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

1993 ◽  
Vol 265 (4) ◽  
pp. C997-C1005 ◽  
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.

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

1993 ◽  
Vol 264 (4) ◽  
pp. C794-C802 ◽  
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.

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.

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

1996 ◽  
Vol 271 (1) ◽  
pp. C74-C84 ◽  
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.

scholarly journals Zn2+ Slows Down CaV3.3 Gating Kinetics: Implications for Thalamocortical Activity

2007 ◽  
Vol 98 (4) ◽  
pp. 2274-2284 ◽  
Author(s):  
M. Cataldi ◽  
V. Lariccia ◽  
V. Marzaioli ◽  
A. Cavaccini ◽  
G. Curia ◽  
...  
Keyword(s):  
Neuronal Excitability ◽  
Whole Cell ◽  
Hek 293 ◽  
Gating Kinetics ◽  
Epileptiform Discharges ◽  
Whole Cell Patch Clamp ◽  
Recovery From Inactivation ◽  
293 Cells ◽  
Channel Opening ◽  
Current Activation

We employed whole cell patch-clamp recordings to establish the effect of Zn2+ on the gating the brain specific, T-type channel isoform CaV3.3 expressed in HEK-293 cells. Zn2+ (300 μM) modified the gating kinetics of this channel without influencing its steady-state properties. When inward Ca2+ currents were elicited by step depolarizations at voltages above the threshold for channel opening, current inactivation was significantly slowed down while current activation was moderately affected. In addition, Zn2+ slowed down channel deactivation but channel recovery from inactivation was only modestly changed. Zn2+ also decreased whole cell Ca2+ permeability to 45% of control values. In the presence of Zn2+, Ca2+ currents evoked by mock action potentials were more persistent than in its absence. Furthermore, computer simulation of action potential generation in thalamic reticular cells performed to model the gating effect of Zn2+ on T-type channels (while leaving the kinetic parameters of voltage-gated Na+ and K+ unchanged) revealed that Zn2+ increased the frequency and the duration of burst firing, which is known to depend on T-type channel activity. In line with this finding, we discovered that chelation of endogenous Zn2+ decreased the frequency of occurrence of ictal-like epileptiform discharges in rat thalamocortical slices perfused with medium containing the convulsant 4-aminopyridine (50 μM). These data demonstrate that Zn2+ modulates CaV3.3 channel gating thus leading to increased neuronal excitability. We also propose that endogenous Zn2+ may have a role in controlling thalamocortical oscillations.

Electrophysiological properties of cultured outer medullary collecting duct cells

1992 ◽  
Vol 263 (6) ◽  
pp. F1004-F1010 ◽  
Author(s):  
C. A. Pappas ◽  
B. M. Koeppen
Keyword(s):  
Collecting Duct ◽  
Extracellular Fluid ◽  
Outward Current ◽  
Membrane Voltage ◽  
Disulfonic Acid ◽  
Channel Blockers ◽  
Dependent Manner ◽  
Whole Cell ◽  
Electrophysiological Properties ◽  
Medullary Collecting Duct

Whole cell patch-clamp techniques were used to characterize the electrophysiological properties of cells from the inner stripe portion of the rabbit outer medullary collecting duct (OMCDi) grown in primary culture. With pipette and bathing solutions mimicking intracellular and extracellular fluid, the resting membrane voltage was -30 to -40 mV. The whole cell conductance exhibited slight outward rectification, and at the resting membrane voltage the cell conductance averaged 2.58 +/- 0.49 nS (n = 17). The major conductive ion species was Cl-. The Cl- conductance was also found to have a significant permeability to HCO3- and was inhibited by the Cl(-)-channel blockers diphenylamine carboxylic acid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. A small K+ conductance was also present, but no Na+ conductance was detected. Current generated by the H(+)-adenosinetriphosphatase (H(+)-ATPase) was quantitated. This current was dependent on the presence of ATP in the pipette. Dicyclohexylcarbodiimide, N-ethylmaleimide, and bafilomycin A1, inhibitors of the vacuolar H(+)-ATPase, also reduced this outward current in an ATP-dependent manner. The inhibitor-sensitive component of the outward current, a measure of the current generated by the H(+)-ATPase, was in the range of 35-100 pA/cell.

