Transmembrane chloride currents in human atrial myocytes

The present study was designed to evaluate the presence of basal, swelling-induced, and cAMP-dependent Cl- currents in human atrial myocytes studied with the whole cell patch-clamp technique. Under basal conditions, a small outwardly rectifying background conductance was noted that reversed close to 0 mV and was not altered by Cl- replacement. Isoproterenol (1 microM), forskolin (3 microM), and 8-bromoadenosine 3',5'-cyclic monophosphate (50 microM) did not increase membrane conductance, even when responsiveness to isoproterenol was confirmed by an increase in Ca2+ current and when perforated-patch techniques (nystatin) were used. Exposure to hyposmotic solutions increased cell volume and induced a whole cell conductance that showed outward rectification, was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (100 microM), and responded to changes in Cl- gradient in a fashion consistent with a Cl(-)-selective conductance, with estimated relative permeabilities of 1, 0.25, and 0.07 for Cl-, methanesulfonate, and aspartate, respectively. The results suggest that human atrial cells lack basal and adenosine 3',5'-cyclic monophosphate-dependent Cl- current but manifest a substantial Cl- conductance in the presence of cell swelling.

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Characterization of a swelling-induced chloride conductance in cultured rat epididymal cells

AJP Cell Physiology ◽

10.1152/ajpcell.1993.265.4.c997 ◽

1993 ◽

Vol 265 (4) ◽

pp. C997-C1005 ◽

Cited By ~ 9

Author(s):

H. C. Chan ◽

W. O. Fu ◽

Y. W. Chung ◽

S. J. Huang ◽

T. S. Zhou ◽

...

Keyword(s):

Kinase Inhibitor ◽

Disulfonic Acid ◽

Channel Blockers ◽

Induced Current ◽

Whole Cell ◽

Cyclic Monophosphate ◽

Whole Cell Patch Clamp ◽

Current Activation ◽

Ethanesulfonic Acid ◽

Cl Conductance

Swelling-induced Cl- conductance in cultured rat epididymal cells was characterized using whole cell patch-clamp techniques. Activation of whole cell current with an outwardly rectifying current-potential relationship was observed in cells exposed to hyposmotic solutions. This current was determined, from the observed current-reversal potentials at different Cl- concentrations, to be Cl- selective. The anion selectivity sequence of the swelling-induced Cl- conductance was I- approximately NO3- approximately Br- > Cl- > 2-(N-morpholino)ethanesulfonic acid. The swelling-induced Cl- conductance was reversibly inhibited by different Cl- channel blockers. Unlike diphenylamine-2-carboxylate or 5-nitro-2-(3-phenylpropylamino)-benzoate, which showed voltage-independent blockade, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid showed a marked voltage-dependent blockade of the volume-sensitive Cl- current, with a greater effect at depolarizing voltages. The swelling-induced Cl- conductance appeared to be different from the Ca(2+)- or adenosine 3',5'-cyclic monophosphate-activated Cl- conductances on the basis of the following observations: 1) swelling-induced current activation was seen even in the presence of kinase inhibitor (H-8) or absence of external free Ca2+, and 2) further increase in current activation could be produced by swelling after Ca(2+)- or adenosine 3',5'-cyclic monophosphate-induced current activation. The swelling-induced Cl- conductance may be involved in regulating epithelial cell volume as well as serving other important epididymal functions such as facilitating transepithelial secretion of organic compounds.

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Whole cell membrane currents in cultured pig endocardial endothelial cells

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1995.268.5.h2036 ◽

1995 ◽

Vol 268 (5) ◽

pp. H2036-H2047 ◽

Cited By ~ 2

Author(s):

P. F. Fransen ◽

M. J. Demolder ◽

D. L. Brutsaert

Keyword(s):

Endothelial Cells ◽

Vascular Endothelium ◽

Cell Swelling ◽

Disulfonic Acid ◽

Patch Clamp Technique ◽

Pipette Solution ◽

Whole Cell ◽

K Current ◽

Endocardial Endothelial Cells ◽

Inwardly Rectifying

The whole cell mode of the patch-clamp technique was applied to cultured endocardial endothelial cells from the porcine right ventricle to study their electrophysiological properties. With isotonic pipette and bathing solutions (300-310 mosmol/kgH2O), single endocardial endothelial cells had resting membrane potentials ranging from -20 to -90 mV (mean = -55 +/- 20 mV, n = 48). In voltage-clamp experiments, the main membrane current was an inwardly rectifying K+ current with all characteristics described for the inwardly rectifying K+ current in vascular endothelium. Outward currents at positive clamp potentials were small, but when cell swelling was induced by means of a hypertonic pipette or hypotonic bathing solution and ATP (5 mM) was present in the pipette solution, a large outwardly rectifying current developed. This volume-activated current was insensitive to extracellular K+ or Na+ concentration variations but sensitive to changes in extracellular Cl- concentrations. It was inhibited in the presence of 4,4'-diisothiocyanostilbene-2,2 disulfonic acid (100-300 microM) and flufenamic acid (50-100 microM). Volume-activated Cl- channels are different from the stretch-activated cationic channels described in vascular endothelium and might be involved in the regulation of cell volume or the response to mechanical stretch.

