scholarly journals Small-conductance Ca2+-dependent K+ channels activated by ATP in murine colonic smooth muscle

1997 ◽  
Vol 273 (6) ◽  
pp. C2010-C2021 ◽  
Author(s):  
S. D. Koh ◽  
G. M. Dick ◽  
K. M. Sanders
Keyword(s):  
Smooth Muscle ◽  
Pyridoxal Phosphate ◽  
Single Channel ◽  
Outward Current ◽  
Disulfonic Acid ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Outward Currents ◽  
Murine Colon ◽  
Small Conductance

The patch-clamp technique was used to determine the ionic conductances activated by ATP in murine colonic smooth muscle cells. Extracellular ATP, UTP, and 2-methylthioadenosine 5′-triphosphate (2-MeS-ATP) increased outward currents in cells with amphotericin B-perforated patches. ATP (0.5–1 mM) did not affect whole cell currents of cells dialyzed with solutions containing ethylene glycol-bis(β-aminoethyl ether)- N, N, N′, N′-tetraacetic acid. Apamin (3 × 10−7M) reduced the outward current activated by ATP by 32 ± 5%. Single channel recordings from cell-attached patches showed that ATP, UTP, and 2-MeS-ATP increased the open probability of small-conductance, Ca2+-dependent K+ channels with a slope conductance of 5.3 ± 0.02 pS. Caffeine (500 μM) enhanced the open probability of the small-conductance K+ channels, and ATP had no effect after caffeine. Pyridoxal phosphate 6-azophenyl-2′,4′-disulfonic acid tetrasodium (PPADS, 10−4 M), a nonselective P2 receptor antagonist, prevented the increase in open probability caused by ATP and 2-MeS-ATP. PPADS had no effect on the response to caffeine. ATP-induced hyperpolarization in the murine colon may be mediated by P2y-induced release of Ca2+ from intracellular stores and activation of the 5.3-pS Ca2+-activated K+ channels.

scholarly journals Effect of Auxin (IAA) on the Fast Vacuolar (FV) Channels in Red Beet (Beta vulgaris L.) Taproot Vacuoles

2020 ◽  
Vol 21 (14) ◽  
pp. 4876
Author(s):  
Zbigniew Burdach ◽  
Agnieszka Siemieniuk ◽  
Waldemar Karcz
Keyword(s):  
Single Channel ◽  
Outward Current ◽  
K Channel ◽  
Single Channels ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Inward Current ◽  
Bath Solution ◽  
Outward Currents ◽  
Inward Currents

In contrast to the well-studied effect of auxin on the plasma membrane K+ channel activity, little is known about the role of this hormone in regulating the vacuolar K+ channels. Here, the patch-clamp technique was used to investigate the effect of auxin (IAA) on the fast-activating vacuolar (FV) channels. It was found that the macroscopic currents displayed instantaneous currents, which at the positive potentials were about three-fold greater compared to the one at the negative potentials. When auxin was added to the bath solution at a final concentration of 1 µM, it increased the outward currents by about 60%, but did not change the inward currents. The imposition of a ten-fold vacuole-to-cytosol KCl gradient stimulated the efflux of K+ from the vacuole into the cytosol and reduced the K+ current in the opposite direction. The addition of IAA to the bath solution with the 10/100 KCl gradient decreased the outward current and increased the inward current. Luminal auxin reduced both the outward and inward current by approximately 25% compared to the control. The single channel recordings demonstrated that cytosolic auxin changed the open probability of the FV channels at the positive voltages to a moderate extent, while it significantly increased the amplitudes of the single channel outward currents and the number of open channels. At the positive voltages, auxin did not change the unitary conductance of the single channels. We suggest that auxin regulates the activity of the fast-activating vacuolar (FV) channels, thereby causing changes of the K+ fluxes across the vacuolar membrane. This mechanism might serve to tightly adjust the volume of the vacuole during plant cell expansion.

