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

1993 ◽  
Vol 265 (1) ◽  
pp. C156-C165 ◽  
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.

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

1997 ◽  
Vol 273 (5) ◽  
pp. F680-F697 ◽  
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.

Whole cell membrane currents in cultured pig endocardial endothelial cells

1995 ◽  
Vol 268 (5) ◽  
pp. H2036-H2047 ◽  
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.

scholarly journals Evidence That the Product of the Human X-Linked Cgd Gene, Gp91-phox, Is a Voltage-Gated H+ Pathway

1999 ◽  
Vol 114 (6) ◽  
pp. 771-786 ◽  
Author(s):  
Lydia M. Henderson ◽  
Robert W. Meech
Keyword(s):  
Cell Membrane ◽  
Time Course ◽  
Lucifer Yellow ◽  
Chinese Hamster ◽  
Reversal Potential ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Whole Cell ◽  
Histidine Residues ◽  
Voltage Gated

Expression of gp91-phox in Chinese hamster ovary (CHO91) cells is correlated with the presence of a voltage-gated H+ conductance. As one component of NADPH oxidase in neutrophils, gp91-phox is responsible for catalyzing the production of superoxide (O2·2). Suspensions of CHO91 cells exhibit arachidonate-activatable H+ fluxes (Henderson, L.M., G. Banting, and J.B. Chappell. 1995. J. Biol. Chem. 270:5909–5916) and we now characterize the electrical properties of the pathway. Voltage-gated currents were recorded from CHO91 cells using the whole-cell configuration of the patch-clamp technique under conditions designed to exclude a contribution from ions other than H+. As in other voltage-gated proton currents (Byerly, L., R. Meech, and W. Moody. 1984. J. Physiol. 351:199–216; DeCoursey, T.E., and V.V. Cherny. 1993. Biophys. J. 65:1590–1598), a lowered external pH (pHo) shifted activation to more positive voltages and caused the tail current reversal potential to shift in the manner predicted by the Nernst equation. The outward currents were also reversibly inhibited by 200 μM zinc. Voltage-gated currents were not present immediately upon perforating the cell membrane, but showed a progressive increase over the first 10–20 min of the recording period. This time course was consistent with a gradual shift in activation to more negative potentials as the pipette solution, pH 6.5, equilibrated with the cell contents (reported by Lucifer yellow included in the patch pipette). Use of the pH-sensitive dye 2′7′ bis-(2-carboxyethyl)-5(and 6) carboxyfluorescein (BCECF) suggested that the final intracellular pH (pHi) was ∼6.9, as though pHi was largely determined by endogenous cellular regulation. Arachidonate (20 μM) increased the amplitude of the currents by shifting activation to more negative voltages and by increasing the maximally available conductance. Changes in external Cl− concentration had no effect on either the time scale or the appearance of the currents. Examination of whole cell currents from cells expressing mutated versions of gp91-phox suggest that: (a) voltage as well as arachidonate sensitivity was retained by cells with only the NH2-terminal 230 amino acids, (b) histidine residues at positions 111, 115, and 119 on a putative membrane-spanning helical region of the protein contribute to H+ permeation, (c) histidine residues at positions 111 and 119 may contribute to voltage gating, (d) the histidine residue at position 115 is functionally important for H+ selectivity. Mechanisms of H+ permeation through gp91-phox include the possible protonation/deprotonation of His-115 as it is exposed alternatively to the interior and exterior faces of the cell membrane (see Starace, D.M., E. Stefani, and F. Bezanilla. 1997. Neuron. 19:1319–1327) and the transfer of protons across an “H-X-X-X-H-X-X-X-H” motif lining a conducting pore.

scholarly journals Characterization of the calcium-activated chloride channel in isolated guinea-pig hepatocytes.

1994 ◽  
Vol 104 (2) ◽  
pp. 357-373 ◽  
Author(s):  
S Koumi ◽  
R Sato ◽  
T Aramaki
Keyword(s):  
Guinea Pig ◽  
Single Channel ◽  
Hill Coefficient ◽  
Disulfonic Acid ◽  
Dependent Manner ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Cl Channels ◽  
Inside Out

