scholarly journals The Tyrosine Kinase p56lck Mediates Activation of Swelling-induced Chloride Channels in Lymphocytes

1998 ◽  
Vol 141 (1) ◽  
pp. 281-286 ◽  
Author(s):  
Albrecht Lepple-Wienhues ◽  
Ildikò Szabò ◽  
Tilmann Laun ◽  
Nubia Kristen Kaba ◽  
Erich Gulbins ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Tyrosine Kinase ◽  
Single Channel ◽  
Regulatory Volume Decrease ◽  
Cell Swelling ◽  
Osmotic Swelling ◽  
Whole Cell ◽  
Herbimycin A ◽  
Unitary Conductance ◽  
Human T Cell

Osmotic cell swelling activates Cl− channels to achieve anion efflux. In this study, we find that both the tyrosine kinase inhibitor herbimycin A and genetic knockout of p56lck, a src-like tyrosine kinase, block regulatory volume decrease (RVD) in a human T cell line. Activation of a swelling-activated chloride current (ICl−swell) by osmotic swelling in whole-cell patch-clamp experiments is blocked by herbimycin A and lavendustin. Osmotic activation of ICl−swell is defective in p56lck-deficient cells. Retransfection of p56lck restores osmotic current activation. Furthermore, tyrosine kinase activity is sufficient for activation of ICl−swell. Addition of purified p56lck to excised patches activates an outwardly rectifying chloride channel with 31 pS unitary conductance. Purified p56lck washed into the cytoplasm activates ICl−swell in native and p56lck-deficient cells even when hypotonic intracellular solutions lead to cell shrinkage. When whole-cell currents are activated either by swelling or by p56lck, slow single-channel gating events can be observed revealing a unitary conductance of 25–28 pS. In accordance with our patch-clamp data, osmotic swelling increases activity of immunoprecipitated p56lck. We conclude that osmotic swelling activates ICl−swell in lymphocytes via the tyrosine kinase p56lck.

scholarly journals Regulation of the Epithelial Na+ Channel by Membrane Tension

1998 ◽  
Vol 112 (2) ◽  
pp. 97-111 ◽  
Author(s):  
Mouhamed S. Awayda ◽  
Muthangi Subramanyam
Keyword(s):  
Cytochalasin B ◽  
Single Channel ◽  
Direct Injection ◽  
Expression System ◽  
Cell Swelling ◽  
Na Channel ◽  
Bathing Solution ◽  
Membrane Tension ◽  
Whole Cell ◽  
Epithelial Na Channel

The sensitivity of αβγ rat epithelial Na+ channel (rENaC) to osmotically or mechanically induced changes of membrane tension was investigated in the Xenopus oocyte expression system, using both dual electrode voltage clamp and cell-attached patch clamp methodologies. ENaC whole-cell currents were insensitive to mechanical cell swelling caused by direct injection of 90 or 180 nl of 100-mM KCl. Similarly, ENaC whole-cell currents were insensitive to osmotic cell swelling caused by a 33% decrease of bathing solution osmolarity. The lack of an effect of cell swelling on ENaC was independent of the status of the actin cytoskeleton, as ENaC remained insensitive to osmotic and mechanical cell swelling in oocytes pretreated with cytochalasin B for 2–5 h. This apparent insensitivity of ENaC to increased cell volume and changes of membrane tension was also observed at the single channel level in membrane patches subjected to negative or positive pressures of 5 or 10 in. of water. However, and contrary to the lack of an effect of cell swelling, ENaC currents were inhibited by cell shrinking. A 45-min incubation in a 260-mosmol solution (a 25% increase of solution osmolarity) caused a decrease of ENaC currents (at −100 mV) from −3.42 ± 0.34 to −2.02 ± 0.23 μA (n = 6). This decrease of current with cell shrinking was completely blocked by pretreatment of oocytes with cytochalasin B, indicating that these changes of current are not likely related to a direct effect of cell shrinking. We conclude that αβγ rENaC is not directly mechanosensitive when expressed in a system that can produce a channel with identical properties to those found in native epithelia.

