scholarly journals Measurement of Na(+)-K+ pump current in isolated rabbit ventricular myocytes using the whole-cell voltage-clamp technique. Inhibition of the pump by oxidant stress.

1993 ◽  
Vol 72 (1) ◽  
pp. 91-101 ◽  
Author(s):  
M J Shattock ◽  
H Matsuura
Keyword(s):  
Voltage Clamp ◽  
Ventricular Myocytes ◽  
Oxidant Stress ◽  
Cell Voltage ◽  
Pump Current ◽  
Whole Cell ◽  
Voltage Clamp Technique ◽  
Clamp Technique ◽  
Whole Cell Voltage Clamp ◽  
Rabbit Ventricular Myocytes

Steady-state twitch Ca2+ fluxes and cytosolic Ca2+ buffering in rabbit ventricular myocytes

1996 ◽  
Vol 270 (1) ◽  
pp. C192-C199 ◽  
Author(s):  
L. M. Delbridge ◽  
J. W. Bassani ◽  
D. M. Bers
Keyword(s):  
Steady State ◽  
Sarcoplasmic Reticulum ◽  
Voltage Clamp ◽  
Ventricular Myocytes ◽  
Cell Voltage ◽  
Whole Cell ◽  
Rabbit Ventricular Myocytes ◽  
The Mean ◽  
State Contraction ◽  
Caffeine Contractures

Intracellular Ca2+ ([Ca2+]i) transients and transsarcolemmal Ca2+ currents were measured in indo 1-loaded isolated rabbit ventricular myocytes during whole cell voltage clamp to quantitate the components of cytosolic Ca2+ influx and to describe the dynamic aspects of cytosolic Ca2+ buffering during steady-state contraction (0.5 Hz, 22 degrees C). Sarcolemmal Ca2+ influx was directly measured from the integrated Ca2+ current (Ica) recorded during the clamp (158 +/- 10 attomoles; amol). Sarcoplasmic reticulum (SR) Ca2+ content was determined from the integrated electrogenic Na+/Ca2+ exchange current (Ix) induced during rapid application and sustained exposure of cells to caffeine to elicit the release of the SR Ca2+ load (1,208 +/- 170 amol). The mean steady-state SR Ca2+ load was calculated to be 87 +/- 13 microM (mumol/l nonmitochondrial cytosolic volume). Ca2+ influx via Ica represented approximately 14% of the stored SR Ca2+ and 23% of the total cytosolic Ca2+ flux during a twitch (47 +/- 6 microM). Comparison of electrophysiologically measured Ca2+ fluxes with Ca2+ transients yields apparent buffering values of 60 for caffeine contractures and 110 for twitches (delta Ca2+ total/delta Ca2+ free). This is consistent with the occurrence of "active" buffering of cytosolic Ca2+ by SR Ca2+ uptake during the twitch.

Effects of endothelin 1 on calcium and sodium currents in isolated human cardiac myocytes

1995 ◽  
Vol 73 (12) ◽  
pp. 1774-1783 ◽  
Author(s):  
Tzu-Hurng Cheng ◽  
Chung-Yi Chang ◽  
Jeng Wei ◽  
Cheng-I Lin
Keyword(s):  
Voltage Clamp ◽  
Cardiac Myocytes ◽  
Ventricular Myocytes ◽  
Cell Voltage ◽  
Cardiac Cells ◽  
Sodium Currents ◽  
Atrial Myocytes ◽  
Whole Cell ◽  
Voltage Clamp Technique ◽  
Endothelin 1

We have used the whole-cell voltage-clamp technique to study the effects of endothelin 1 (ET-1, 10 nM) on L-type Ca2+ currents and voltage-dependent Na+ inward currents in human cardiac cells. Myocytes were enzymatically isolated from atrial specimens obtained during open-heart surgery and from human ventricular tissues of explanted hearts. Extracellular application of ET-1 decreased the peak amplitude of Ca2+ currents by 26 ± 6% (n = 13) in atrial myocytes and by 19 ± 3% (n = 8) in ventricular myocytes. In three atrial cells, treatment with 1 μM BQ123 prevented the decrease in Ca2+ currents induced by ET-1. When GTP (0.2 mM) was added to the dialyzing pipette solution, ET-1 still caused a small decline by 12 ± 5% (n = 16), in peak Ca2+ currents, in atrial myocytes. When Ca2+ currents were increased (+210 ± 19%) by a β-adrenoceptor agonist (0.1 μM isoproterenol) or by the phosphodiesterase inhibitor isobutylmethylxanthine (10 μM), ET-1 reduced Ca2+ currents by 35 ± 6% (n = 4) and 30 ± 4% (n = 5), respectively. In human ventricular myocytes in the presence of 1 μM isoproterenol, which increased the peak Ca2+ currents by 150 ± 30%, ET-1 also induced a drastic reduction in Ca2+ currents, by 40 ± 11% (n = 5). The tetrodotoxin-sensitive Na+ currents measured in the presence of 5 mM [Na]o were significantly enhanced (+28 ± 7%) by ET-1 in five atrial myocytes. The stimulatory effect of ET-1 on Na+ currents was partially reversible. The present findings in human cardiac cells show that ET-1 did not enhance the Ca2+ currents in the absence or presence of internal GTP. The positive inotropic actions induced by ET-1 in human heart may be mediated mainly by signal-transduction pathways other than the G-protein – adenylyl cyclase – cAMP system.Key words: endothelin 1, human cardiac myocytes, whole-cell voltage-clamp technique, calcium currents, sodium currents.

