Isoflurane Decreases ATP Sensitivity of Guinea Pig Cardiac Sarcolemmal KATPChannel at Reduced Intracellular pH

2003 ◽  
Vol 98 (2) ◽  
pp. 396-403 ◽  
Author(s):  
Anna Stadnicka ◽  
Zeljko J. Bosnjak
Keyword(s):  
Guinea Pig ◽  
Intracellular Ph ◽  
Single Channel ◽  
Volatile Anesthetics ◽  
Dependent Manner ◽  
Channel Activity ◽  
Open Probability ◽  
Concentration Dependent Manner ◽  
Channel Interaction ◽  
Channel Opening

Background Volatile anesthetics can protect the myocardium against ischemic injury by opening the adenosine triphosphate (ATP)-sensitive potassium (K(atp)) channels. However, direct evidence for anesthetic-channel interaction is still limited, and little is known about the role K(atp) channel modulators play in this effect. Because pH is one of the regulators of K(atp) channels, the authors tested the hypothesis that intracellular pH (pHi) modulates the direct interaction of isoflurane with the cardiac K(atp) channel. Methods The effects of isoflurane on sarcolemmal K(atp) channels were investigated at pHi 7.4 and pHi 6.8 in excised inside-out membrane patches from ventricular myocytes of guinea pig hearts. Results At pHi 7.4, intracellular ATP (1-1,000 microm) inhibited K(atp) channels and decreased channel open probability (Po) in a concentration-dependent manner with an IC(50) of 8 +/- 1.5 microm, and isoflurane (0.5 mm) either had no effect or decreased channel activity. Lowering pHi from 7.4 to 6.8 enhanced channel opening by increasing Po and reduced channel sensitivity to ATP, with IC shifting from 8 +/- 1.2 to 45 +/- 5.6 microm. When applied to the channels activated at pHi 6.8, isoflurane (0.5 mm) increased Po and further reduced ATP sensitivity, shifting IC(50) to 110 +/- 10.0 microm. Conclusions Changes in pHi appear to modulate isoflurane interaction with the cardiac K(atp) channel. At pHi 6.8, which itself facilitates channel opening, isoflurane enhances channel activity by increasing Po and reduces sensitivity to inhibition by ATP without changing the unitary amplitude of single channel current or the conductance. These results support the hypothesis of direct isoflurane-K(atp) channel interaction that may play a role in cardioprotection by volatile anesthetics.

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.

Modification of the gating of the cardiac sarcoplasmic reticulum Ca(2+)-release channel by H2O2 and dithiothreitol

1994 ◽  
Vol 267 (3) ◽  
pp. H1010-H1016 ◽  
Author(s):  
A. Boraso ◽  
A. J. Williams
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Single Channel ◽  
Inhibitory Response ◽  
Dependent Manner ◽  
Open Probability ◽  
Release Channel ◽  
Concentration Dependent Manner ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Gating Mechanism ◽  
Cytosolic Side

The effect of hydrogen peroxide (H2O2) on the sheep cardiac sarcoplasmic reticulum (SR) Ca(2+)-release channel has been investigated under voltage-clamp conditions after incorporation of native membrane vesicles into planar phospholipid bilayers. In the presence of micromolar activating calcium concentrations on the cytosolic side of the membrane, H2O2 (3-5 mM) increased open probability of the channels. H2O2 did not affect the conductance of the channel or the response to activating compounds, such as ATP and caffeine. H2O2 did not alter the inhibitory response to magnesium or the modification of channels by ryanodine. At subactivating calcium concentrations (approximately 45 pM) on the cytosolic side of the membrane, 5 mM H2O2 was still able to open the channel. Analysis of single-channel open and closed lifetimes suggested that H2O2 had a direct effect on the gating mechanism of the channel. Open probability of the SR Ca(2+)-release channel is reduced by millimolar concentrations of dithiothreitol, a sulfhydryl-protecting compound, in a concentration-dependent manner. In conclusion, it is probable that H2O2 activates the SR Ca(2+)-release channel via an oxidation of cysteine thiol groups in the channel protein.