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cAMP-regulated whole cell chloride currents in pancreatic duct cells

AJP Cell Physiology ◽

10.1152/ajpcell.1993.264.3.c591 ◽

1993 ◽

Vol 264 (3) ◽

pp. C591-C602 ◽

Cited By ~ 94

Author(s):

M. A. Gray ◽

S. Plant ◽

B. E. Argent

Keyword(s):

Pancreatic Duct ◽

Single Cells ◽

Duct Cell ◽

Disulfonic Acid ◽

Apical Plasma Membrane ◽

Patch Clamp Technique ◽

Chloride Currents ◽

Whole Cell ◽

Duct Cells ◽

Pancreatic Duct Cells

Using the whole cell configuration of the patch-clamp technique, we have identified an adenosine 3',5'-cyclic monophosphate (cAMP)-regulated chloride conductance in pancreatic duct cells. Basal whole cell currents in single isolated cells were very low (approximately 5 pA/pF) but could be stimulated 17-fold by elevation of intracellular cAMP. The cAMP-activated currents exhibited 1) a high chloride selectivity, 2) a near linear current-voltage relationship, 3) time and voltage independence, 4) block by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and 5) an anion selectivity sequence based on permeability ratios of SCN > NO3 > Br > Cl > I > HCO3 > F > ClO4 > gluconate. Currents in single cells ran down within a few minutes; however, stable chloride currents could be recorded from duct cell clusters in which four or five cells were in electrical communication. We present evidence suggesting that these cAMP-regulated currents are carried by cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels. Physiologically, these CFTR channels act in parallel with chloride-bicarbonate exchangers to facilitate bicarbonate secretion across the apical plasma membrane of the duct cell.

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Swelling and cAMP on hyperpolarization-activated Cl- conductance in rat Leydig cells

AJP Cell Physiology ◽

10.1152/ajpcell.1996.271.1.c74 ◽

1996 ◽

Vol 271 (1) ◽

pp. C74-C84 ◽

Cited By ~ 6

Author(s):

J. F. Noulin ◽

E. Fayolle-Julien ◽

J. F. Desaphy ◽

J. P. Poindessault ◽

M. Joffre

Keyword(s):

Leydig Cells ◽

Membrane Conductance ◽

Disulfonic Acid ◽

Intracellular Camp ◽

Patch Clamp Technique ◽

Osmotic Swelling ◽

Bath Solution ◽

Whole Cell Patch Clamp ◽

Current Activation ◽

Cl Conductance

We have used the whole cell patch-clamp technique to characterize changes in membrane conductance induced by osmotic swelling in mature rat Leydig cells dialyzed with ATP (control cells) or adenosine 3',5'-cyclic monophosphate (cAMP) plus ATP (cAMP cells). A spontaneous current activation occurs in both groups in isosmotic conditions (300/295 mosM in/out). This development is entirely counteracted in control cells and partly inhibited in cAMP cells by exposing them to a hyperosmotic (350 mosM) bath solution, and these currents increase again in a hyposmotic (205 mosM) bath solution. These currents are sensitive to 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, a Cl- channel blocker. Taken together, the results indicate that, in the control cells (ATP alone) as well as in the presence of intracellular cAMP, osmotic swelling activates the background hyperpolarization-activated Cl- conductance, osmotic swelling and cAMP appearing to act synergistically.

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Cl− and K+ conductances activated by cell swelling in primary cultures of rabbit distal bright convoluted tubules

AJP Renal Physiology ◽

10.1152/ajprenal.1997.273.5.f680 ◽

1997 ◽

Vol 273 (5) ◽

pp. F680-F697 ◽

Cited By ~ 9

Author(s):

I. Rubera ◽

M. Tauc ◽

C. Poujeol ◽

M. T. Bohn ◽

M. Bidet ◽

...