Nitric oxide mediates outward potassium currents in opossum esophageal circular smooth muscle

1996 ◽  
Vol 270 (6) ◽  
pp. G932-G938 ◽  
Author(s):  
J. Jury ◽  
K. R. Boev ◽  
E. E. Daniel
Keyword(s):  
Nitric Oxide ◽  
Smooth Muscle ◽  
Circular Muscle ◽  
Outward Current ◽  
Equilibrium Potential ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Whole Cell ◽  
Circular Smooth Muscle ◽  
Outward Currents

Single smooth muscle cells from the opossum body circular muscle were isolated and whole cell currents were characterized by the whole cell patch-clamp technique. When the cells were held at -50 mV and depolarized to 70 mV in 20-mV increments, initial small inactivating inward currents were evoked (-30 to 30 mV) followed by larger sustained outward currents. Depolarization from a holding potential of -90 mV evoked an initial fast inactivating outward current sensitive to 4-aminopyridine but not to high levels of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). The outward currents reversed near K+ equilibrium potential and were abolished when KCl was replaced by CsCl in the pipette solution. The sustained outward current was inhibited by quinine and cesium. High EGTA in the pipette solution reduced but did not abolish the sustained outward currents, suggesting that both Ca(2+)-dependent and -independent currents were evoked. The nitric oxide (NO)-releasing agents Sin-1 and sodium nitroprusside increased outward K+ currents. High levels of EGTA in the pipette solution abolished the increase in outward current induced by Sin-1. The presence of cyclopiazonic acid, an inhibitor of the sarcoplasmic reticulum (SR) Ca2+ pump, blocked the effects of NO-releasing agents. We conclude that NO release activates K+ outward currents in opossum esophagus circular muscle, which may depend on Ca2+ release from the SR stores.

Transient potassium currents in avian sensory neurons

1990 ◽  
Vol 63 (4) ◽  
pp. 725-737 ◽  
Author(s):  
S. K. Florio ◽  
C. D. Westbrook ◽  
M. R. Vasko ◽  
R. J. Bauer ◽  
J. L. Kenyon
Keyword(s):  
Single Channel ◽  
Outward Current ◽  
Potassium Currents ◽  
Delayed Rectifier ◽  
Transient Outward Current ◽  
Single Channels ◽  
Patch Clamp Technique ◽  
Small Component ◽  
Whole Cell ◽  
Outward Currents

1. We used the patch-clamp technique to study voltage-activated transient potassium currents in freshly dispersed and cultured chick dorsal root ganglion (DRG) cells. Whole-cell and cell-attached patch currents were recorded under conditions appropriate for recording potassium currents. 2. In whole-cell experiments, 100-ms depolarizations from normal resting potentials (-50 to -70 mV) elicited sustained outward currents that inactivated over a time scale of seconds. We attribute this behavior to a component of delayed rectifier current. After conditioning hyperpolarizations to potentials negative to -80 mV, depolarizations elicited transient outward current components that inactivated with time constants in the range of 8-26 ms. We attribute this behavior to a transient outward current component. 3. Conditioning hyperpolarizations increased the rate of activation of the net outward current implying that the removal of inactivation of the transient outward current allows it to contribute to early outward current during depolarizations from negative potentials. 4. Transient current was more prominent on the day the cells were dispersed and decreased with time in culture. 5. In cell-attached patches, single channels mediating outward currents were observed that were inactive at resting potentials but were active transiently during depolarizations to potentials positive to -30 mV. The probability of channels being open increased rapidly (peaking within approximately 6 ms) and then declined with a time constant in the range of 13-30 ms. With sodium as the main extracellular cation, single-channel conductances ranged from 18 to 32 pS. With potassium as the main extracellular cation, the single-channel conductance was approximately 43 pS, and the channel current reversed near 0 mV, as expected for a potassium current. 6. We conclude that the transient potassium channels mediate the component of transient outward current seen in the whole-cell experiments. This current is a relatively small component of the net current during depolarizations from normal resting potentials, but it can contribute significant outward current early in depolarizations from hyperpolarized potentials.

scholarly journals A Comparison of the Effect of Lead (Pb) on the Slow Vacuolar (SV) and Fast Vacuolar (FV) Channels in Red Beet (Beta vulgaris L.) Taproot Vacuoles

