Calmodulin inhibition of human RyR2 channels requires phosphorylation of RyR2-S2808 or RyR2-S2814

Kafa Walweel, Nieves Gomez-Hurtado, Robyn T. Rebbeck, Ye Wint Oo, Nicole A. Beard, Peter Molenaar, Cris dos Remedios, Dirk F. van Helden, Razvan L. Cornea, Björn C. Knollmann, Derek R. Laver

Research output: Contribution to journalArticle

Abstract

Calmodulin (CaM) is a Ca-binding protein that binds to, and can directly inhibit cardiac ryanodine receptor calcium release channels (RyR2). Animal studies have shown that RyR2 hyperphosphorylation reduces CaM binding to RyR2 in failing hearts, but data are lacking on how CaM regulates human RyR2 and how this regulation is affected by RyR2 phosphorylation. Physiological concentrations of CaM (100 nM) inhibited the diastolic activity of RyR2 isolated from failing human hearts by ~50% but had no effect on RyR2 from healthy human hearts. Using FRET between donor-FKBP12.6 and acceptor-CaM bound to RyR2, we determined that CaM binds to RyR2 from healthy human heart with a K d = 121 ± 14 nM. Ex-vivo phosphorylation/dephosphorylation experiments suggested that the divergent CaM regulation of healthy and failing human RyR2 was caused by differences in RyR2 phosphorylation by protein kinase A and Ca-CaM-dependent kinase II. Ca 2+ -spark measurements in murine cardiomyocytes harbouring RyR2 phosphomimetic or phosphoablated mutants at S2814 and S2808 suggest that phosphorylation of residues corresponding to either human RyR2-S2808 or S2814 is both necessary and sufficient for RyR2 regulation by CaM. Our results challenge the current concept that CaM universally functions as a canonical inhibitor of RyR2 across species. Rather, CaM's biological action on human RyR2 appears to be more nuanced, with inhibitory activity only on phosphorylated RyR2 channels, which occurs during exercise or in patients with heart failure.

LanguageEnglish
Pages96-106
Number of pages11
JournalJournal of Molecular and Cellular Cardiology
Volume130
DOIs
Publication statusPublished - 1 May 2019

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Ryanodine Receptor Calcium Release Channel
Calmodulin
Phosphorylation
Calcium-Calmodulin-Dependent Protein Kinases

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Walweel, Kafa ; Gomez-Hurtado, Nieves ; Rebbeck, Robyn T. ; Oo, Ye Wint ; Beard, Nicole A. ; Molenaar, Peter ; dos Remedios, Cris ; van Helden, Dirk F. ; Cornea, Razvan L. ; Knollmann, Björn C. ; Laver, Derek R. / Calmodulin inhibition of human RyR2 channels requires phosphorylation of RyR2-S2808 or RyR2-S2814. In: Journal of Molecular and Cellular Cardiology. 2019 ; Vol. 130. pp. 96-106.
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title = "Calmodulin inhibition of human RyR2 channels requires phosphorylation of RyR2-S2808 or RyR2-S2814",
abstract = "Calmodulin (CaM) is a Ca-binding protein that binds to, and can directly inhibit cardiac ryanodine receptor calcium release channels (RyR2). Animal studies have shown that RyR2 hyperphosphorylation reduces CaM binding to RyR2 in failing hearts, but data are lacking on how CaM regulates human RyR2 and how this regulation is affected by RyR2 phosphorylation. Physiological concentrations of CaM (100 nM) inhibited the diastolic activity of RyR2 isolated from failing human hearts by ~50{\%} but had no effect on RyR2 from healthy human hearts. Using FRET between donor-FKBP12.6 and acceptor-CaM bound to RyR2, we determined that CaM binds to RyR2 from healthy human heart with a K d = 121 ± 14 nM. Ex-vivo phosphorylation/dephosphorylation experiments suggested that the divergent CaM regulation of healthy and failing human RyR2 was caused by differences in RyR2 phosphorylation by protein kinase A and Ca-CaM-dependent kinase II. Ca 2+ -spark measurements in murine cardiomyocytes harbouring RyR2 phosphomimetic or phosphoablated mutants at S2814 and S2808 suggest that phosphorylation of residues corresponding to either human RyR2-S2808 or S2814 is both necessary and sufficient for RyR2 regulation by CaM. Our results challenge the current concept that CaM universally functions as a canonical inhibitor of RyR2 across species. Rather, CaM's biological action on human RyR2 appears to be more nuanced, with inhibitory activity only on phosphorylated RyR2 channels, which occurs during exercise or in patients with heart failure.",
keywords = "Calcium sparks, Calmodulin, Cardiomyopathy, Lipid bilayer, Phosphorylation, Ryanodine receptor",
author = "Kafa Walweel and Nieves Gomez-Hurtado and Rebbeck, {Robyn T.} and Oo, {Ye Wint} and Beard, {Nicole A.} and Peter Molenaar and {dos Remedios}, Cris and {van Helden}, {Dirk F.} and Cornea, {Razvan L.} and Knollmann, {Bj{\"o}rn C.} and Laver, {Derek R.}",
year = "2019",
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Walweel, K, Gomez-Hurtado, N, Rebbeck, RT, Oo, YW, Beard, NA, Molenaar, P, dos Remedios, C, van Helden, DF, Cornea, RL, Knollmann, BC & Laver, DR 2019, 'Calmodulin inhibition of human RyR2 channels requires phosphorylation of RyR2-S2808 or RyR2-S2814', Journal of Molecular and Cellular Cardiology, vol. 130, pp. 96-106. https://doi.org/10.1016/j.yjmcc.2019.03.018

