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Late cardiac sodium current can be assessed using automated patch-clamp.

Chevalier M, Amuzescu B, Gawali V, Todt H, Knott T, Scheel O, Abriel H - F1000Res (2014)

Bottom Line: We show that ranolazine at 10 and 30 μM in 3 min applications reduced the late Na (+) current to 75.0 ± 2.7% (mean ± SEM, n = 17) and 58.4 ± 3.5% ( n = 18) of initial levels, respectively, while a 5 min application of veratridine 1 μM resulted in a reversible current increase to 269.1 ± 16.1% ( n = 28) of initial values.Using fluctuation analysis, we observed that ranolazine 30 μM decreased mean open probability p from 0.6 to 0.38 without modifying the number of active channels n, while veratridine 1 μM increased n 2.5-fold without changing p.This effect is attributable to inactivation removal in Nav1.5 channels, since significant inhibitory effects on hERG current were detected at higher concentrations in hERG-expressing HEK293 cells, with a 28.9 ± 6.0% inhibition (mean ± SD, n = 10) with 50 μM veratridine.       

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Research, University of Bern, Bern, 3010, Switzerland.

ABSTRACT
The cardiac late Na (+) current is generated by a small fraction of voltage-dependent Na (+) channels that undergo a conformational change to a burst-gating mode, with repeated openings and closures during the action potential (AP) plateau. Its magnitude can be augmented by inactivation-defective mutations, myocardial ischemia, or prolonged exposure to chemical compounds leading to drug-induced (di)-long QT syndrome, and results in an increased susceptibility to cardiac arrhythmias. Using CytoPatch™ 2 automated patch-clamp equipment, we performed whole-cell recordings in HEK293 cells stably expressing human Nav1.5, and measured the late Na (+) component as average current over the last 100 ms of 300 ms depolarizing pulses to -10 mV from a holding potential of -100 mV, with a repetition frequency of 0.33 Hz. Averaged values in different steady-state experimental conditions were further corrected by the subtraction of current average during the application of tetrodotoxin (TTX) 30 μM. We show that ranolazine at 10 and 30 μM in 3 min applications reduced the late Na (+) current to 75.0 ± 2.7% (mean ± SEM, n = 17) and 58.4 ± 3.5% ( n = 18) of initial levels, respectively, while a 5 min application of veratridine 1 μM resulted in a reversible current increase to 269.1 ± 16.1% ( n = 28) of initial values. Using fluctuation analysis, we observed that ranolazine 30 μM decreased mean open probability p from 0.6 to 0.38 without modifying the number of active channels n, while veratridine 1 μM increased n 2.5-fold without changing p. In human iPSC-derived cardiomyocytes, veratridine 1 μM reversibly increased APD90 2.12 ± 0.41-fold (mean ± SEM, n = 6). This effect is attributable to inactivation removal in Nav1.5 channels, since significant inhibitory effects on hERG current were detected at higher concentrations in hERG-expressing HEK293 cells, with a 28.9 ± 6.0% inhibition (mean ± SD, n = 10) with 50 μM veratridine.       

No MeSH data available.


Related in: MedlinePlus

Activation by veratridine of the late Na+ current in Nav1.5-expressing cells.A. Time course ofINa late (average transmembrane current during the last 100 ms of a 300-ms depolarizing pulse to -10 mV from a holding potential of -100 mV) during a typical experiment with repeated applications of veratridine 1 μM and a final application of TTX 30 μM.B. Overlap of individual sweeps showing the peak and late component ofINa during the application of veratridine 1 μM, the co-application with ranolazine 10 μM, and with TTX 30 μM. The peak and lateINa component are shown separately in inserts using magnified time and voltage scales, respectively.
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f1: Activation by veratridine of the late Na+ current in Nav1.5-expressing cells.A. Time course ofINa late (average transmembrane current during the last 100 ms of a 300-ms depolarizing pulse to -10 mV from a holding potential of -100 mV) during a typical experiment with repeated applications of veratridine 1 μM and a final application of TTX 30 μM.B. Overlap of individual sweeps showing the peak and late component ofINa during the application of veratridine 1 μM, the co-application with ranolazine 10 μM, and with TTX 30 μM. The peak and lateINa component are shown separately in inserts using magnified time and voltage scales, respectively.

Mentions: Using automated voltage-clamp protocols applied to human Nav1.5-expressing HEK293 cells in the above mentioned conditions, we routinely recorded, in high-quality seal conditions (both seal and membrane resistance > 1 GΩ), and with stability for at least 20 min, whole-cell Na+ currents. These currents were elicited by membrane depolarization to -10 mV from a holding potential of -100 mV, required for the proper removal from inactivation of cardiac Na+ channels. The late Na+ current component (INa late) was automatically computed and plotted as time average over the last 100 ms of the 300-ms depolarizing pulse of each sweep.Figure 1A shows the time course of 5 averaged experiments including repeated applications of veratridine 1 μM with different durations (2 min, 1 min, 5 min), as well as a 1-min application of TTX 30 μM, resulting in the rapid complete block of late Na+ current. The average current level during TTX application in each experiment was subsequently subtracted from all other averaged steady-state levels to obtain unbiased estimations ofINa late in different experimental conditions. In general, reversibility was good and rapid upon TTX wash-out.