Swelling-activated K+ transport via two functionally distinct pathways in eel erythrocytes

1996 ◽  
Vol 270 (1) ◽  
pp. R61-R70 ◽  
Author(s):  
J. D. Bursell ◽  
K. Kirk
Keyword(s):  
Regulatory Volume Decrease ◽  
Anguilla Anguilla ◽  
Cell Swelling ◽  
European Eel ◽  
Pharmacological Properties ◽  
Osmotic Swelling ◽  
Diverse Range ◽  
K Transport ◽  
Volume Decrease ◽  
In Cells

Following osmotic swelling, erythrocytes from the European eel, Anguilla anguilla, underwent a regulatory volume decrease. This was prevented by replacement of Na+ with K+ in the suspending medium, consistent with a role for the (normally outward) electrochemical K+ gradient in the volume-regulatory response. The effect of cell swelling on K- transport in these cells was investigated using 86Rb+ as a tracer for K+. Osmotic swelling resulted in an increase in ouabain-insensitive K+ transport that was highest for cells in Cl- and Br- media but which was also significant in I- and NO3- media. Treatment of eel erythrocytes suspended in isotonic Cl- or Br- (but not I- or NO3-) media with the sulfhydryl reagent N-ethylmaleimide (NEM) resulted in a large increase in K+ transport. A quantitative comparison of the pharmacological properties of the “Cl(-)-dependent” NEM-activated pathway with those of the “Cl(-)-independent” pathway mediating swelling-activated K+ transport in cells in Cl(-)-free (NO3- containing) media showed there to be significant differences between them. By contrast, the pharmacological properties of the Cl(-)-independent swelling-activated K+ pathway were indistinguishable from those of the pathway responsible for the swelling-activated transport of taurine, the major organic osmolyte in these cells. A pharmacological analysis of ouabain-insensitive K+ transport in cells swollen in a hypotonic Cl(-)-containing medium showed there to be two components, one with the characteristics of the NEM-activated system, the other showing the characteristics of the Cl(-)-independent swelling-activated pathway. The data are consistent with the presence of two functionally distinct swelling-activated K+ transport mechanisms in eel erythrocytes: a KCl cotransporter that is activated under isotonic conditions by NEM and a Cl(-)-independent, broad-specificity channel that accommodates a diverse range of organic and inorganic solutes.

Patch-clamp study of single-channel and whole-cell K+ currents in guinea pig pancreatic acinar cells

1988 ◽  
Vol 255 (3) ◽  
pp. G275-G285 ◽  
Author(s):  
K. Suzuki ◽  
O. H. Petersen
Keyword(s):  
Patch Clamp ◽  
Guinea Pig ◽  
Single Channel ◽  
Pancreatic Acinar Cells ◽  
Intact Cells ◽  
Membrane Area ◽  
Pancreatic Acini ◽  
Whole Cell ◽  
Single Channel Currents ◽  
K Currents

K+ channels in the plasma membrane of isolated guinea pig pancreatic acini were studied by patch-clamp single-channel and whole-cell current recording techniques. Three types of K+-permeable pores were found in excised patch experiments: Ca2+-activated nonselective cation channels with a unit conductance of approximately 25 pS that could be inhibited by ATP acting on the membrane inside, and two kinds of Ca2+- and voltage-activated K+-selective channels with unit conductances (in symmetrical K+-rich solutions) of about 200 and 30 pS, respectively. In intact cells, pentagastrin activation of currents through the 30 pS K+-selective pores was demonstrated. In these experiments pentagastrin was added to the bath solution and had no direct contact with the electrically isolated membrane area from which the single-channel currents were recorded, suggesting that the activation is mediated via an intracellular messenger system. Pentagastrin stimulation of voltage-gated K+ currents was also observed in whole-cell recording experiments. Results from these experiments suggest that in the stimulated condition the membrane electrical properties were dominated by the 30 pS K+-selective channels.

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.

