Role of protein phosphatases in the run down of guinea pig cardiac Cav1.2 Ca2+ channels

2016 ◽  
Vol 310 (10) ◽  
pp. C773-C779 ◽  
Author(s):  
Lifeng Yu ◽  
Jianjun Xu ◽  
Etsuko Minobe ◽  
Asako Kameyama ◽  
Lei Yang ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Protein Phosphatase ◽  
Ventricular Myocytes ◽  
Protein Phosphatases ◽  
Slow Phase ◽  
Patch Clamp Technique ◽  
Time Constants ◽  
Open Time ◽  
Protein Phosphatase Type

This study aimed to investigate protein phosphatases involved in the run down of Cav1.2 Ca2+ channels. Single ventricular myocytes obtained from adult guinea pig hearts were used to record Ca2+ channel currents with the patch-clamp technique. Calmodulin (CaM) and ATP were used to restore channel activity in inside-out patches. Inhibitors of protein phosphatases were applied to investigate the role of phosphatases. The specific protein phosphatase type 1 (PP1) inhibitor (PP1 inhibitor-2) and protein phosphatase type 2A (PP2A) inhibitor (fostriecin) abolished the slow run down of Cav1.2 Ca2+ channels, which was evident as the time-dependent attenuation of the reversing effect of CaM/ATP on the run down. However, protein phosphatase type 2B (PP2B, calcineurin) inhibitor cyclosporine A together with cyclophilin A had no effect on the channel run down. Furthermore, PP1 inhibitor-2 mainly prolonged the open time constants of the channel, specifically, the slow open time. Fostriecin primarily shortened the slow close time constants. Our data suggest that PP1 and PP2A were involved in the slow phase of Cav1.2 Ca2+ channel run down. In addition, they exerted different effects on the open-close kinetics of the channel. All above support the view that PP1 and PP2A may dephosphorylate distinct phosphorylation sites on the Cav1.2 Ca2+ channel.

scholarly journals On the mechanism of rectification of the isoproterenol-activated chloride current in guinea-pig ventricular myocytes.

1993 ◽  
Vol 102 (5) ◽  
pp. 871-895 ◽  
Author(s):  
J L Overholt ◽  
M E Hobert ◽  
R D Harvey
Keyword(s):  
Guinea Pig ◽  
Ventricular Myocytes ◽  
Reversal Potential ◽  
Rate Theory ◽  
Constant Field ◽  
Patch Clamp Technique ◽  
Site Model ◽  
Outward Rectification ◽  
Current Equation

The whole cell configuration of the patch clamp technique was used to investigate the mechanism underlying rectification of the isoproterenol-activated chloride (Cl-) current in isolated guinea pig ventricular myocytes. When extracellular Cl- was replaced with either bromide (Br-), glutamate (Glut), iodide (I-), isethionate (Iseth), or nitrate (NO3-), the magnitude of the shift in reversal potential of the macroscopic current suggested the following selectivity sequence: NO3- > Br- > or = Cl- > or = I- > Iseth > or = Glut. This information was used to investigate the role of permeant ions in rectification of this current. Consistent with previous observations, when the concentration of intracellular Cl- (Cli-) was less than the concentration of extracellular Cl- (Clo-) (40 mM Cli-/150 mM Clo-) the current exhibited outward rectification, but when Cli- was increased to equal that outside (150 Cli-/150 Clo-), the current no longer rectified. Rectification in the presence of asymmetrical concentrations of permeant ions on either side of the membrane is predicted by constant field theory, as described by the Goldman-Hodgkin-Katz current equation. However, when the Cl- gradient was reversed (150 Cli-/40 Clo-) the current did not rectify in the opposite direction, and in the presence of lower symmetrical concentrations of Cl- inside and out (40 Cli-/40 Clo-), outward rectification did not disappear. Reducing Cli- by equimolar replacement with glutamate caused a concentration dependent increase in the degree of rectification. However, when Cli- was replaced with more permeant anions (NO3- and Br-), rectification was not observed. These results can be explained by a single binding site model based on Eyring rate theory, indicating that rectification is a function of the concentration and the permeability of the anions in the intracellular solution.