Whole cell Cl- conductances in mouse choroid plexus epithelial cells do not require CFTR expression

1997 ◽  
Vol 272 (6) ◽  
pp. C1899-C1907 ◽  
Author(s):  
J. D. Kibble ◽  
C. Garner ◽  
W. H. Colledge ◽  
S. Brown ◽  
H. Kajita ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Choroid Plexus ◽  
Disulfonic Acid ◽  
Wild Type ◽  
Pipette Solution ◽  
Whole Cell ◽  
Whole Cell Patch Clamp ◽  
Significant Difference ◽  
Choroid Plexus Epithelial

Whole cell patch-clamp studies were performed with tissue isolated from the cystic fibrosis (CF) transgenic Cftrm1cam mouse, to determine whether anion currents in choroid plexus epithelial cells require the expression of cystic fibrosis transmembrane conductance regulator (CFTR). Inclusion of 0.25 mM adenosine 3',5'-cyclic monophosphate (cAMP) and 375 nM protein kinase A (PKA) in the pipette solution caused a significant activation of a Cl(-)-selective, inward-rectifying conductance in cells from wild-type and CF mice. The small, outward currents observed in wild-type and CF animals, however, were not activated by cAMP-PKA. There were no significant differences in the size of currents between wild-type, heterozygote, and CF cells in the presence or absence of cAMP-PKA. A second whole cell conductance was activated when cells from wild-type mice were swollen. These volume-activated currents were Cl- selective and exhibited outward rectification. They were Ca2+ independent and ATP dependent and blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid. The volume-activated channels were also activated in CF mutant cells, and there was no significant difference in the size of the volume-activated currents between wild-type, heterozygote, and CF cells. It is concluded that CFTR neither contributes to the whole cell conductance nor regulates the other anion conductances in choroid plexus epithelial cells.

Swelling-activated efflux of taurine and other organic osmolytes in endothelial cells

1997 ◽  
Vol 273 (1) ◽  
pp. C214-C222 ◽  
Author(s):  
V. G. Manolopoulos ◽  
T. Voets ◽  
P. E. Declercq ◽  
G. Droogmans ◽  
B. Nilius
Keyword(s):  
Endothelial Cells ◽  
Anion Channel ◽  
Disulfonic Acid ◽  
Channel Blockers ◽  
Organic Osmolytes ◽  
Dependent Manner ◽  
Anion Channels ◽  
Whole Cell Patch Clamp ◽  
Osmolyte Efflux ◽  
Dose Dependent

We used a combined biochemical, pharmacological, and electrophysiological approach to study the effects of hyposmotic swelling on organic osmolyte efflux in endothelial cells (EC). In [3H]taurine-loaded monolayers of calf pulmonary artery EC (CPAEC), hyposmolality activated time- and dose-dependent effluxes of [3H]taurine. Swelling-activated [3H]taurine efflux (Jtau swell)in CPAEC was inhibited by the anion channel blockers tamoxifen, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), fenamates, and also quinine (in a pH-dependent manner), ATP, and the phospholipase A2 inhibitor 4-bromophenacyl bromide. In contrast, Jtau swell was partly or totally insensitive to bumetanide, forskolin, phorbol 12-myristate 13-acetate, and staurosporine. Swelling also activated myo-[3H]inositol efflux that was blocked by tamoxifen, NPPB, DIDS, and niflumic acid. Moreover, the cellular content of taurine and other amino acids was significantly reduced in osmotically activated CPAEC. Finally, in whole cell patch-clamp experiments, taurine, glycine, aspartate, and glutamate exhibited significant permeability for swelling-activated anion channels. In conclusion, hyposmotic swelling activates efflux of taurine and other organic osmolytes in EC. In addition, our results suggest that anion channels may provide a pathway for swelling-activated efflux of organic osmolytes in EC.

Persistent Na+ conductance in medium-sized neostriatal neurons: characterization using infrared videomicroscopy and whole cell patch-clamp recordings

1995 ◽  
Vol 74 (3) ◽  
pp. 1343-1348 ◽  
Author(s):  
C. Cepeda ◽  
S. H. Chandler ◽  
L. W. Shumate ◽  
M. S. Levine
Keyword(s):  
Patch Clamp ◽  
Sodium Current ◽  
Repetitive Firing ◽  
Channel Blockers ◽  
Potential Generation ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp ◽  
Outward Currents ◽  
Ramp Voltage