2021 ◽  
Vol 22 (23) ◽  
pp. 12621
Author(s):  
Agnieszka Siemieniuk ◽  
Zbigniew Burdach ◽  
Waldemar Karcz
Keyword(s):  
Single Channel ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Outward Currents ◽  
Inward Currents ◽  
Sv Channels ◽  
The One ◽  
The Impact

Little is known about the effect of lead on the activity of the vacuolar K+ channels. Here, the patch-clamp technique was used to compare the impact of lead (PbCl2) on the slow-activating (SV) and fast-activating (FV) vacuolar channels. It was revealed that, under symmetrical 100-mM K+, the macroscopic currents of the SV channels exhibited a typical slow activation and a strong outward rectification of the steady-state currents, while the macroscopic currents of the FV channels displayed instantaneous currents, which, at the positive potentials, were about three-fold greater compared to the one at the negative potentials. When PbCl2 was added to the bath solution at a final concentration of 100 µM, it decreased the macroscopic outward currents of both channels but did not change the inward currents. The single-channel recordings demonstrated that cytosolic lead causes this macroscopic effect by a decrease of the single-channel conductance and decreases the channel open probability. We propose that cytosolic lead reduces the current flowing through the SV and FV channels, which causes a decrease of the K+ fluxes from the cytosol to the vacuole. This finding may, at least in part, explain the mechanism by which cytosolic Pb2+ reduces the growth of plant cells.

scholarly journals Heterogeneity and Remodeling of Ion Currents in Cultured Right Atrial Fibroblasts From Patients With Sinus Rhythm or Atrial Fibrillation

2021 ◽  
Vol 12 ◽  
Author(s):  
Dorothee Jakob ◽  
Alexander Klesen ◽  
Elisa Darkow ◽  
Fabian A. Kari ◽  
Friedhelm Beyersdorf ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Patch Clamp ◽  
Sinus Rhythm ◽  
Cardiac Electrophysiology ◽  
Single Channel ◽  
Right Atrial ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Cultured Fibroblasts ◽  
Outward Currents

Cardiac fibroblasts express multiple voltage-dependent ion channels. Even though fibroblasts do not generate action potentials, they may influence cardiac electrophysiology by electrical coupling via gap junctions with cardiomyocytes, and through fibrosis. Here, we investigate the electrophysiological phenotype of cultured fibroblasts from right atrial appendage tissue of patients with sinus rhythm (SR) or atrial fibrillation (AF). Using the patch-clamp technique in whole-cell mode, we observed steady-state outward currents exhibiting either no rectification or inward and/or outward rectification. The distributions of current patterns between fibroblasts from SR and AF patients were not significantly different. In response to depolarizing voltage pulses, we measured transient outward currents with fast and slow activation kinetics, an outward background current, and an inward current with a potential-dependence resembling that of L-type Ca2+ channels. In cell-attached patch-clamp mode, large amplitude, paxilline-sensitive single channel openings were found in ≈65% of SR and ∼38% of AF fibroblasts, suggesting the presence of “big conductance Ca2+-activated K+ (BKCa)” channels. The open probability of BKCa was significantly lower in AF than in SR fibroblasts. When cultured in the presence of paxilline, the shape of fibroblasts became wider and less spindle-like. Our data confirm previous findings on cardiac fibroblast electrophysiology and extend them by illustrating differential channel expression in human atrial fibroblasts from SR and AF tissue.