Macroscopic and unitary currents through Ca(2+)-activated Cl- channels were examined in enzymatically isolated guinea-pig hepatocytes using whole-cell, excised outside-out and inside-out configurations of the patch-clamp technique. When K+ conductances were blocked and the intracellular Ca2+ concentration ([Ca2+]i) was set at 1 microM (pCa = 6), membrane currents were observed under whole-cell voltage-clamp conditions. The reversal potential of the current shifted by approximately 60 mV per 10-fold change in the external Cl- concentration. In addition, the current did not appear when Cl- was omitted from the internal and external solutions, indicating that the current was Cl- selective. The current was activated by increasing [Ca2+]i and was inactivated in Ca(2+)-free, 5 mM EGTA internal solution (pCa > 9). The current was inhibited by bath application of 9-anthracenecarboxylic acid (9-AC) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) in a voltage-dependent manner. In single channel recordings from outside-out patches, unitary current activity was observed, whose averaged slope conductance was 7.4 +/- 0.5 pS (n = 18). The single channel activity responded to extracellular Cl- changes as expected for a Cl- channel current. The open time distribution was best described by a single exponential function with mean open lifetime of 97.6 +/- 10.4 ms (n = 11), while at least two exponentials were required to fit the closed time distributions with a time constant for the fast component of 21.5 +/- 2.8 ms (n = 11) and that for the slow component of 411.9 +/- 52.0 ms (n = 11). In excised inside-out patch recordings, channel open probability was sensitive to [Ca2+]i. The relationship between [Ca2+]i and channel activity was fitted by the Hill equation with a Hill coefficient of 3.4 and the half-maximal activation was 0.48 microM. These results suggest that guinea-pig hepatocytes possess Ca(2+)-activated Cl- channels.

scholarly journals Mobilization of intracellular calcium by intracellular flash photolysis of caged dihydrosphingosine in cultured neonatal rat sensory neurones.

1998 ◽  
Vol 45 (2) ◽  
pp. 311-326 ◽  
Author(s):  
A Ayar ◽  
N M Thatcher ◽  
U Zehavi ◽  
D R Trentham ◽  
R H Scott
Keyword(s):  
Flash Photolysis ◽  
Reversal Potential ◽  
Neonatal Rat ◽  
Xenon Lamp ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Sensory Neurones ◽  
Whole Cell ◽  
Inward Currents ◽  
Current Activation

The ability of dihydrosphingosine to release Ca2+ from intracellular stores in neurones was investigated by combining the whole cell patch clamp technique with intracellular flash photolysis of caged, N-(2-nitrobenzyl)dihydrosphingosine. The caged dihydrosphingosine (100 microM) was applied to the intracellular environment via the CsCl-based patch pipette solution which also contained 0.3% dimethylformamide and 2 mM dithiothreitol. Cultured dorsal root ganglion neurones from neonatal rats were voltage clamped at -90 mV and inward whole cell Ca2+-activated currents were recorded in response to intracellular photorelease of dihydrosphingosine. Intracellular photorelease of dihydrosphingosine (about 5 microM) was achieved using a Xenon flash lamp. Inward Ca2+-activated currents were evoked in 50 out of 57 neurones, the mean delay to current activation following photolysis was 82+/-13 s. The responses were variable with neurones showing transient, oscillating or sustained inward currents. High voltage-activated Ca2+ currents evoked by 100 ms voltage step commands to 0 mV were not attenuated by photorelease of dihydrosphingosine. Controls showed that alone a flash from the Xenon lamp did not activate currents, and that the unphotolysed caged dihydrosphingosine, and intracellular photolysis of 2-(2-nitrobenzylamino) propanediol also did not evoke responses. The dihydrosphingosine current had a reversal potential of -11+/-3 mV (n = 11), and was carried by two distinct Cl- and cation currents which were reduced by 85% and about 20% following replacement of monovalent cations with N-methyl-D-glucamine or application of the Cl- channel blocker niflumic acid (10 microM) respectively. The responses to photoreleased dihydrosphingosine were inhibited by intracellular application of 20 mM EGTA, 10 microM ryanodine or extracellular application of 10 microM dantrolene, but persisted when Ca2+ free saline was applied to the extracellular environment. Intracellular application of uncaged dihydrosphingosine evoked responses which were attenuated by photolysis of the caged Ca2+ chelator Diazo-2. Experiments also suggested that extracellular application of dihydrosphingosine can activate membrane conductances. We conclude that dihydrosphingosine directly or indirectly mobilises Ca2+ from ryanodine-sensitive intracellular stores in cultured sensory neurones.

