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.

Mechanism of the negative inotropic effect of adenosine in guinea pig atrial myocytes

1994 ◽  
Vol 267 (6) ◽  
pp. H2420-H2429
Author(s):  
D. Wang ◽  
L. Belardinelli
Keyword(s):  
Guinea Pig ◽  
Negative Inotropic Effect ◽  
Inotropic Effect ◽  
Dependent Manner ◽  
Atrial Myocytes ◽  
Patch Clamp Technique ◽  
Concentration Dependent Manner ◽  
Whole Cell Patch Clamp ◽  
K Current ◽  
Highly Correlated

The ionic basis of the negative inotropic effect of adenosine on guinea pig atrial myocytes was studied. Membrane potentials and currents were measured using a whole cell patch-clamp technique. The contractility was assessed by video quantitation of cell twitch amplitude. Adenosine shortened action potential duration [measured at 90% repolarization (APD90)] and decreased twitch amplitude in a concentration-dependent manner. The maximal effects of adenosine (100 microM) were to reduce APD90 from 102 +/- 14 to 34 +/- 8 ms (n = 11) and twitch amplitude from 4.3 +/- 0.9 to 1.5 +/- 0.4 microns (n = 8). The concentration of adenosine that caused one-half of the maximal reductions of twitch amplitude and of APD90 was 0.6 microM. Reductions in APD90 and in twitch amplitude were parallel and highly correlated (r = 0.98). Decreases in twitch amplitude by adenosine could be mimicked by application of voltage-clamp pulses with durations similar to the durations of action potentials in the presence of adenosine. Clamp pulse could reverse adenosine-induced but not cadmium chloride-induced decreases in twitch amplitude. Adenosine activated the inwardly rectifying K+ current (IK,Ado), but did not significantly decrease the L-type Ca2+ current (ICa,L). Adenosine reduced the effects of BAY K 8644 on APD90 and twitch amplitude but did not attenuate the BAY K-induced increase in ICa,L. The effects of adenosine on APD90 and twitch amplitude could be reversed after activation of IK,Ado was inhibited by intracellular application of cesium and tetraethylammonium chloride.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Involvement of A pertussis Toxin Sensitive G-Protein in the Inhibition of Inwardly Rectifying K+Currents by Platelet-Activating Factor in Guinea-Pig Atrial Cardiomyocytes

1994 ◽  
Vol 3 (1) ◽  
pp. 45-51
Author(s):  
M. Gollasch ◽  
T. Kleppisch ◽  
D. Krautwurst ◽  
D. Lewinsohn ◽  
J. Hescheler
Keyword(s):  
Cyclic Amp ◽  
Guinea Pig ◽  
G Protein ◽  
Pertussis Toxin ◽  
Platelet Activating Factor ◽  
Resting Membrane Potential ◽  
Patch Clamp Technique ◽  
Guinea Pig Heart ◽  
Ventricular Cells ◽  
Inwardly Rectifying

Platelet-activating factor (PAF) inhibits single inwardly rectifying K+channels in guinea-pig ventricular cells. There is currently little information as to the mechanism by which these channels are modulated. The effect of PAF on quasi steady-state inwardly rectifying K+currents (presumably of the IK1type) of auricular, atrial and ventricular cardiomyocytes from guinea-pig were studied. Applying the patch-clamp technique in the whole-cell configuration, PAF (10 nM) reduced the K+currents in all three cell types. The inhibitory effect of PAF occurred within seconds and was reversible upon wash-out. It was almost completely abolished by the PAF receptor antagonist BN 50730. Intracellular infusion of atrial cells with guanine 5′-(β-thio)diphosphate (GDPS) or pretreatment of cells with pertussis toxin abolished the PAF dependent reduction of the currents. Neither extracellularly applied isoproterenol nor intracellularly applied adenosine 3′,5′-cyclic monophosphate (cyclic AMP) attenuated the PAF effect. In multicellular preparations of auricles, PAF (10 nM) induced arrhythmias. The arrhythmogenic activity was also reduced by BN 50730. The data indicate that activated PAF receptors inhibit inwardly rectifying K+currents via a pertussis toxin sensitive G-protein without involvement of a cyclic AMP-dependent step. Since IK1is a major component in stabilizing the resting membrane potential, the observed inhibition of this type of channel could play an important role in PAF dependent arrhythmogenesis in guinea-pig heart.

scholarly journals Glibenclamide Specifically Blocks ATP-Sensitive K<+ Channel Current in Atrial Myocytes of Guinea Pig Heart

1990 ◽  
Vol 54 (4) ◽  
pp. 473-477 ◽  
Author(s):  
Eiji HAMADA ◽  
Reiko TAKIKAWA ◽  
Hiroyuki ITO ◽  
Mari IGUCHI ◽  
Akira TERANO ◽  
...  
Keyword(s):  
Guinea Pig ◽  
K Channel ◽  
Atrial Myocytes ◽  
Guinea Pig Heart ◽  
Channel Current