scholarly journals Computer modeling of whole-cell voltage-clamp analyses to delineate guidelines for good practice of manual and automated patch-clamp

2020 ◽  
Author(s):  
Jérôme Montnach ◽  
Maxime Lorenzini ◽  
Adrien Lesage ◽  
Isabelle Simon ◽  
Sébastien Nicolas ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Voltage Clamp ◽  
Good Practice ◽  
Cell Voltage ◽  
Current Amplitude ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Clamp Technique ◽  
Whole Cell Voltage Clamp

ABSTRACTThe patch-clamp technique has contributed to major advances in the characterization of ion channels. The recent development of high throughput patch-clamp provides a new momentum to the field. However, whole-cell voltage-clamp technique presents certain limits that need to be considered for robust data generation. One major caveat is that current amplitude profoundly impacts the precision of the analyzed characteristics of the ion current under study. For voltagegated channels, the higher the current amplitude is, the less precise the characteristics of voltagedependence are. Similarly, in ion channel pharmacology, the characteristics of dose-response curves are hindered by high current amplitudes. In addition, the recent development of high throughput patch-clamp technique is often associated with the generation of stable cell lines demonstrating high current amplitudes. It is therefore critical to set the limits for current amplitude recordings to avoid inaccuracy in the characterization of channel properties or drug actions, such limits being different from one channel to another. In the present study, we use kinetic models of a voltage-gated sodium channel and a voltage-gated potassium channel to edict simple guidelines for good practice of whole-cell voltage-clamp recordings.

Reexamination of the Stoichiometry of Na+/Ca2+ Exchange with Whole-Cell Voltage Clamp of Guinea Pig Ventricular Myocytes

2006 ◽  
Vol 976 (1) ◽  
pp. 154-156 ◽  
Author(s):  
MASAMITSU HINATA ◽  
HISAO YAMAMURA ◽  
LIBING LI ◽  
YASUHIDE WATANABE ◽  
TOMOKAZU WATANO ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Voltage Clamp ◽  
Ventricular Myocytes ◽  
Cell Voltage ◽  
Whole Cell ◽  
Whole Cell Voltage Clamp

scholarly journals Computer modeling of whole-cell voltage-clamp analyses to delineate guidelines for good practice of manual and automated patch-clamp

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jérôme Montnach ◽  
Maxime Lorenzini ◽  
Adrien Lesage ◽  
Isabelle Simon ◽  
Sébastien Nicolas ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Voltage Clamp ◽  
Good Practice ◽  
Cell Voltage ◽  
Current Amplitude ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Clamp Technique ◽  
Whole Cell Voltage Clamp

AbstractThe patch-clamp technique and more recently the high throughput patch-clamp technique have contributed to major advances in the characterization of ion channels. However, the whole-cell voltage-clamp technique presents certain limits that need to be considered for robust data generation. One major caveat is that increasing current amplitude profoundly impacts the accuracy of the biophysical analyses of macroscopic ion currents under study. Using mathematical kinetic models of a cardiac voltage-gated sodium channel and a cardiac voltage-gated potassium channel, we demonstrated how large current amplitude and series resistance artefacts induce an undetected alteration in the actual membrane potential and affect the characterization of voltage-dependent activation and inactivation processes. We also computed how dose–response curves are hindered by high current amplitudes. This is of high interest since stable cell lines frequently demonstrating high current amplitudes are used for safety pharmacology using the high throughput patch-clamp technique. It is therefore critical to set experimental limits for current amplitude recordings to prevent inaccuracy in the characterization of channel properties or drug activity, such limits being different from one channel type to another. Based on the predictions generated by the kinetic models, we draw simple guidelines for good practice of whole-cell voltage-clamp recordings.

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