Swelling-induced and depolarization-induced C1-channels in normal and cystic fibrosis epithelial cells

1991 ◽  
Vol 261 (4) ◽  
pp. C658-C674 ◽  
Author(s):  
C. K. Solc ◽  
J. J. Wine
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Current Density ◽  
Single Channel ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Channel Activity ◽  
Whole Cell ◽  
Cl Channels ◽  
Single Channel Currents

Cl- currents induced by cell swelling were characterized at the whole cell and single-channel levels in primary cultures of normal and cystic fibrosis (CF) epithelial cells and in the T84 cell line. Currents recorded in normal and CF cells were indistinguishable. At 22-24 degrees C with isotonic CsCl in the pipette, initial whole cell outward current density at 100 mV in unswollen cells was 2-4 pA/pF. The current density increased with time during whole cell recording up to 100 pA/pF in isotonic solutions and up to 200 pA/pF in a hypotonic bath, though values typically ranged between 10 and 70 pA/pF. Currents were outwardly rectifying, active at negative voltages, started to inactivate above approximately 40 mV, and were blocked by 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS). Single Cl- channels (approximately 50 pS near 0 mV) with an outwardly rectifying current-voltage relation were recorded in cell-attached and outside-out patches from swollen cells. The channels were mostly open at negative voltages and inactivated at positive voltages with a voltage dependence similar to the whole cell currents. Channel activity decreased rapidly (channel rundown) after seal formation. After swelling-induced channel activity had ceased, outwardly rectifying, depolarization-induced Cl- channels (ORDIC channels) were activated in some patches. The swelling-induced and ORDIC single-channel currents were similar, but some consistent differences were observed. ORDIC channels were often closed at resting voltages (-70 to -50 mV), while swelling-induced channels were always open in this voltage range. In addition, ORDIC channels started to inactivate at more positive voltages (approximately 90 vs. approximately 50 mV), rectified more, and had smaller conductances (approximately 25 pS near 0 mV), shorter mean open durations (approximately 70 vs. approximately 350 ms), and more open-channel noise than swelling-induced channels. The two types of currents might arise from separate channel proteins or from a single channel molecule in different states.

Transmembrane chloride currents in human atrial myocytes

1996 ◽  
Vol 270 (2) ◽  
pp. C500-C507 ◽  
Author(s):  
G. R. Li ◽  
J. Feng ◽  
Z. Wang ◽  
S. Nattel
Keyword(s):  
Membrane Conductance ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Atrial Myocytes ◽  
Patch Clamp Technique ◽  
Chloride Currents ◽  
Whole Cell ◽  
Human Atrial Myocytes ◽  
Cyclic Monophosphate ◽  
Whole Cell Patch Clamp

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.

Method for manipulation of cytosolic pH in cells clamped in the whole cell or perforated-patch configurations

1994 ◽  
Vol 267 (4) ◽  
pp. C1152-C1159 ◽  
Author(s):  
S. Grinstein ◽  
R. Romanek ◽  
O. D. Rotstein
Keyword(s):  
Single Cells ◽  
Reversal Potential ◽  
Patch Clamp Technique ◽  
Intact Cells ◽  
Pipette Solution ◽  
Whole Cell ◽  
Perforated Patch ◽  
Cell Configuration ◽  
Patch Configuration ◽  
In Cells

A number of methods have been developed to manipulate the intracellular pH (pHi) of intact cells. However, such methods are not applicable when cells are studied using the patch-clamp technique, due to the continuity of the cell interior with the recording pipette. The perfused-pipette method can be used to modify pHi in the whole cell configuration, but this approach is slow, technically demanding, and not useful in the case of the perforated-patch configuration. In this report, we introduce a simple procedure that enables the investigator to predictably and reversibly alter pHi in cells clamped in either the whole cell or perforated-patch modes. The method is based on the provision of a virtually unlimited reservoir of an intracellular H+ (equivalent) donor/acceptor system, by inclusion of large concentrations of permeable weak electrolytes in the pipette solution. This system not only provides a means for the imposition and maintenance of a chosen pHi but, by changing the external concentration of the weak electrolyte, enables the investigator to rapidly and reversibly change pHi or the transmembrane delta pH during the course of an experiment. The effectiveness of the procedure was validated in peritoneal macrophages by two methods: 1) direct measurement of pHi in single cells by fluorescence ratio determinations and 2) estimation of the reversal potential of H(+)-selective currents. The pHi clamping procedure is shown to be effective using either organic or inorganic weak bases in the whole cell configuration. In addition, because NH+4/NH3 can readily permeate the pores formed by nystatin or amphotericin, the method is also shown to apply to the perforated-patch configuration.