Properties of veratridine-modified single Na+ channels in guinea pig ventricular myocytes

1993 ◽  
Vol 264 (2) ◽  
pp. H454-H463 ◽  
Author(s):  
A. Sunami ◽  
T. Sasano ◽  
A. Matsunaga ◽  
Z. Fan ◽  
T. Swanobori ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Voltage Dependence ◽  
Single Channel ◽  
Ventricular Myocytes ◽  
Simultaneous Occurrence ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Open Time ◽  
High Event ◽  
Event Times

Modification of single Na+ channels by the alkaloid neurotoxin veratridine was investigated in guinea pig ventricular myocytes using the cell-attached configuration of the patch-clamp technique. Pipette application of veratridine (50 microM) induced long-lasting openings with two different single-channel conductances of 7.6 and 3.0 pS, in addition to normal type of short openings with a single-channel conductance of 16 pS. The veratridine-modified high- and low-conductance channels appeared commonly, and they could coexist with the normal one in the same patch. The open-time distributions for the high- and low-conductance channels could be fitted by a single exponential. The mean open time for the high- and low-conductance events ranged between 19.1 ms at -120 mV and 86.0 ms at -10 mV and between 4.5 ms at -120 mV and 16.2 ms at -10 mV, respectively. The closed-time distributions for the two conductance channels consisted of at least two components, and their values and voltage dependence were similar. External Ca2+ block resulted in an apparent reduction of unitary current amplitudes with a similar voltage dependence and affinity for Ca2+ in the high- and low-conductance channels. However, the low-conductance channel was more resistant to tetrodotoxin than the high one. The probability of simultaneous occurrence of the high and low events was equal to the product of the probabilities of occurrence of the high event times that of the low event. Furthermore, we observed modified channel openings after a normal opening for the two conductance channels and a modified one turning into a normal one for the high-conductance channel. It is concluded that veratridine induces the two different types of modified Na+ channels in cardiac myocytes and these are correlated with normal openings.

Effects of estrogen on action potential and membrane currents in guinea pig ventricular myocytes

1999 ◽  
Vol 277 (2) ◽  
pp. H826-H833 ◽  
Author(s):  
Seiko Tanabe ◽  
Toshio Hata ◽  
Masayasu Hiraoka
Keyword(s):  
Guinea Pig ◽  
Action Potential ◽  
Action Potentials ◽  
Voltage Dependence ◽  
Ventricular Myocytes ◽  
Membrane Currents ◽  
Patch Clamp Technique ◽  
17Β Estradiol ◽  
Electrophysiological Effects ◽  
Ionic Basis

To explore a possible ionic basis for the prolonged Q-T interval in women compared with that in men, we investigated the electrophysiological effects of estrogen in isolated guinea pig ventricular myocytes. Action potentials and membrane currents were recorded using the whole cell configuration of the patch-clamp technique. Application of 17β-estradiol (10–30 μM) significantly prolonged the action potential duration (APD) at 20% (APD20) and 90% repolarization (APD90) at stimulation rates of 0.1–2.0 Hz. In the presence of 30 μM 17β-estradiol, APD20 and APD90 at 0.1 Hz were prolonged by 46.2 ± 17.1 and 63.4 ± 11.7% of the control ( n = 5), respectively. In the presence of 30 μM 17β-estradiol the peak inward Ca2+ current ( I CaL) was decreased to 80.1 ± 2.5% of the control ( n = 4) without a shift in its voltage dependence. Application of 30 μM 17β-estradiol decreased the rapidly activating component of the delayed outward K+ current ( I Kr) to 63.4 ± 8% and the slowly activating component ( I Ks) to 65.8 ± 8.7% with respect to the control; the inward rectifier K+ current was barely affected. The results suggest that 17β-estradiol prolonged APD mainly by inhibiting the I Kcomponents I Krand I Ks.

Force measurements from voltage-clamped guinea pig ventricular myocytes

1990 ◽  
Vol 258 (2) ◽  
pp. H452-H459 ◽  
Author(s):  
N. Shepherd ◽  
M. Vornanen ◽  
G. Isenberg
Keyword(s):  
Guinea Pig ◽  
Time Course ◽  
Ventricular Myocytes ◽  
Isometric Force ◽  
Contractile Force ◽  
Patch Clamp Technique ◽  
Thin Glass ◽  
Tonic Component ◽  
Time To Peak ◽  
Whole Cell Patch Clamp

We describe the first observations of isolated mammalian guinea pig ventricular myocytes that combine measurements of contractile force with the voltage-clamp method. The myocytes were attached by poly-L-lysine to the beveled ends of a pair of thin glass rods having a compliance of 0.76 m/N. The contractile force of a cell caused a 1- to 3-microm displacement of the rods; the motion of which was converted to an output voltage by phototransistors. By the use of the whole cell patch-clamp technique, the cells were depolarized at 1 Hz with 200-ms-long clamp pulses from -45 to +5 mV (35 degrees C, 3.6 mM CaCl2). Isometric force began after a latency of 7 +/- 2 ms, peaked at 93 +/- 21 ms, and relaxed (90%) at 235 +/- 63 ms. The time course of force was always faster than that of isotonic shortening (time to peak 154 +/- 18 ms). With 400-ms-long depolarizations, a tonic component was recorded as either sustained force or sustained shortening that decayed on repolarization. Substitution of Ca by Sr in the bath increased the inward current through Ca channels but slowed down the time course of force development. The results are consistent with the hypothesis that activator calcium derives mainly from internal stores and that Ca release needs Ca entry through channels.

