Phosphorylation of the voltage-gated Na+ (NaV) channel NaV1.5 regulates cardiac excitability, yet the phosphorylation sites regulating its function and the underlying mechanisms remain largely unknown. Using a systematic, quantitative phosphoproteomic approach, we analyzed NaV1.5 channel complexes purified from nonfailing and failing mouse left ventricles, and we identified 42 phosphorylation sites on NaV1.5. Most sites are clustered, and three of these clusters are highly phosphorylated. Analyses of phosphosilent and phosphomimetic NaV1.5 mutants revealed the roles of three phosphosites in regulating NaV1.5 channel expression and gating. The phosphorylated serines S664 and S667 regulate the voltage dependence of channel activation in a cumulative manner, whereas the nearby S671, the phosphorylation of which is increased in failing hearts, regulates cell surface NaV1.5 expression and peak Na+ current. No additional roles could be assigned to the other clusters of phosphosites. Taken together, our results demonstrate that ventricular NaV1.5 is highly phosphorylated and that the phosphorylation-dependent regulation of NaV1.5 channels is highly complex, site specific, and dynamic.
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January 07 2021
Proteomic and functional mapping of cardiac NaV1.5 channel phosphorylation sites
Maxime Lorenzini,
Maxime Lorenzini
1
Université de Nantes, Centre national de la recherche scientifique, Institut National de la Santé et de la Recherche Médicale, l’Institut du thorax, Nantes, France
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Sophie Burel,
Sophie Burel
1
Université de Nantes, Centre national de la recherche scientifique, Institut National de la Santé et de la Recherche Médicale, l’Institut du thorax, Nantes, France
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Adrien Lesage,
Adrien Lesage
1
Université de Nantes, Centre national de la recherche scientifique, Institut National de la Santé et de la Recherche Médicale, l’Institut du thorax, Nantes, France
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Emily Wagner,
Emily Wagner
2
Department of Biomedical Engineering, Washington University in Saint Louis, St. Louis, MO
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Camille Charrière,
Camille Charrière
1
Université de Nantes, Centre national de la recherche scientifique, Institut National de la Santé et de la Recherche Médicale, l’Institut du thorax, Nantes, France
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Pierre-Marie Chevillard,
Pierre-Marie Chevillard
1
Université de Nantes, Centre national de la recherche scientifique, Institut National de la Santé et de la Recherche Médicale, l’Institut du thorax, Nantes, France
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Bérangère Evrard,
Bérangère Evrard
1
Université de Nantes, Centre national de la recherche scientifique, Institut National de la Santé et de la Recherche Médicale, l’Institut du thorax, Nantes, France
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Dan Maloney,
Dan Maloney
3
Bioinformatics Solutions Inc., Waterloo, Ontario, Canada
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Kiersten M. Ruff,
Kiersten M. Ruff
2
Department of Biomedical Engineering, Washington University in Saint Louis, St. Louis, MO
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Rohit V. Pappu,
Rohit V. Pappu
2
Department of Biomedical Engineering, Washington University in Saint Louis, St. Louis, MO
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Stefan Wagner,
Stefan Wagner
4
Department of Internal Medicine II, University Heart Center, University Hospital Regensburg, Regensburg, Germany
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Jeanne M. Nerbonne,
Jeanne M. Nerbonne
5
Department of Developmental Biology, Washington University Medical School, St. Louis, MO
6
Department of Medicine, Washington University Medical School, St. Louis, MO
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Jonathan R. Silva,
Jonathan R. Silva
2
Department of Biomedical Engineering, Washington University in Saint Louis, St. Louis, MO
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R. Reid Townsend,
R. Reid Townsend
6
Department of Medicine, Washington University Medical School, St. Louis, MO
7
Department of Cell Biology and Physiology, Washington University Medical School, St. Louis, MO
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Lars S. Maier,