Keyword(s):

Osmotic Shock ◽

Ion Selectivity ◽

Primary Cultures ◽

Regulatory Volume Decrease ◽

Ionic Currents ◽

Cell Swelling ◽

Disulfonic Acid ◽

Patch Clamp Technique ◽

Pipette Solution ◽

Whole Cell

Ionic currents induced by cell swelling were characterized in primary cultures of rabbit distal bright convoluted tubule (DCTb) by the whole cell patch-clamp technique. Cl− currents were produced spontaneously by whole cell recording with an isotonic pipette solution or by exposure to a hypotonic stress. Initial Cl− currents exhibited outwardly rectifying current-voltage relationship, whereas steady-state currents showed strong decay with depolarizing pulses. The ion selectivity sequence was I−= Br− > Cl− ≫ glutamate. Currents were inhibited by 0.1 mM 5-nitro-2-(3-phenylpropylamino)benzoic acid and 1 mM 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid and strongly blocked by 1 mM diphenylamine-2-carboxylate. Currents were insensitive to intracellular Ca2+but required the presence of extracellular Ca2+. They were not activated in cells pretreated with 200 nM staurosporine, 50 μM LaCl3, 10 μM nifedipine, 100 μM verapamil, 5 μM tamoxifen, and 50 μM dideoxyforskolin. Staurosporine, tamoxifen, verapamil, or the absence of external Ca2+ was without effect on the fully developed Cl−currents. Osmotic shock also activated K+ currents in Cl−-free conditions. These currents were time independent, activated at depolarized potentials, and inhibited by 5 mM BaCl2. The activation of Cl− and K+ currents by an osmotic shock may be implicated in regulatory volume decrease in DCTb cells.

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Whole cell Cl- currents in human neutrophils induced by cell swelling

AJP Cell Physiology ◽

10.1152/ajpcell.1993.265.1.c156 ◽

1993 ◽

Vol 265 (1) ◽

pp. C156-C165 ◽

Cited By ~ 47

Author(s):

J. S. Stoddard ◽

J. H. Steinbach ◽

L. Simchowitz

Keyword(s):

Regulatory Volume Decrease ◽

Reversal Potential ◽

Cell Swelling ◽

Disulfonic Acid ◽

Human Neutrophils ◽

Patch Clamp Technique ◽

Transport Pathway ◽

Pipette Solution ◽

Whole Cell ◽

Cl Channels

The properties of the conductive Cl- transport pathway underlying regulatory volume decrease (RVD) in human neutrophils were investigated using the whole cell patch-clamp technique. Cell swelling was induced during whole cell recordings by making the patch pipette solution hyperosmotic (approximately 20%) relative to the bath by addition of sucrose. Immediately after establishment of the whole cell configuration, no measurable Cl- currents were evident. Over a period of several minutes the outwardly rectifying Cl- current that developed displayed no apparent voltage dependence of activation and did not inactivate with time during voltage steps over the range of -80 to +80 mV. Reduction of Cl- currents by application of suction to the interior of the pipette implied that the swelling-induced Cl- channels are activated by membrane stretch. Based on reversal potential measurements, the volume-induced Cl- conductance was found to discriminate poorly among Cl-, Br-, I-, and NO3-, to possess a finite permeability to glucuronate (Pglucuronate/PCl approximately 0.1) and to be impermeable to cations. Single-channel conductance was estimated to be 1.5 pS from analysis of the variance of membrane current fluctuations. The activated Cl- currents were blocked by 100 microM of the compound MK-447 analogue A (inhibitor constant Ki = 37 microM) and by 200 microM 3,5-diiodosalicylate, 500 microM 4-acetamido-4'-iodothiocyanostilbene-2,2'-disulfonic acid, and 200 microM UK-5099. These results suggest that the initial event triggering RVD in neutrophils may be activation of stretch sensitive Cl- channels in the plasma membrane.

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CRISPR/Cas9 Mediated Knock Down of δ-ENaC Blunted the TNF-Induced Activation of ENaC in A549 Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22041858 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1858

Author(s):

Waheed Shabbir ◽

Nermina Topcagic ◽

Mohammed Aufy ◽

Murat Oz

Keyword(s):

A549 Cells ◽

Na Channel ◽

Na Current ◽

Patch Clamp Technique ◽

Whole Cell ◽

Knock Down ◽

Whole Cell Patch Clamp ◽

Glycosylation Sites ◽

Epithelial Na Channel

Tumor necrosis factor (TNF) is known to activate the epithelial Na+ channel (ENaC) in A549 cells. A549 cells are widely used model for ENaC research. The role of δ-ENaC subunit in TNF-induced activation has not been studied. In this study we hypothesized that δ-ENaC plays a major role in TNF-induced activation of ENaC channel in A549 cells which are widely used model for ENaC research. We used CRISPR/Cas 9 approach to knock down (KD) the δ-ENaC in A549 cells. Western blot and immunofluorescence assays were performed to analyze efficacy of δ-ENaC protein KD. Whole-cell patch clamp technique was used to analyze the TNF-induced activation of ENaC. Overexpression of wild type δ-ENaC in the δ-ENaC KD of A549 cells restored the TNF-induced activation of whole-cell Na+ current. Neither N-linked glycosylation sites nor carboxyl terminus domain of δ-ENaC was necessary for the TNF-induced activation of whole-cell Na+ current in δ-ENaC KD of A549 cells. Our data demonstrated that in A549 cells the δ-ENaC plays a major role in TNF-induced activation of ENaC.