Existence of a transient outward K+ current in guinea pig cardiac myocytes

2000 ◽  
Vol 279 (1) ◽  
pp. H130-H138 ◽  
Author(s):  
Gui-Rong Li ◽  
Baofeng Yang ◽  
Haiying Sun ◽  
Clive M. Baumgarten
Keyword(s):  
Guinea Pig ◽  
Ventricular Myocytes ◽  
Outward Current ◽  
Resting Potential ◽  
Transient Outward Current ◽  
Time Constants ◽  
Zero Current ◽  
Steady State Inactivation ◽  
K Current ◽  
External K

A novel transient outward K+current that exhibits inward-going rectification ( I to.ir) was identified in guinea pig atrial and ventricular myocytes. I to.ir was insensitive to 4-aminopyridine (4-AP) but was blocked by 200 μmol/l Ba2+or removal of external K+. The zero current potential shifted 51–53 mV/decade change in external K+. I to.ir density was twofold greater in ventricular than in atrial myocytes, and biexponential inactivation occurs in both types of myocytes. At −20 mV, the fast inactivation time constants were 7.7 ± 1.8 and 6.1 ± 1.2 ms and the slow inactivation time constants were 85.1 ± 14.8 and 77.3 ± 10.4 ms in ventricular and atrial cells, respectively. The midpoints for steady-state inactivation were −36.4 ± 0.3 and −51.6 ± 0.4 mV, and recovery from inactivation was rapid near the resting potential (time constants = 7.9 ± 1.9 and 8.8 ± 2.1 ms, respectively). I to.ir was detected in Na+-containing and Na+-free solutions and was not blocked by 20 nmol/l saxitoxin. Action potential clamp revealed that I to.ir contributed an outward current that activated rapidly on depolarization and inactivated by early phase 2 in both tissues. Although it is well known that 4-AP-sensitive transient outward current is absent in guinea pig, this Ba2+-sensitive and 4-AP-insensitive K+ current has been overlooked.

PKC regulation of cardiac CFTR Cl− channel function in guinea pig ventricular myocytes

1998 ◽  
Vol 275 (1) ◽  
pp. C293-C302 ◽  
Author(s):  
Lisa M. Middleton ◽  
Robert D. Harvey
Keyword(s):  
Protein Kinase ◽  
Patch Clamp ◽  
Guinea Pig ◽  
Ventricular Myocytes ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Patch Clamp Technique ◽  
Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Perforated Patch Clamp

The role of protein kinase C (PKC) in regulating the protein kinase A (PKA)-activated Cl− current conducted by the cardiac isoform of the cystic fibrosis transmembrane conductance regulator (cCFTR) was studied in guinea pig ventricular myocytes using the whole cell patch-clamp technique. Although stimulation of endogenous PKC with phorbol 12,13-dibutyrate (PDBu) alone did not activate this Cl− current, even when intracellular dialysis was limited with the perforated patch-clamp technique, activation of PKC did elicit a significant response in the presence of PKA-dependent activation of the current by the β-adrenergic receptor agonist isoproterenol. PDBu increased the magnitude of the Cl− conductance activated by a supramaximally stimulating concentration of isoproterenol by 21 ± 3.3% ( n = 9) when added after isoproterenol and by 36 ± 16% ( n= 14) when introduced before isoproterenol. 4α-Phorbol 12,13-didecanoate, a phorbol ester that does not activate PKC, did not mimic these effects. Preexposure to chelerythrine or bisindolylmaleimide, two highly selective inhibitors of PKC, significantly reduced the magnitude of the isoproterenol-activated Cl− current by 79 ± 7.7% ( n = 11) and 52 ± 10% ( n = 8), respectively. Our results suggest that although acute activation of endogenous PKC alone does not significantly regulate cCFTR Cl− channel activity in native myocytes, it does potentiate PKA-dependent responses, perhaps most dramatically demonstrated by basal PKC activity, which may play a pivotal role in modulating the function of these channels.

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