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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

Effects of ranolazine, TTX and veratridine on the late Na+ current in Nav1.5-expressing cells.A. Time plots ofINa late in four typical experiments. Intervals of application of ranolazine 10 or 30 μM, TTX 30 μM, and veratridine 1 μM are indicated. Right panels:INa late traces from 260 to 270 ms at the time points marked with arrows and numbered in the left panels.B andC. Effects of ranolazine 10 and 30 μM, respectively, on relative levels of TTX-sensitive late Na+ current.D. Effect of veratridine 1 μM. Mean values are indicated for each condition, and error bars represent SEM.
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f2: Effects of ranolazine, TTX and veratridine on the late Na+ current in Nav1.5-expressing cells.A. Time plots ofINa late in four typical experiments. Intervals of application of ranolazine 10 or 30 μM, TTX 30 μM, and veratridine 1 μM are indicated. Right panels:INa late traces from 260 to 270 ms at the time points marked with arrows and numbered in the left panels.B andC. Effects of ranolazine 10 and 30 μM, respectively, on relative levels of TTX-sensitive late Na+ current.D. Effect of veratridine 1 μM. Mean values are indicated for each condition, and error bars represent SEM.

Mentions: Under control conditions, a 5-min application of veratridine 1 μM induced an increase of the late Na+ current by 269.1 ± 16.1% (mean ± SEM,n = 28) of initial values (Table 1 andFigure 2).Figure 1B shows representative current traces of selected sweeps, as well as magnified inserts of the peak and late phase, within an individual experiment. The application of veratridine 1 μM increased the late current amplitude by more than two-fold relative to the TTX 30 μM reference trace, without any noticeable effect on peak current, while the co-application of ranolazine 10 μM reduced the peak current with apparently no effect on the late component. Systematic experiments with ranolazine applied alone demonstratedINa late reduction to 75.0 ± 2.7% (mean ± SEM,n = 17) of initial values at 10 μM, and 58.4 ± 3.5% (mean ± SEM,n = 18) at 30 μM (Table 1 andFigure 2).


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)

Effects of ranolazine, TTX and veratridine on the late Na+ current in Nav1.5-expressing cells.A. Time plots ofINa late in four typical experiments. Intervals of application of ranolazine 10 or 30 μM, TTX 30 μM, and veratridine 1 μM are indicated. Right panels:INa late traces from 260 to 270 ms at the time points marked with arrows and numbered in the left panels.B andC. Effects of ranolazine 10 and 30 μM, respectively, on relative levels of TTX-sensitive late Na+ current.D. Effect of veratridine 1 μM. Mean values are indicated for each condition, and error bars represent SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Effects of ranolazine, TTX and veratridine on the late Na+ current in Nav1.5-expressing cells.A. Time plots ofINa late in four typical experiments. Intervals of application of ranolazine 10 or 30 μM, TTX 30 μM, and veratridine 1 μM are indicated. Right panels:INa late traces from 260 to 270 ms at the time points marked with arrows and numbered in the left panels.B andC. Effects of ranolazine 10 and 30 μM, respectively, on relative levels of TTX-sensitive late Na+ current.D. Effect of veratridine 1 μM. Mean values are indicated for each condition, and error bars represent SEM.
Mentions: Under control conditions, a 5-min application of veratridine 1 μM induced an increase of the late Na+ current by 269.1 ± 16.1% (mean ± SEM,n = 28) of initial values (Table 1 andFigure 2).Figure 1B shows representative current traces of selected sweeps, as well as magnified inserts of the peak and late phase, within an individual experiment. The application of veratridine 1 μM increased the late current amplitude by more than two-fold relative to the TTX 30 μM reference trace, without any noticeable effect on peak current, while the co-application of ranolazine 10 μM reduced the peak current with apparently no effect on the late component. Systematic experiments with ranolazine applied alone demonstratedINa late reduction to 75.0 ± 2.7% (mean ± SEM,n = 17) of initial values at 10 μM, and 58.4 ± 3.5% (mean ± SEM,n = 18) at 30 μM (Table 1 andFigure 2).

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