TY - JOUR
T1 - Proteomic and functional mapping of cardiac NaV1.5 channel phosphorylation sites
AU - Lorenzini, Maxime
AU - Burel, Sophie
AU - Lesage, Adrien
AU - Wagner, Emily
AU - Charriere, Camille
AU - Chevillard, Pierre Marie
AU - Evrard, Bérangere
AU - Maloney, Dan
AU - Ruff, Kiersten M.
AU - Pappu, Rohit V.
AU - Wagner, Stefan
AU - Nerbonne, Jeanne M.
AU - Silva, Jonathan R.
AU - Townsend, R. Reid
AU - Maier, Lars S.
AU - Marionneau, Céline
N1 - Funding Information:
This work was supported by the Agence Nationale de la Recherche (ANR-15-CE14-0006-01 and ANR-16-CE92-0013-01 to C. Marionneau), the Deutsche Forschungsgemeinschaft (Ma 1982/ 5-1 to L.S. Maier), and the National Institutes of Health (R01-HL148803 to C. Marionneau, R.V. Pappu, and J.R. Silva; R01-HL034161 and R01-HL142520 to J.M. Nerbonne; and 5R01NS056114 to R.V. Pappu). The MS experiments were performed at the Washington University Proteomics Shared Resource (WU-PSR), which is supported in part by the WU Institute of Clinical and Translational Sciences (National Center for Advancing Translational Sciences grant UL1 TR000448), the Mass Spectrometry Research Resource (National Institute of General Medical Sciences grant P41 GM103422), and the Siteman Comprehensive Cancer Center Support Grant (National Cancer Institute grant P30 CA091842). M. Lorenzini was supported by a Groupe de Réflexion sur la Recherche Cardiovasculaire–Société Française de Cardiologie predoctoral fellowship (SFC/GRRC2018). S. Burel was supported by a Fondation Lefoulon Delalande postdoctoral fellowship. The content of the research reported is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies. The authors declare no competing financial interests.
Publisher Copyright:
© 2021 Rockefeller University Press. All rights reserved.
PY - 2021
Y1 - 2021
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85100279600&partnerID=8YFLogxK
U2 - 10.1085/JGP.202012646
DO - 10.1085/JGP.202012646
M3 - Article
C2 - 33410863
AN - SCOPUS:85100279600
VL - 153
JO - Journal of General Physiology
JF - Journal of General Physiology
SN - 0022-1295
IS - 2
M1 - e202012646
ER -