scholarly journals On the Interaction of Neomycin with the Slow Vacuolar Channel of Arabidopsis thaliana

2006 ◽  
Vol 127 (3) ◽  
pp. 329-340 ◽  
Author(s):  
Joachim Scholz-Starke ◽  
Armando Carpaneto ◽  
Franco Gambale
Keyword(s):  
Arabidopsis Thaliana ◽  
Single Channel ◽  
Aminoglycoside Antibiotic ◽  
Dependent Manner ◽  
Open Probability ◽  
Mesophyll Cells ◽  
Concentration Dependent Manner ◽  
Slow Vacuolar Channel ◽  
Sv Channel

This study investigates the interaction of the aminoglycoside antibiotic neomycin with the slow vacuolar (SV) channel in vacuoles from Arabidopsis thaliana mesophyll cells. Patch-clamp experiments in the excised patch configuration revealed a complex pattern of neomycin effects on the channel: applied at concentrations in the submicromolar to millimolar range neomycin (a) blocked macroscopic SV currents in a voltage- and concentration-dependent manner, (b) slowed down activation and deactivation kinetics of the channel, and most interestingly, (c) at concentrations above 10 μM, neomycin shifted the SV activation threshold towards negative membrane potentials, causing a two-phasic activation at high concentrations. Single channel experiments showed that neomycin causes these macroscopic effects by combining a decrease of the single channel conductance with a concomitant increase of the channel's open probability. Our results clearly demonstrate that the SV channel can be activated at physiologically relevant tonoplast potentials in the presence of an organic effector molecule. We therefore propose the existence of a cellular equivalent regulating the activity of the SV channel in vivo.

Large-conductance Ca2+-activated potassium channel in mitochondria of endothelial EA.hy926 cells

2013 ◽  
Vol 304 (11) ◽  
pp. H1415-H1427 ◽  
Author(s):  
Piotr Bednarczyk ◽  
Agnieszka Koziel ◽  
Wieslawa Jarmuszkiewicz ◽  
Adam Szewczyk
Keyword(s):  
Potassium Channel ◽  
Mitochondrial Membrane ◽  
Single Channel ◽  
Dependent Manner ◽  
Channel Activity ◽  
Inner Mitochondrial Membrane ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Α Subunit ◽  
Ea.Hy926 Cells

In the present study, we describe the existence of a large-conductance Ca2+-activated potassium (BKCa) channel in the mitochondria of the human endothelial cell line EA.hy926. A single-channel current was recorded from endothelial mitoplasts (i.e., inner mitochondrial membrane) using the patch-clamp technique in the mitoplast-attached mode. A potassium-selective current was recorded with a mean conductance equal to 270 ± 10 pS in a symmetrical 150/150 mM KCl isotonic solution. The channel activity, which was determined as the open probability, increased with the addition of calcium ions and the potassium channel opener NS1619. Conversely, the activity of the channel was irreversibly blocked by paxilline and iberiotoxin, BKCa channel inhibitors. The open-state probability was found to be voltage dependent. The substances known to modulate BKCa channel activity influenced the bioenergetics of mitochondria isolated from human endothelial EA.hy926 cells. In isolated mitochondria, 100 μM Ca2+, 10 μM NS1619, and 0.5 μM NS11021 depolarized the mitochondrial membrane potential and stimulated nonphosphorylating respiration. These effects were blocked by iberiotoxin and paxilline in a potassium-dependent manner. Under phosphorylating conditions, NS1619-induced, iberiotoxin-sensitive uncoupling diverted energy from ATP synthesis during the phosphorylating respiration of the endothelial mitochondria. Immunological analysis with antibodies raised against proteins of the plasma membrane BKCa channel identified a pore-forming α-subunit and an auxiliary β2-subunit of the channel in the endothelial mitochondrial inner membrane. In conclusion, we show for the first time that the inner mitochondrial membrane in human endothelial EA.hy926 cells contains a large-conductance calcium-dependent potassium channel with properties similar to those of the surface membrane BKCa channel.