Purinergic activation of spontaneous transient outward currents in guinea pig taenia colonic myocytes

2000 ◽  
Vol 278 (2) ◽  
pp. C352-C362 ◽  
Author(s):  
In Deok Kong ◽  
Sang Don Koh ◽  
Kenton M. Sanders
Keyword(s):  
Guinea Pig ◽  
Pyridoxal Phosphate ◽  
Receptor Blocker ◽  
Disulfonic Acid ◽  
Sk Channels ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Outward Currents ◽  
Relaxation Responses ◽  
Spontaneous Transient Outward Currents

Spontaneous transient outward currents (STOCs) were recorded from smooth muscle cells of the guinea pig taenia coli using the whole cell patch-clamp technique. STOCs were resolved at potentials positive to −50 mV. Treating cells with caffeine (1 mM) caused a burst of outward currents followed by inhibition of STOCs. Replacing extracellular Ca2+ with equimolar Mn2+ caused STOCs to “run down.” Iberiotoxin (200 nM) or charybdotoxin (ChTX; 200 nM) inhibited large-amplitude STOCs, but small-amplitude “mini-STOCs” remained in the presence of these drugs. Mini-STOCs were reduced by apamin (500 nM), an inhibitor of small-conductance Ca2+-activated K+ channels (SK channels). Application of ATP or 2-methylthioadenosine 5′-triphosphate (2-MeS-ATP) increased the frequency of STOCs. The effects of 2-MeS-ATP persisted in the presence of charybdotoxin but were blocked by combination of ChTX (200 nM) and apamin (500 nM). 2-MeS-ATP did not increase STOCs in the presence of pyridoxal phosphate 6-azophenyl-2′,4′-disulfonic acid, a P2 receptor blocker. Similarly, pretreatment of cells with U-73122 (1 μM), an inhibitor of phospholipase C (PLC), abolished the effects of 2-MeS-ATP. Xestospongin C, an inositol 1,4,5-trisphosphate (IP3) receptor blocker, attenuated STOCs, but these events were not affected by ryanodine. The data suggest that purinergic activation through P2Y receptors results in localized Ca2+ release via PLC- and IP3-dependent mechanisms. Release of Ca2+ is coupled to STOCs, which are composed of currents mediated by large-conductance Ca2+-activated K+ channels and SK channels. The latter are thought to mediate hyperpolarization and relaxation responses of gastrointestinal muscles to inhibitory purinergic stimulation.

Potassium current in circular smooth muscle of human jejunum activated by fenamates

1993 ◽  
Vol 265 (5) ◽  
pp. G873-G879 ◽  
Author(s):  
G. Farrugia ◽  
J. L. Rae ◽  
M. G. Sarr ◽  
J. H. Szurszewski
Keyword(s):  
Smooth Muscle ◽  
Membrane Potential ◽  
Outward Current ◽  
Major Determinant ◽  
Flufenamic Acid ◽  
Blood Levels ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Circular Smooth Muscle ◽  
Normal Human

Thirty-eight cells, freshly isolated from circular smooth muscle of normal human jejunum obtained from nine patients undergoing weight-reduction surgery for morbid obesity, were patch clamped using a perforated patch-clamp technique. A highly potassium-selective voltage-dependent outward current was present in all cells. The current was carried by a 220-pS channel that activated near -75 mV and reached unit open probability at about +10 mV. Blockade of the current by quinidine (50 microM) and tetraethylammonium (25 mM) was accompanied by membrane depolarization to 0 to -3 mV, suggesting that this current was the major determinant of the membrane potential. Flufenamic and mefenamic acid at concentrations comparable with blood levels reached when these drugs are used in clinical therapy as nonsteroidal anti-inflammatory agents, activated the potassium outward current and hyperpolarized the membrane potential. The shift in the membrane potential for 250 microM flufenamic acid was -36 +/- 24 (SD) mV. Activation was rapid (seconds) and reversible. It was concluded that normal human jejunal circular smooth muscle cells have a highly potassium-selective outward current, which is the major determinant of the membrane potential and which is activated by fenamates.