Lars S. Maier
4
Department of Internal Medicine II, University Heart Center, University Hospital Regensburg, Regensburg, Germany
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Céline Marionneau
1
Université de Nantes, Centre national de la recherche scientifique, Institut National de la Santé et de la Recherche Médicale, l’Institut du thorax, Nantes, France
Correspondence to Céline Marionneau: celine.marionneau@univ-nantes.fr
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Maxime Lorenzini
1
Université de Nantes, Centre national de la recherche scientifique, Institut National de la Santé et de la Recherche Médicale, l’Institut du thorax, Nantes, France
Sophie Burel
1
Université de Nantes, Centre national de la recherche scientifique, Institut National de la Santé et de la Recherche Médicale, l’Institut du thorax, Nantes, France
Adrien Lesage
1
Université de Nantes, Centre national de la recherche scientifique, Institut National de la Santé et de la Recherche Médicale, l’Institut du thorax, Nantes, France
Emily Wagner
2
Department of Biomedical Engineering, Washington University in Saint Louis, St. Louis, MO
Camille Charrière
1
Université de Nantes, Centre national de la recherche scientifique, Institut National de la Santé et de la Recherche Médicale, l’Institut du thorax, Nantes, France
Pierre-Marie Chevillard
1
Université de Nantes, Centre national de la recherche scientifique, Institut National de la Santé et de la Recherche Médicale, l’Institut du thorax, Nantes, France
Bérangère Evrard
1
Université de Nantes, Centre national de la recherche scientifique, Institut National de la Santé et de la Recherche Médicale, l’Institut du thorax, Nantes, France
Dan Maloney
3
Bioinformatics Solutions Inc., Waterloo, Ontario, Canada
Kiersten M. Ruff
2
Department of Biomedical Engineering, Washington University in Saint Louis, St. Louis, MO
Rohit V. Pappu
2
Department of Biomedical Engineering, Washington University in Saint Louis, St. Louis, MO
Stefan Wagner
4
Department of Internal Medicine II, University Heart Center, University Hospital Regensburg, Regensburg, Germany
Jeanne M. Nerbonne
5
Department of Developmental Biology, Washington University Medical School, St. Louis, MO
6
Department of Medicine, Washington University Medical School, St. Louis, MO
Jonathan R. Silva
2
Department of Biomedical Engineering, Washington University in Saint Louis, St. Louis, MO
R. Reid Townsend
6
Department of Medicine, Washington University Medical School, St. Louis, MO
7
Department of Cell Biology and Physiology, Washington University Medical School, St. Louis, MO
Lars S. Maier
4
Department of Internal Medicine II, University Heart Center, University Hospital Regensburg, Regensburg, Germany
Céline Marionneau
1
Université de Nantes, Centre national de la recherche scientifique, Institut National de la Santé et de la Recherche Médicale, l’Institut du thorax, Nantes, France
Correspondence to Céline Marionneau: celine.marionneau@univ-nantes.fr
Received:
May 02 2020
Revision Received:
October 23 2020
Accepted:
December 03 2020
Online Issn: 1540-7748
Print Issn: 0022-1295
Funding:
Agence Nationale de la Recherche
(ANR-15-CE14-0006-01, ANR-16-CE92-0013-01)
Deutsche Forschungsgemeinschaft
(Ma 1982/5-1)
Fondation Lefoulon Delalande
(NO AWARD)
Institute of Clinical and Translational Sciences
(NO AWARD)
National Cancer Institute
(P30 CA091842)
National Center for Advancing Translational Sciences
(UL1 TR000448)
National Institute of General Medical Sciences
(P41 GM103422)
National Institutes of Health
(R01-HL148803, R01-HL034161, R01-HL142520, 5R01NS056114)
Société Française de Cardiologie
(SFC/GRRC2018)
© 2021 Lorenzini et al.
2021
This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).
J Gen Physiol (2021) 153 (2): e202012646.
Article history
Received:
May 02 2020
Revision Received:
October 23 2020
Accepted:
December 03 2020
Citation
Maxime Lorenzini, Sophie Burel, Adrien Lesage, Emily Wagner, Camille Charrière, Pierre-Marie Chevillard, Bérangère Evrard, Dan Maloney, Kiersten M. Ruff, Rohit V. Pappu, Stefan Wagner, Jeanne M. Nerbonne, Jonathan R. Silva, R. Reid Townsend, Lars S. Maier, Céline Marionneau; Proteomic and functional mapping of cardiac NaV1.5 channel phosphorylation sites. J Gen Physiol 1 February 2021; 153 (2): e202012646. doi: https://doi.org/10.1085/jgp.202012646
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