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Late cardiac sodium current can be assessed using automated patch-clamp
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 mu...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
F1000Research
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4215750/ https://www.ncbi.nlm.nih.gov/pubmed/25383189 http://dx.doi.org/10.12688/f1000research.5544.1 |
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author | Chevalier, Morgan Amuzescu, Bogdan Gawali, Vaibhavkumar Todt, Hannes Knott, Thomas Scheel, Olaf Abriel, Hugues |
author_facet | Chevalier, Morgan Amuzescu, Bogdan Gawali, Vaibhavkumar Todt, Hannes Knott, Thomas Scheel, Olaf Abriel, Hugues |
author_sort | Chevalier, Morgan |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-4215750 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | F1000Research |
record_format | MEDLINE/PubMed |
spelling | pubmed-42157502014-11-06 Late cardiac sodium current can be assessed using automated patch-clamp Chevalier, Morgan Amuzescu, Bogdan Gawali, Vaibhavkumar Todt, Hannes Knott, Thomas Scheel, Olaf Abriel, Hugues F1000Res Research Article 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. F1000Research 2014-10-16 /pmc/articles/PMC4215750/ /pubmed/25383189 http://dx.doi.org/10.12688/f1000research.5544.1 Text en Copyright: © 2014 Chevalier M et al. http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. http://creativecommons.org/publicdomain/zero/1.0/ Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication). |
spellingShingle | Research Article Chevalier, Morgan Amuzescu, Bogdan Gawali, Vaibhavkumar Todt, Hannes Knott, Thomas Scheel, Olaf Abriel, Hugues Late cardiac sodium current can be assessed using automated patch-clamp |
title | Late cardiac sodium current can be assessed using automated patch-clamp |
title_full | Late cardiac sodium current can be assessed using automated patch-clamp |
title_fullStr | Late cardiac sodium current can be assessed using automated patch-clamp |
title_full_unstemmed | Late cardiac sodium current can be assessed using automated patch-clamp |
title_short | Late cardiac sodium current can be assessed using automated patch-clamp |
title_sort | late cardiac sodium current can be assessed using automated patch-clamp |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4215750/ https://www.ncbi.nlm.nih.gov/pubmed/25383189 http://dx.doi.org/10.12688/f1000research.5544.1 |
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