Ion channels in rabbit cultured fibroblasts

1986 ◽  
Vol 227 (1246) ◽  
pp. 1-16 ◽  
Keyword(s):  
Calcium Concentration ◽  
Voltage Dependence ◽  
Single Channel ◽  
Outward Current ◽  
Patch Clamp Technique ◽  
Cultured Fibroblasts ◽  
Whole Cell ◽  
Whole Cell Patch Clamp ◽  
Outward Currents ◽  
High Conductance

Large outward currents are recorded with the whole-cell patch-clamp technique on depolarization of rabbit cultured fibroblasts. Our findings suggest that these outward currents consist of two voltage-dependent components, one of which also depends on cytoplasmic calcium concentration. Total replacement of external Cl - by the large anion ascorbate does not affect the amplitude of the currents, indicating that both components must be carried by K + . Consistent with these findings with whole-cell currents, in single channel recordings from fibroblasts we found that most patches contain high-conductance potassium-selective channels whose activation depends on both membrane potential and the calcium concentration at the cytoplasmic surface of the membrane. In a smaller number of patches, a second population of high-conductance calcium-independent potassium channels is observed having different voltage-dependence. The calcium- and voltage-dependence suggest that these two channels correspond with the two components of outward current seen in the whole-cell recordings. The single channel conductance of both channels in symmetrical KCl (150 mM) is 260-270 pS. Both channels are highly selective for K + over both Na + and Cl - . The conductance of the channels when outward current is carried by Rb + is considerably smaller than when it is carried by K + . Some evidence is adduced to support the hypothesis that these potassium channel populations may be involved in the control of cell proliferation.

Effect of purinergic blockers on outward current in isolated smooth muscle cells of the sheep bladder

1996 ◽  
Vol 270 (3) ◽  
pp. C969-C973 ◽  
Author(s):  
K. D. Cotton ◽  
M. A. Hollywood ◽  
K. D. Thornbury ◽  
N. G. McHale
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Outward Current ◽  
Muscle Cells ◽  
Maximal Response ◽  
Physiological Salt Solution ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Cibacron Blue

Freshly dispersed cells from sheep urinary bladder were voltage clamped using the whole cell and inside-out patch-clamp technique. Cibacron and Basilen blue increased outward current in a dose-dependent manner with a half-maximal response at 10(-5)M. Suramin, in concentrations to 10(-3)M, had no such effect. The Cibacron blue response was abolished in Ca2+ -free physiological salt solution, suggesting that it was acting on a Ca2+ -dependent current. Similarly, the Cibacron blue-sensitive current was significantly attenuated by charybdotoxin. Cibacron blue did not modulate inward current nor were its effects modified by caffeine or heparin, suggesting that its effect on outward current was not secondary to an increase in intracellular Ca2+. Application of 10(-4)M Cibacron blue to the inside membrane of excised patches caused a rapid increase in open probability of a large conductance (300 pS) K+ channel. These results suggest that Cibacron blue is a potent activator of a Ca2+ -dependent outward current in bladder smooth muscle cells in addition to its action as a purinergic blocker.

Chemical modification of Ca(2+)-activated potassium channels of GH3 anterior pituitary cells

1992 ◽  
Vol 263 (5) ◽  
pp. C1081-C1087 ◽  
Author(s):  
A. M. Frace ◽  
D. C. Eaton
Keyword(s):  
Single Channel ◽  
Gh3 Cells ◽  
Disulfonic Acid ◽  
State Transitions ◽  
Trinitrobenzene Sulfonic Acid ◽  
Voltage Sensitivity ◽  
Pituitary Cells ◽  
Amino Groups ◽  
Open Probability ◽  
Long Duration

The effects of amino group specific reagents were examined on single, large-conductance, Ca(2+)-activated, K+ channels in excised membrane patches from GH3 cells. The reagents used include trinitrobenzene sulfonic acid, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and its 4-acetamido derivative, and sulfophenyl-isothiocyanate. These reagents react covalently with peptide terminal amino groups and the epsilon amino groups of lysine residues, thereby removing positive charge. Internal application of 0.1-1.0 mM reagent to inside-out patches irreversibly increases channel open probability. Single-channel conductance and voltage sensitivity are not affected by modification. Analysis of channel openings and closures shows that the increase in open probability is predominantly due to the loss of long-duration closures of the channel; however, the lengths of long-duration openings are increased. After the modification in the presence of Ca2+ was performed, the channel open probability remains large, regardless of the internal Ca2+ concentration. Transitions among several open and closed states of the modified channel are present in the absence of Ca2+, suggesting that many state transitions are not directly dependent on Ca2+ binding or dissociation.

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