Muscarinic inhibition of cardiac norepinephrine and neuropeptide Y release during ischemia and reperfusion

1994 ◽  
Vol 267 (6) ◽  
pp. R1552-R1558 ◽  
Author(s):  
A. Haunstetter ◽  
M. Haass ◽  
X. Yi ◽  
C. Kruger ◽  
W. Kubler
Keyword(s):  
Guinea Pig ◽  
Neuropeptide Y ◽  
Stellate Ganglion ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Muscarinic Agonists ◽  
Concentration Dependent Manner ◽  
Guinea Pig Heart ◽  
Specific Antagonist ◽  
Stop Flow

It was the aim of the present study to characterize the modulatory effect of muscarinic agonists on the overflow of norepinephrine and neuropeptide Y (NPY) from the in situ perfused guinea pig heart, induced by electrical stimulation of the left stellate ganglion (6 Hz, 5 V, 1 min). The muscarinic agonists oxotremorine (0.01-1 microM) and carbachol (0.1-10 microM) reduced norepinephrine and NPY overflow in a concentration-dependent manner to approximately 30% of control. The inhibitory effect of carbachol was antagonized by the unspecific muscarinic antagonist atropine (1 microM) but not by the nicotinic antagonist hexamethonium (100 microM). The M2-specific antagonist AF-DX-116BS was 25 times more potent than the M1-specific antagonist pirenzepine in antagonizing the inhibitory effect of carbachol [50% inhibitory concentration (IC50) = 0.2 microM for AF-DX-116BS; IC50 = 5.0 microM for pirenzepine]. These findings indicate that presynaptic muscarinic inhibition of stimulated norepinephrine and NPY release from the guinea pig heart is mediated mainly by activation of M2 receptors. As early as 2 min after stop-flow ischemia, the inhibitory effect of carbachol (10 microM) on the stimulation-evoked overflow of norepinephrine and NPY was lost. On reperfusion with oxygenated buffer after 10 min of stop-flow ischemia the inhibitory effect of carbachol (10 microM) on stimulation-induced norepinephrine and NPY overflow recovered within 3 min.

Acetylcholine-sensitive potassium channels in human atrial myocytes

1990 ◽  
Vol 259 (6) ◽  
pp. H1730-H1735 ◽  
Author(s):  
R. Sato ◽  
I. Hisatome ◽  
J. A. Wasserstrom ◽  
C. E. Arentzen ◽  
D. H. Singer
Keyword(s):  
Single Channel ◽  
K Channel ◽  
Channel Activity ◽  
Atrial Myocytes ◽  
Open Probability ◽  
Human Atrial Myocytes ◽  
Open Time ◽  
Patch Pipette ◽  
Single Channel Activity ◽  
Gamma S

Single channel recording techniques were used to study acetylcholine (ACh)-sensitive K+ channel activity in human atrial myocytes isolated from specimens obtained during corrective cardiac surgery. Under conditions of cell-attached patch, the presence of ACh in the patch pipette activated K+ channels. Single channel activity occurred in periodic bursts. The channels exhibited a slope conductance of 46 +/- 2 pS inwardly (means +/- SD, n = 4). During a burst, both open and closed time histograms were fitted by a single exponential curve, suggesting the existence of one open and one closed state during a burst. Open probability increased directly with ACh concentration without affecting open time. The channel could be activated by GTP and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) (in the presence and absence of ACh in the pipette, respectively). Slope conductance, the response to GTP and GTP gamma S, and the independence of activation from Ca2+ were similar to those for other species. In contrast, sensitivity to ACh appeared diminished compared with frog atrial myocytes.

G protein-mediated inhibition of inwardly rectifying K+ channels in guinea pig chromaffin cells

1993 ◽  
Vol 265 (4) ◽  
pp. C946-C956 ◽  
Author(s):  
M. Inoue ◽  
I. Imanaga
Keyword(s):  
Guinea Pig ◽  
G Protein ◽  
Chromaffin Cells ◽  
Negative Slope ◽  
Equilibrium Potential ◽  
Patch Clamp Technique ◽  
K Channels ◽  
K Current ◽  
Gamma S ◽  
Inwardly Rectifying