Propofol Inhibits Cardiac L-Type Calcium Current in Guinea Pig Ventricular Myocytes

1996 ◽  
Vol 84 (3) ◽  
pp. 626-635 ◽  
Author(s):  
Ching-Yue Yang ◽  
Chih-Shung Wong ◽  
Chuan-Cheng Yu ◽  
Hsiang-Ning Luk ◽  
Cheng-I Lin
Keyword(s):  
Steady State ◽  
Guinea Pig ◽  
Time Course ◽  
Single Channel ◽  
Ventricular Myocytes ◽  
Hill Coefficient ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Inactivation Curve ◽  
Whole Cell

Background Propofol may exert negative inotropic and chronotropic actions in the heart. Single-channel studies show that propofol affects the kinetics of opening and closing of cardiac L-type calcium channels (ICa(L)) without altering channel conductance. The aim of this study was to investigate the mechanisms of depressant effects of propofol on cardiac whole-cell ICa(L). Methods Single ventricular myocytes were freshly dissciated from guinea pig hearts using enzymatic isolation. One-suction electrode voltage-clamp technique (whole-cell mode) was used. LCa(L) was separated from other contaminated ionic currents. Propofol was applied in the commercial 10% Intralipid emulsion formula (Zeneca, UK). Results In isolated cardiomyocytes, propofol significantly inhibited whole-cell ICa(L) in a concentration-dependent manner (K D = 52.0 microM; Hill coefficient = 1.3). The solvent (Intralipid) did not affect ICa(L). Propofol decreased ICa(L) at all potentials tested along the voltage axis and reduced the slope conductance. The threshold potential for activation and the peak potential of the current-voltage relationship were not changed by propofol. The steady-state activation curves overlapped in the absence and the presence of 56 microM propofol. In contrast, the steady-state inactivation curve was shifted in the hyperpolarizing direction. The time course of the recovery from inactivation was delayed by 56 microM propofol. The blocking action on ICa(L) of propofol shows marked resting block and use-dependent block. Propofol caused more pronounced inhibition at a higher stimulation frequency. The effect of propofol on the inactivation process was even more clear on ICa(L). Conclusions The authors conclude tha propofol, at supratherapeutic concentrations, inhibits cardiac ICa(L). This inhibition is mainly due to a shift of inactivation curve and a reduction in slope conductance.

Activation by Membrane Stretch and Depolarization of an Epithelial Monovalent Cation Channel from Teleost Intestine

1992 ◽  
Vol 169 (1) ◽  
pp. 87-104
Author(s):  
WENHAN CHANG ◽  
CHRISTOPHER A. LORETZ
Keyword(s):  
Single Channel ◽  
Applied Pressure ◽  
Cell Volume Regulation ◽  
Monovalent Cation ◽  
Dependent Manner ◽  
Membrane Tension ◽  
Channel Activity ◽  
Open Probability ◽  
Membrane Stretch ◽  
Inside Out

The intestine of euryhaline teleosts is an important osmoregulatory organ which actively absorbs Na+, Cl− and water from the lumen. This ion-transporting epithelium experiences a variety of physical stimuli resulting from variations in luminal osmolality and distension and from peristaltic contractions. Using patchclamp techniques in the inside-out configuration, single stretch-activated channels (SA channels) were identified and characterized. These SA channels had a conductance of about 67 pS in symmetrical solutions containing 140 mmoll−1 NaCl and were permeable to both Na+ and K+ (PNa/PK≈0.83) but not to anions. In excised, inside-out membrane patches, channel activity could be enhanced in the absence of membrane tension by strong depolarization of the membrane potential (Vm) to between 0 mV and + 90 mV, with Vo [Vm at which the single-channel open probability (Po)=0.5] at + 25.7 mV. In the presence of membrane tension, the voltage-dependence of channel activity was shifted into the physiological range of Vm. Each kPa (10 cmH2O) of applied pressure (ΔP) generated the same effect on Po as a membrane depolarization of 49 mV. Membrane tension also increased the single-channel current and single-channel conductance in a dose-dependent manner. The kinetic data suggest that this channel has two open states and three closed states. Both stretch- and depolarization-induced increases in Po were attributed to prolongation of the lifetime of the longer open state. Possible physiological roles for this channel include the cellular uptake of Na+ from the lumen as part of the salt and water absorptive process or a yet undefined involvement in cell volume regulation.

