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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

The late Na+ current presents fluctuations due to the opening and closure of individual channels.A. Time course of a typical experiment in Nav1.5-expressing cells showing the effects of ranolazine 30 μM, TTX 30 μM, and veratridine 1 μM onINa late.B. Averaged Fourier transforms of multiple traces recorded in different conditions in the experiment shown inA: 1. initial control; 2. ranolazine 30 μM; 3. TTX 30 μM; 4. veratridine 1 μM; 5. veratridine 50 μM.C. Fluctuation analysis of individual traces recorded during the five distinct periods shown inA allowed the distinction of individual channel gating events and the computation of the average open probabilityp and the average number of channelsn for each condition.
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f3: The late Na+ current presents fluctuations due to the opening and closure of individual channels.A. Time course of a typical experiment in Nav1.5-expressing cells showing the effects of ranolazine 30 μM, TTX 30 μM, and veratridine 1 μM onINa late.B. Averaged Fourier transforms of multiple traces recorded in different conditions in the experiment shown inA: 1. initial control; 2. ranolazine 30 μM; 3. TTX 30 μM; 4. veratridine 1 μM; 5. veratridine 50 μM.C. Fluctuation analysis of individual traces recorded during the five distinct periods shown inA allowed the distinction of individual channel gating events and the computation of the average open probabilityp and the average number of channelsn for each condition.

Mentions: Figure 3 shows the power density spectra of Fourier-transformed traces recorded under different conditions, as numbered in the upper time course of the experiments. Presumably, the opening and closure of Nav1.5 channels in the late gating mode conformation produces a Lorentzian component with corner frequency above 2 KHz. However, due to the reduced number of individual signal sources, the plateau spectral power density was small and difficult to distinguish. A better approach to fluctuation analysis is by the computation of single-channel parameters such as the mean open probabilityp and the average number of channelsn, starting from the well-known estimations of current varianceσ2 and macroscopic currentI25:


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)

The late Na+ current presents fluctuations due to the opening and closure of individual channels.A. Time course of a typical experiment in Nav1.5-expressing cells showing the effects of ranolazine 30 μM, TTX 30 μM, and veratridine 1 μM onINa late.B. Averaged Fourier transforms of multiple traces recorded in different conditions in the experiment shown inA: 1. initial control; 2. ranolazine 30 μM; 3. TTX 30 μM; 4. veratridine 1 μM; 5. veratridine 50 μM.C. Fluctuation analysis of individual traces recorded during the five distinct periods shown inA allowed the distinction of individual channel gating events and the computation of the average open probabilityp and the average number of channelsn for each condition.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: The late Na+ current presents fluctuations due to the opening and closure of individual channels.A. Time course of a typical experiment in Nav1.5-expressing cells showing the effects of ranolazine 30 μM, TTX 30 μM, and veratridine 1 μM onINa late.B. Averaged Fourier transforms of multiple traces recorded in different conditions in the experiment shown inA: 1. initial control; 2. ranolazine 30 μM; 3. TTX 30 μM; 4. veratridine 1 μM; 5. veratridine 50 μM.C. Fluctuation analysis of individual traces recorded during the five distinct periods shown inA allowed the distinction of individual channel gating events and the computation of the average open probabilityp and the average number of channelsn for each condition.
Mentions: Figure 3 shows the power density spectra of Fourier-transformed traces recorded under different conditions, as numbered in the upper time course of the experiments. Presumably, the opening and closure of Nav1.5 channels in the late gating mode conformation produces a Lorentzian component with corner frequency above 2 KHz. However, due to the reduced number of individual signal sources, the plateau spectral power density was small and difficult to distinguish. A better approach to fluctuation analysis is by the computation of single-channel parameters such as the mean open probabilityp and the average number of channelsn, starting from the well-known estimations of current varianceσ2 and macroscopic currentI25:

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