Late cardiac sodium current can be assessed using automated patch-clamp.

Chevalier M, Amuzescu B, Gawali V, Todt H, Knott T, Scheel O, Abriel H - F1000Res (2014)

Activation by veratridine of the late Na+ current in Nav1.5-expressing cells.A. Time course ofINa late (average transmembrane current during the last 100 ms of a 300-ms depolarizing pulse to -10 mV from a holding potential of -100 mV) during a typical experiment with repeated applications of veratridine 1 μM and a final application of TTX 30 μM.B. Overlap of individual sweeps showing the peak and late component ofINa during the application of veratridine 1 μM, the co-application with ranolazine 10 μM, and with TTX 30 μM. The peak and lateINa component are shown separately in inserts using magnified time and voltage scales, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4215750&req=5

f1: Activation by veratridine of the late Na+ current in Nav1.5-expressing cells.A. Time course ofINa late (average transmembrane current during the last 100 ms of a 300-ms depolarizing pulse to -10 mV from a holding potential of -100 mV) during a typical experiment with repeated applications of veratridine 1 μM and a final application of TTX 30 μM.B. Overlap of individual sweeps showing the peak and late component ofINa during the application of veratridine 1 μM, the co-application with ranolazine 10 μM, and with TTX 30 μM. The peak and lateINa component are shown separately in inserts using magnified time and voltage scales, respectively.
Mentions: Using automated voltage-clamp protocols applied to human Nav1.5-expressing HEK293 cells in the above mentioned conditions, we routinely recorded, in high-quality seal conditions (both seal and membrane resistance > 1 GΩ), and with stability for at least 20 min, whole-cell Na+ currents. These currents were elicited by membrane depolarization to -10 mV from a holding potential of -100 mV, required for the proper removal from inactivation of cardiac Na+ channels. The late Na+ current component (INa late) was automatically computed and plotted as time average over the last 100 ms of the 300-ms depolarizing pulse of each sweep.Figure 1A shows the time course of 5 averaged experiments including repeated applications of veratridine 1 μM with different durations (2 min, 1 min, 5 min), as well as a 1-min application of TTX 30 μM, resulting in the rapid complete block of late Na+ current. The average current level during TTX application in each experiment was subsequently subtracted from all other averaged steady-state levels to obtain unbiased estimations ofINa late in different experimental conditions. In general, reversibility was good and rapid upon TTX wash-out.

Bottom Line: We show that ranolazine at 10 and 30 μM in 3 min applications reduced the late Na (+) current to 75.0 ± 2.7% (mean ± SEM, n = 17) and 58.4 ± 3.5% ( n = 18) of initial levels, respectively, while a 5 min application of veratridine 1 μM resulted in a reversible current increase to 269.1 ± 16.1% ( n = 28) of initial values.Using fluctuation analysis, we observed that ranolazine 30 μM decreased mean open probability p from 0.6 to 0.38 without modifying the number of active channels n, while veratridine 1 μM increased n 2.5-fold without changing p.This effect is attributable to inactivation removal in Nav1.5 channels, since significant inhibitory effects on hERG current were detected at higher concentrations in hERG-expressing HEK293 cells, with a 28.9 ± 6.0% inhibition (mean ± SD, n = 10) with 50 μM veratridine.       

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Research, University of Bern, Bern, 3010, Switzerland.

ABSTRACT
The cardiac late Na (+) current is generated by a small fraction of voltage-dependent Na (+) channels that undergo a conformational change to a burst-gating mode, with repeated openings and closures during the action potential (AP) plateau. Its magnitude can be augmented by inactivation-defective mutations, myocardial ischemia, or prolonged exposure to chemical compounds leading to drug-induced (di)-long QT syndrome, and results in an increased susceptibility to cardiac arrhythmias. Using CytoPatch™ 2 automated patch-clamp equipment, we performed whole-cell recordings in HEK293 cells stably expressing human Nav1.5, and measured the late Na (+) component as average current over the last 100 ms of 300 ms depolarizing pulses to -10 mV from a holding potential of -100 mV, with a repetition frequency of 0.33 Hz. Averaged values in different steady-state experimental conditions were further corrected by the subtraction of current average during the application of tetrodotoxin (TTX) 30 μM. We show that ranolazine at 10 and 30 μM in 3 min applications reduced the late Na (+) current to 75.0 ± 2.7% (mean ± SEM, n = 17) and 58.4 ± 3.5% ( n = 18) of initial levels, respectively, while a 5 min application of veratridine 1 μM resulted in a reversible current increase to 269.1 ± 16.1% ( n = 28) of initial values. Using fluctuation analysis, we observed that ranolazine 30 μM decreased mean open probability p from 0.6 to 0.38 without modifying the number of active channels n, while veratridine 1 μM increased n 2.5-fold without changing p. In human iPSC-derived cardiomyocytes, veratridine 1 μM reversibly increased APD90 2.12 ± 0.41-fold (mean ± SEM, n = 6). This effect is attributable to inactivation removal in Nav1.5 channels, since significant inhibitory effects on hERG current were detected at higher concentrations in hERG-expressing HEK293 cells, with a 28.9 ± 6.0% inhibition (mean ± SD, n = 10) with 50 μM veratridine.       

No MeSH data available.


Related in: MedlinePlus