Properties of inwardly directed rectification and its G protein-mediated inhibition in guinea pig chromaffin cells were studied using the whole cell version of the patch-clamp technique. The current-voltage (I-V) relationship for plateau currents in response to a 50-ms pulse showed an inwardly directed rectification between -80 and -140 mV and a negative slope at more negative potentials in normal solution. Replacement of Na+ with N-methyl-D-glucamine (NMDG) in the perfusate did not alter the plateau I-V relationship between -110 and -130 mV but did abolish the negative slope below -140 mV. The zero current or resting membrane potential in the NMDG solution was in fair agreement with the equilibrium potential for K+. The chord conductance-voltage relationship showed a good fit with the Boltzmann equation and shifted along the voltage axis by an approximate change in driving force on K+ when K+ concentration was increased. External Cs+ and Ba2+ produced a voltage-dependent inhibition of the inwardly directed rectification. These results indicate that inwardly rectifying (IR) K+ channels are mediating an inwardly directed rectification. Intracellular dialysis with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) produced a complete suppression of this IR K+ channel, irrespective of treatment with pertussis toxin. Adding GTP or guanosine 5'-O-(2-thiodiphosphate) to the patch solution resulted in a decrease in GTP gamma S inhibition of the K+ current. Internal application of vanadate was without effect. Time course of the inhibition of the IR K+ current coincided in part with that of inactivation of a nonselective cation current. In conclusion, IR K+ channels in the chromaffin cell are subject to G protein-mediated inhibition.

A large conductance K(+)-selective channel of guinea pig villus enterocytes is Ca2+ independent

1992 ◽  
Vol 262 (2) ◽  
pp. G369-G374 ◽  
Author(s):  
G. M. Mintenig ◽  
A. S. Monaghan ◽  
F. V. Sepulveda
Keyword(s):  
Guinea Pig ◽  
Single Channel ◽  
K Channel ◽  
Constant Field ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Current Voltage ◽  
Selective Channel ◽  
K Concentration ◽  
Current Voltage Relationship

The presence of K(+)-selective channels has been probed in enterocytes isolated from guinea pig small intestinal villi by the patch-clamp technique. A channel with a single-channel conductance of approximately 130 pS was observed in excised inside-out patches bathed in symmetrical K+. A change in the K+ concentration in the intracellular aspect of the membrane altered the current-voltage relationship as expected from the constant-field equation when it is assumed that K+ is the only permeant ion. A change in Cl- concentration was without effect. Neither the activity of the channel nor its conductance was altered by addition of ATP or Ba2+ to the intracellular side of the patches. Changes in the free Ca2+ concentration were also without effect. The channel's open probability showed no voltage dependence and appeared only occasionally active in cell-attached patches where it had a linear current-voltage relation. The K+ channel described, which cannot be readily classified in any of the known classes of K+ channels, might provide an exit pathway for K+ recycling in guinea pig villus enterocytes.

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.

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 G protein control of potassium channel activity in a mast cell line.

1990 ◽  
Vol 95 (2) ◽  
pp. 205-227 ◽  
Author(s):  
M A McCloskey ◽  
M D Cahalan
Keyword(s):  
Cell Line ◽  
G Protein ◽  
Pertussis Toxin ◽  
Inward Rectifier ◽  
K Channel ◽  
Half Time ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
K Channels ◽  
Gamma S

Using the patch-clamp technique, we studied regulation of potassium channels by G protein activators in the histamine-secreting rat basophilic leukemia (RBL-2H3) cell line. These cells normally express inward rectifier K+ channels, with a macroscopic whole-cell conductance in normal Ringer ranging from 1 to 16 nS/cell. This conductance is stabilized by including ATP or GTP in the pipette solution. Intracellular dialysis with any of three different activators of G proteins (GTP gamma S, GppNHp, or AlF-4) completely inhibited the inward rectifier K+ conductance with a half-time for decline averaging approximately 300 s after "break-in" to achieve whole-cell recording. In addition, with a half-time averaging approximately 200 s, G protein activators induced the appearance of a novel time-independent outwardly rectifying K+ conductance, which reached a maximum of 1-14 nS. The induced K+ channels are distinct from inward rectifier channels, having a smaller single-channel conductance of approximately 8 pS in symmetrical 160 mM K+, and being more sensitive to block by quinidine, but less sensitive to block by Ba2+. The induced K+ channels were also highly permeable to Rb+ but not to Na+ or Cs+. The current was not activated by the second messengers Ca2+, inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, or by cyclic AMP-dependent phosphorylation. Pretreatment of cells with pertussis toxin (0.1 microgram/ml for 12-13 h) prevented this current's induction both by guanine nucleotides and aluminum fluoride, but had no effect on the decrease in inward rectifier conductance. Since GTP gamma S is known to stimulate secretion from patch-clamped rat peritoneal mast cells, it is conceivable that K+ channels become inserted into the plasma membrane from secretory granules. However, total membrane capacitance remained nearly constant during appearance of the K+ channels, suggesting that secretion induced by GTP gamma S was minimal. Furthermore, pertussis toxin had no effect on secretion triggered by antigen, and triggering of secretion before electrical recording failed to induce the outward K+ current. Finally, GTP gamma S activated the K+ channel in excised inside-out patches of membrane. We conclude that two different GTP-binding proteins differentially regulate two subsets of K+ channels, causing the inward rectifier to close and a novel K+ channel to open when activated.

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