1. In the present study we investigate the expression of a persistent Na+ conductance (INaP) in identified medium-sized neostriatal neurons. Nomarski optics and infrared videomicroscopy were used for cell visualization and identification in thick slices (350 microns). Current- and voltage-clamp recordings were obtained utilizing whole cell patch-clamp methodology. 2. Application of depolarizing ramp voltage commands from a holding potential of -70 mV induced a slow, noninactivating inward current that occurred before and independent of the rapidly inactivating sodium current that subserves action potential generation. INaP began to activate at potentials less negative than -70 mV and peaked at -34 +/- 1 (SE) mV. Its average peak amplitude was -100 +/- 17 pA. INaP was abolished by tetrodotoxin (TTX, 0.5-1 microM) or an Na(+)-free solution. In contrast, it was not affected by Ca2+ channel blockers. Depolarizing ramp commands also induced tetraethylammonium-sensitive outward currents. 3. Dopamine (DA) (20-100 microM) produced a significant reduction of INaP. 4. These results demonstrate the existence of a TTX-sensitive persistent Na+ conductance in medium-sized neostriatal neurons. This conductance is modulated by DA and could play a role in the generation of rhythmic oscillations and in supporting repetitive firing.

Stilbenes stimulate T84 Cl- secretion by elevating Ca2+

1993 ◽  
Vol 264 (2) ◽  
pp. G325-G333 ◽  
Author(s):  
D. J. Brayden ◽  
M. E. Krouse ◽  
T. Law ◽  
J. J. Wine
Keyword(s):  
Time Course ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Disulfonic Acid ◽  
T84 Cells ◽  
Cl Secretion ◽  
Heat Stable ◽  
Whole Cell Patch Clamp ◽  
Sustained Inhibition ◽  
Cl Conductance

Basolateral but not apical application of 10-200 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) or 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) to T84 monolayers produced a transient increase in short-circuit current (Isc), followed by a sustained inhibition. 4,4'-Dinitrostilbene-2,2'-disulfonic acid (DNDS) had no effect. The increase in Isc produced by DIDS represents Cl- secretion and appears to result from Ca2+ elevation, because in all respects except time course the response to DIDS mimicked the response to the Ca(2+)-elevating agent thapsigargin. Fura-2 measurements established that thapsigargin elevates Ca2+ in T84 cells, but Ca2+ responses to DIDS could not be established directly because DIDS absorbs strongly at the critical wavelengths. Responses to DIDS and thapsigargin were 1) blocked by bumetanide; 2) not blocked by basolateral Ba2+; 3) completely nonadditive; 4) strongly synergistic with basal levels of Isc or with Isc increases produced by elevating adenosine 3',5'-cyclic monophosphate (cAMP; with forskolin) or guanosine 3',5'-cycxlic monophosphate (with heat-stable enterotoxin); and 5) reversibly abolished by removal of basolateral Ca2+. Interactions between Ca2+ and cAMP-elevating agents strongly support a model of Cl- secretion in which apical Cl- conductance is activated by cyclic nucleotides but not by Ca2+ while basolateral K+ channels are activated by Ca2+. In contrast with this mechanism, whole cell patch-clamp recordings of nonconfluent T84 cells indicated that DIDS and other Ca(2+)-elevating agents stimulated an increase in Cl- conductance. Thus increases in cytosolic free Ca2+ in nonconfluent T84 cells activate conductances that differ from those in confluent monolayers.

Calcium-activated, voltage-dependent, non-selective cation currents in endosperm plasma membrane from higher plants

1989 ◽  
Vol 237 (1287) ◽  
pp. 213-231 ◽  
Keyword(s):  
Plasma Membranes ◽  
Single Channel ◽  
Higher Plants ◽  
Outward Current ◽  
Monovalent Cation ◽  
Channel Blockers ◽  
Whole Cell ◽  
Whole Cell Patch Clamp ◽  
Single Burst ◽  
Subsequent Exposure

Single-channel and whole-cell patch-clamp techniques were used to characterize the electrophysiological behaviour of plasma membranes from freshly isolated, non-enzyme-treated endosperm protoplasts. A non-selective monovalent cation channel with a single-channel conductance of 22 pS in solutions with physiological potassium concentrations was observed in inside-out patches. The channel passes outward current at depolarized potentials and is highly selective for cations over anions, but discriminates poorly between lithium, sodium, potassium, rubidium and caesium ions. Specific potassium channel blockers were ineffective. The channel kinetics were apparently complex, with burst-like openings and rapid closures within a single burst. Single-channel openings were more frequent both for depolarizing pulses and maintained positive potentials. Channel activity was also increased by elevated cytoplasmic concentrations of either calcium or barium. Subsequent exposure of patches to low calcium, EGTA-buffered solutions resulted in large decreases in activity. Under whole-cell current clamp, small negative resting potentials were observed. A slowly developing outward current evoked by depolarizing pulses was seen in whole-cell recordings.

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