membrane patches containing several Cl- channels indicated that the channels did not occur as obligatory dimers and that they gated independently of one another. Channel activity could be divided into three categories: normal, which included about 99% of the openings and closings; buzz mode, which included about 1% and consisted of bursts of about 50 brief open and shut intervals; and inactivated shut states which included about 0.01% of the shut intervals and lasted for seconds, and occasionally minutes. The method of maximum likelihood was used to determine the number of significant exponential components required to fit the distributions of open and shut intervals during normal activity. Open interval distributions required at least two components, with time constants of 0.52 and 1.5 ms at -40 mV and 7.6 degrees C. Shut interval distributions required at least five exponential components, with time constants of 0.064, 0.72, 1.9, 12.3 and 350 ms. Kinetic reaction schemes were developed for the normal and buzz mode using maximum likelihood techniques to determine the most likely models and rate constants. In developing these models the effects of limited time resolution and missed events were taken into account. Each model tested typically had two or more sets of equally likely rate constants. Incorrect sets of rate constants resulting from the effect of missed events could be eliminated by analysis of the data with different time resolutions. Normal activity could be accounted for by several different seven-state models with two open and five shut states. As different models could be found that gave identical descriptions of the data, the distributions of open and shut intervals were not sufficient to define a unique model. It was established that no other seven-state models would be found that describe the distributions of open and shut intervals during normal activity better than the most likely presented models.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