Activation of Cl− currents by intracellular chloride in fibroblasts stably expressing the human cystic fibrosis transmembrane conductance regulator

1993 ◽  
Vol 71 (9) ◽  
pp. 645-649 ◽  
Author(s):  
Xiaodong Wang ◽  
Yoshinori Marunaka ◽  
Ludwik Fedorko ◽  
Sascha Dho ◽  
J. Kevin Foskett ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Cyclic Amp ◽  
Patch Clamp ◽  
Single Channel ◽  
Fibroblast Cell ◽  
Mouse Fibroblast ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Whole Cell ◽  
Cystic Fibrosis Transmembrane

The Cl− conductance of a mouse fibroblast cell line (LTK− cells) that was stably transfected with the human CFTR (cystic fibrosis transmembrane conductance regulator) complementary DNA was studied. Single Cl− channel activity was observed only after treatment of the cells with forskolin, the single-channel conductance being 6.2 ± 0.2 pS with a linear current–voltage relationship. In CFTR+ cells, the whole-cell current at +90 mV increased from 7.3 ± 2.7 pA/pF (n = 12) to 46.1 ± 11.2 pA/pF (n = 5) after addition of dibutyryl-cyclic AMP (10−4 M) to the bath. Increasing the intracellular Cl− concentration to 150 mM activated linear Cl− currents in the absence of cyclic AMP in CFTR+ (n = 42) but not in CFTR− cells (n = 4). Similar Cl− current was also activated by high intracellular I− concentration. These results indicate that the CFTR-induced Cl− conductance in LTK− cells can be activated by either cyclic AMP or high intracellular halide concentrations.Key words: cystic fibrosis transmembrane conductance regulator (CFTR), chloride channel, cyclic AMP, whole-cell patch clamp, single-channel patch clamp.

scholarly journals Properties of single-channel and whole cell Cl− currents in guinea pig detrusor smooth muscle cells

2019 ◽  
Vol 316 (5) ◽  
pp. C698-C710 ◽  
Author(s):  
Viktor Yarotskyy ◽  
John Malysz ◽  
Georgi V. Petkov
Keyword(s):  
Smooth Muscle ◽  
Patch Clamp ◽  
Guinea Pig ◽  
Smooth Muscle Cells ◽  
Single Channel ◽  
Muscle Cells ◽  
Niflumic Acid ◽  
Detrusor Smooth Muscle ◽  
Whole Cell ◽  
Whole Cell Patch Clamp

Multiple types of Cl− channels regulate smooth muscle excitability and contractility in vascular, gastrointestinal, and airway smooth muscle cells. However, little is known about Cl− channels in detrusor smooth muscle (DSM) cells. Here, we used inside-out single channel and whole cell patch-clamp recordings for detailed biophysical and pharmacological characterizations of Cl− channels in freshly isolated guinea pig DSM cells. The recorded single Cl− channels displayed unique gating with multiple subconductive states, a fully opened single-channel conductance of 164 pS, and a reversal potential of −41.5 mV, which is close to the ECl of −65 mV, confirming preferential permeability to Cl−. The Cl− channel demonstrated strong voltage dependence of activation (half-maximum of mean open probability, V0.5, ~−20 mV) and robust prolonged openings at depolarizing voltages. The channel displayed similar gating when exposed intracellularly to solutions containing Ca2+-free or 1 mM Ca2+. In whole cell patch-clamp recordings, macroscopic current demonstrated outward rectification, inhibitions by 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) and niflumic acid, and insensitivity to chlorotoxin. The outward current was reversibly reduced by 94% replacement of extracellular Cl− with I−, Br−, or methanesulfonate (MsO−), resulting in anionic permeability sequence: Cl−>Br−>I−>MsO−. While intracellular Ca2+ levels (0, 300 nM, and 1 mM) did not affect the amplitude of Cl− current and outward rectification, high Ca2+ slowed voltage-step current activation at depolarizing voltages. In conclusion, our data reveal for the first time the presence of a Ca2+-independent DIDS and niflumic acid-sensitive, voltage-dependent Cl− channel in the plasma membrane of DSM cells. This channel may be a key regulator of DSM excitability.

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