Ammonium ion enhances the calcium-dependent gating of a mammalian large conductance, calcium-sensitive K+ channel

2001 ◽  
Vol 79 (11) ◽  
pp. 919-923 ◽  
Author(s):  
Andrew P Braun
Keyword(s):  
Single Channel ◽  
Ammonium Ion ◽  
K Channel ◽  
Free Calcium ◽  
Dependent Manner ◽  
Open Probability ◽  
Concentration Dependent Manner ◽  
Hek 293 ◽  
Calcium Dependent ◽  
Single Channel Currents

We observed that the current amplitude and activation of expressed, mouse brain large conductance, calcium-sensitive K+ channels (BKCa channels) may be reversibly enhanced following addition of low concentrations of the weakly permeant cation NH4+ to the cytoplasmic face of the channel in excised, inside-out membrane patches from HEK 293 cells. Conductance-voltage relations were left-shifted along the voltage axis by addition of NH4Cl in a concentration-dependent manner, with an EC50 of 18.5 mM. Furthermore, this effect was observed in the presence of cytosolic free calcium (~1 µM), but was absent in a cytosolic bath solution containing nominally zero free calcium (e.g., 5 mM EGTA only), a condition under which these channels undergo largely voltage-dependent gating. Recordings of single BKCa channel events indicated that NH4+ increased the channel open probability of single channel activity ~3-fold, but did not alter the amplitude of single channel currents. These findings suggest that the calcium-sensitive gating of mammalian BKCa channels may be modified by other ions present in cytosolic solution.Key words: potassium channel, calcium, modulation, electrophysiology.

Clinically Relevant Concentrations of Propofol Have No Effect on Adenosine Triphosphate–sensitive Potassium Channels in Rat Ventricular Myocytes

2002 ◽  
Vol 96 (6) ◽  
pp. 1472-1477 ◽  
Author(s):  
Takashi Kawano ◽  
Shuzo Oshita ◽  
Yasuo Tsutsumi ◽  
Yoshinobu Tomiyama ◽  
Hiroshi Kitahata ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Adenosine Triphosphate ◽  
Single Channel ◽  
Ventricular Myocytes ◽  
Dependent Manner ◽  
Open Probability ◽  
Concentration Dependent Manner ◽  
Rat Ventricular Myocytes ◽  
Mitochondrial Oxidation ◽  
Inside Out

Background Activation of adenosine triphosphate-sensitive potassium (K(ATP)) channels produces cardioprotective effects during ischemia. Because propofol is often used in patients who have coronary artery disease undergoing a wide variety of surgical procedures, it is important to evaluate the direct effects of propofol on K(ATP) channel activities in ventricular myocardium during ischemia. Methods The effects of propofol (0.4-60.1 microg/ml) on both sarcolemmal and mitochondrial K(ATP) channel activities were investigated in single, quiescent rat ventricular myocytes. Membrane currents were recorded using cell-attached and inside-out patch clamp configurations. Flavoprotein fluorescence was measured to evaluate mitochondrial oxidation mediated by mitochondrial K(ATP) channels. Results In the cell-attached configuration, open probability of K(ATP) channels was reduced by propofol in a concentration-dependent manner (EC(50) = 14.2 microg/ml). In the inside-out configurations, propofol inhibited K(ATP) channel activities without changing the single-channel conductance (EC(50) = 11.4 microg/ml). Propofol reduced mitochondrial oxidation in a concentration-dependent manner with an EC(50) of 14.6 microg/ml. Conclusions Propofol had no effect on the sarcolemmal K(ATP) channel activities in patch clamp configurations and the mitochondrial flavoprotein fluorescence induced by diazoxide at clinically relevant concentrations (< 2 microm), whereas it significantly inhibited both K(ATP) channel activities at very high, nonclinical concentrations (> 5.6 microg/ml; 31 microm).

scholarly journals On the mechanism of basal and agonist-induced activation of the G protein-gated muscarinic K+ channel in atrial myocytes of guinea pig heart.