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BICUCULLINE/BACLOFEN-INSENSITIVE GABA RESPONSE IN CRUSTACEAN NEURONES IN CULTURE

Journal of Experimental Biology ◽

10.1242/jeb.191.1.167 ◽

1994 ◽

Vol 191 (1) ◽

pp. 167-193

Author(s):

C Jackel ◽

W Krenz ◽

F Nagy

Keyword(s):

Reversal Potential ◽

Membrane Conductance ◽

Gamma Aminobutyric Acid ◽

Action Potential Firing ◽

Patch Clamp Technique ◽

Conformationally Restricted ◽

Whole Cell Patch Clamp ◽

Gabac Receptor ◽

Potential Firing ◽

Sr 95531

Neurones were dissociated from thoracic ganglia of embryonic and adult lobsters and kept in primary culture. When gamma-aminobutyric acid (GABA) was applied by pressure ejection, depolarizing or hyperpolarizing responses were produced, depending on the membrane potential. They were accompanied by an increase in membrane conductance. When they were present, action potential firing was inhibited. The pharmacological profile and ionic mechanism of GABA-evoked current were investigated under voltage-clamp with the whole-cell patch-clamp technique. The reversal potential of GABA-evoked current depended on the intracellular and extracellular Cl- concentration but not on extracellular Na+ and K+. Blockade of Ca2+ channels by Mn2+ was also without effect. The GABA-evoked current was mimicked by application of the GABAA agonists muscimol and isoguvacine with an order of potency muscimol>GABA>isoguvacine. cis-4-aminocrotonic acid (CACA), a folded and conformationally restricted GABA analogue, supposed to be diagnostic for the vertebrate GABAC receptor, also induced a bicuculline-resistant chloride current, although with a potency about 10 times lower than that of GABA. The GABA-evoked current was largely blocked by picrotoxin, but was insensitive to the GABAA antagonists bicuculline, bicuculline methiodide and SR 95531 at concentrations of up to 100 µmol l-1. Diazepam and phenobarbital did not exert modulatory effects. The GABAB antagonist phaclophen did not affect the GABA-induced current, while the GABAB agonists baclophen and 3-aminopropylphosphonic acid (3-APA) never evoked any response. Our results suggest that lobster thoracic neurones in culture express a chloride-conducting GABA-receptor channel which conforms to neither the GABAA nor the GABAB types of vertebrates but shows a pharmacology close to that of the novel GABAC receptor described in the vertebrate retina.

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Properties of cAMP-dependent and Ca(2+)-dependent whole cell Cl- conductances in rat epididymal cells

AJP Cell Physiology ◽

10.1152/ajpcell.1993.264.4.c794 ◽

1993 ◽

Vol 264 (4) ◽

pp. C794-C802 ◽

Cited By ~ 40

Author(s):

S. J. Huang ◽

W. O. Fu ◽

Y. W. Chung ◽

T. S. Zhou ◽

P. Y. Wong

Keyword(s):

Cystic Fibrosis ◽

Transmembrane Conductance Regulator ◽

Transmembrane Conductance ◽

Whole Cell ◽

Cyclic Monophosphate ◽

Cation Current ◽

Whole Cell Patch Clamp ◽

Current Voltage ◽

Clamp Condition ◽

Cl Conductance

Single rat epididymal cell studied under whole cell patch-clamp condition responded to 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP) (500 microM) and to ionomycin (1 microM) by an increase in whole cell conductance. A major part of the stimulated current was carried by Cl-, although a small part was due to nonselective cation current. After elimination of the cation current component by using impermeant cation, the cells revealed different Cl- conductance properties in response to adenosine 3',5'-cyclic monophosphate (cAMP) and ionomycin. The cAMP-stimulated Cl- conductance was independent of time and voltage and showed a linear current-voltage relationship. The anion permselectivity was NO3- > Br- > Cl- approximately I- >> SO(4)2-. The ionomycin-stimulated Cl- conductance showed marked time and voltage dependency. In contrast to the cAMP-induced anion permselectivity, the ionomycin-induced anion permselectivity was I- > Br- approximately NO3- > Cl- >> SO(4)2-. These results indicate that the epididymal epithelial cells exhibit different Cl- conductances sensitive to cAMP and Ca2+. The cAMP-activated conductance has properties resembling the type associated with the cystic fibrosis transmembrane conductance regulator found in cystic fibrosis-affected epithelia. This finding supports the notion that the epididymis is a cystic fibrosis epithelium.

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