A volume-sensitive chloride conductance in human colonic cell line T84

1989 ◽  
Vol 256 (6) ◽  
pp. C1111-C1119 ◽  
Author(s):  
R. T. Worrell ◽  
A. G. Butt ◽  
W. H. Cliff ◽  
R. A. Frizzell
Keyword(s):  
Cell Line ◽  
Single Channel ◽  
Shape Change ◽  
Regulatory Volume Decrease ◽  
Reversal Potential ◽  
Cell Swelling ◽  
Equilibrium Potential ◽  
T84 Cells ◽  
Pipette Solution ◽  
Cl Conductance

The chloride-secreting colonic cell line, T84, was studied under whole cell patch clamp with Cl as the permeant ion in pipette and bath solutions. Transmembrane current was initially small (approximately 50 pA at +100 mV) but increased steadily to average values of 1-3 nA within 5-10 min. The development of this current was associated with visible cell swelling, either without a shape change or with membrane blebbing. Basal, preswelling current levels were restored by the addition of 50-75 mM sucrose to the bath or when pipette osmolality was reduced by an equivalent amount. These findings suggest that an isosmotic pipette filling solution behaves as if it is hypertonic by approximately 60 mosmol/kgH2O to the bath. Currents traversing the swelling-induced conductance were outwardly rectified and showed activation at hyperpolarizing voltages and inactivation at depolarizing voltages. They were Cl selective because the reversal potential for current flow approached the Cl equilibrium potential when bath [Cl] was varied. Under nonswelling conditions (bath solution, 300 mosmol/kgH2O; pipette solution, 240 mosmol/kgH2O), single-channel steps (approximately 9 pA at +100 mV) could be resolved. The single-channel characteristics were similar to the macroscopic currents recorded from swollen cells, showing inactivation at positive voltages and an outwardly rectified current-voltage relation. Summation of these single-channel events yielded currents that were similar to those from swollen cells, implying that activation of multiple channels with these properties is the basis of the swelling-induced Cl conductance. This volume-sensitive Cl conductance would contribute to a regulatory volume decrease when T84 cells swell. Its relation to the secretory Cl conductance in these cells is unknown.

cAMP- and swelling-activated chloride conductance in rat hepatocytes

1996 ◽  
Vol 271 (1) ◽  
pp. C112-C120 ◽  
Author(s):  
X. J. Meng ◽  
S. A. Weinman
Keyword(s):  
Rat Hepatocytes ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Channel Blockers ◽  
Ionic Selectivity ◽  
Patch Clamp Technique ◽  
Isolated Rat Hepatocytes ◽  
Pipette Solution ◽  
Set Point ◽  
Cl Conductance

An outwardly rectifying Cl- conductance was identified in primary isolated rat hepatocytes, and the whole cell patch-clamp technique was used to characterize its properties and mechanisms of activation. With symmetrical Cl(-)-containing solutions on both sides and adenosine 3',5'-cyclic monophosphate (cAMP; 100 microM) in the pipette solution, a large outwardly rectifying conductance (1,014 +/- 153 pS/pF, n = 20) developed in all cells within 3 min. This cAMP-activated conductance was highly anion selective and slowly inactivated at voltages > 80 mV. It was completely inhibited by the anion channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (200 microM, n = 6) and partially inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (150 microM, n = 7). It displayed a halide selectivity of I- > Br- > Cl-. In the absence of cAMP, a functionally similar conductance was activated by cell swelling. Reduction of bath osmolality from 300 to 250 mosmol/kg increased membrane conductance from 64 +/- 16.4 to 487 +/- 23 pS/pF (n = 4). This swelling-activated conductance was also highly anion selective and had identical halide selectivity and blocker sensitivity as the cAMP-activated conductance. Although cell swelling was not necessary for cAMP activation, cell shrinkage with hyperosmotic bath (350 mosmol/kg), either before or after exposure to cAMP, inhibited the cAMP-activated conductance. By the determination of conductance as a function of bath osmolality in the presence and absence of cAMP, it was observed that cAMP shifted the osmotic set point for conductance activation without changing either the maximum or minimum conductance. In conclusion, both cAMP and cell swelling activate a large outwardly rectifying Cl- conductance in rat hepatocytes. Its ionic selectivity and sensitivity to channel blockers are identical to those seen for swelling-activated Cl- conductances in many cell types. The conductive properties are not those of cystic fibrosis transmembrane conductance regulator-mediated Cl- conductance. cAMP appears to activate this conductance by altering the volume set point of a swelling-activated channel.

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