1991 ◽  
Vol 98 (3) ◽  
pp. 517-533 ◽  
Author(s):  
H Ito ◽  
T Sugimoto ◽  
I Kobayashi ◽  
K Takahashi ◽  
T Katada ◽  
...  
Keyword(s):  
Guinea Pig ◽  
G Protein ◽  
K Channel ◽  
Dependent Manner ◽  
Atrial Myocytes ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Concentration Dependent Manner ◽  
Guinea Pig Heart ◽  
Gamma S

Using the patch clamp technique, we examined the agonist-free, basal interaction between the muscarinic acetylcholine (m-ACh) receptor and the G protein (GK)-gated muscarinic K+ channel (IK.ACh), and the modification of this interaction by ACh binding to the receptor in single atrial myocytes of guinea pig heart. In the whole cell clamp mode, guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma S) gradually increased the IK.ACh current in the absence of agonists (e.g., acetylcholine). This increase was inhibited in cells that were pretreated with islet-activating protein (IAP, pertussis toxin) or N-ethylmaleimide (NEM). In inside-out patches, even in the absence of agonists, intracellular GTP caused openings of IK.ACh in a concentration-dependent manner in approximately 80% of the patches. Channel activation by GTP in the absence of agonist was much less than that caused by GTP-gamma S. The agonist-independent, GTP-induced activation of IK.ACh was inhibited by the A promoter of IAP (with nicotinamide adenine dinucleotide) or NEM. As the ACh concentration was increased, the GTP-induced maximal open probability of IK.ACh was increased and the GTP concentration for the half-maximal activation of IK.ACh was decreased. Intracellular GDP inhibited the GTP-induced openings of IK.ACh in a concentration-dependent fashion. The half-inhibition of IK.ACh openings occurred at a much lower concentration of GDP in the absence of agonists than in the presence of ACh. From these results, we concluded (a) that the interaction between the m-ACh receptor and GK is essential for basal stimulation of IK.ACh, and (b) that ACh binding to the receptor accelerates the turnover of GK and increases GK's affinity to GTP analogues over GDP.

Acute changes in channel density of amphibian diluting segment

1990 ◽  
Vol 259 (6) ◽  
pp. C1005-C1009 ◽  
Author(s):  
A. M. Hurst ◽  
M. Hunter
Keyword(s):  
Intracellular Ph ◽  
Single Channel ◽  
Potassium Conductance ◽  
Distal Tubule ◽  
Channel Activity ◽  
Open Probability ◽  
Diluting Segment ◽  
Potassium Hydrogen ◽  
Distal Tubules ◽  
Acute Changes

Intracellular pH is a well-established modulator of the apical membrane potassium conductance of the amphibian diluting segment (early distal tubule). We investigated the modulation of this apical potassium conductance at the single-channel level in everted early distal tubules of the frog. Alkalinization of the bath fluid increased mean channel open probability (NPo) both in the presence and absence of the potassium-hydrogen ionophore nigericin. Reciprocal changes were seen with acidification. Because these effects were observed in cell-attached patches, where the composition of the fluid in the pipette is assumed to remain constant, the observed changes in channel activity were attributed to changes in intracellular pH. Further analysis of the data revealed that the changes in channel activity were produced exclusively by changes in the functional number of channels within the patch (N). We were unable to detect any significant changes in the single-channel open probability (Po). This suggests that the density of channels within a membrane may be far more dynamic than previously assumed.

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