Calmodulin inhibition of human RyR2 channels requires phosphorylation of RyR2-S2808 or RyR2-S2814. / Walweel, Kafa; Gomez-Hurtado, Nieves; Rebbeck, Robyn T.; Oo, Ye Wint; Beard, Nicole A.; Molenaar, Peter; dos Remedios, Cris; van Helden, Dirk F.; Cornea, Razvan L.; Knollmann, Björn C.; Laver, Derek R.

In: Journal of Molecular and Cellular Cardiology, Vol. 130, 01.05.2019, p. 96-106.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Calmodulin inhibition of human RyR2 channels requires phosphorylation of RyR2-S2808 or RyR2-S2814

AU - Walweel, Kafa

AU - Gomez-Hurtado, Nieves

AU - Rebbeck, Robyn T.

AU - Oo, Ye Wint

AU - Beard, Nicole A.

AU - Molenaar, Peter

AU - dos Remedios, Cris

AU - van Helden, Dirk F.

AU - Cornea, Razvan L.

AU - Knollmann, Björn C.

AU - Laver, Derek R.

PY - 2019/5/1

Y1 - 2019/5/1

N2 - Calmodulin (CaM) is a Ca-binding protein that binds to, and can directly inhibit cardiac ryanodine receptor calcium release channels (RyR2). Animal studies have shown that RyR2 hyperphosphorylation reduces CaM binding to RyR2 in failing hearts, but data are lacking on how CaM regulates human RyR2 and how this regulation is affected by RyR2 phosphorylation. Physiological concentrations of CaM (100 nM) inhibited the diastolic activity of RyR2 isolated from failing human hearts by ~50% but had no effect on RyR2 from healthy human hearts. Using FRET between donor-FKBP12.6 and acceptor-CaM bound to RyR2, we determined that CaM binds to RyR2 from healthy human heart with a K d = 121 ± 14 nM. Ex-vivo phosphorylation/dephosphorylation experiments suggested that the divergent CaM regulation of healthy and failing human RyR2 was caused by differences in RyR2 phosphorylation by protein kinase A and Ca-CaM-dependent kinase II. Ca 2+ -spark measurements in murine cardiomyocytes harbouring RyR2 phosphomimetic or phosphoablated mutants at S2814 and S2808 suggest that phosphorylation of residues corresponding to either human RyR2-S2808 or S2814 is both necessary and sufficient for RyR2 regulation by CaM. Our results challenge the current concept that CaM universally functions as a canonical inhibitor of RyR2 across species. Rather, CaM's biological action on human RyR2 appears to be more nuanced, with inhibitory activity only on phosphorylated RyR2 channels, which occurs during exercise or in patients with heart failure.

AB - Calmodulin (CaM) is a Ca-binding protein that binds to, and can directly inhibit cardiac ryanodine receptor calcium release channels (RyR2). Animal studies have shown that RyR2 hyperphosphorylation reduces CaM binding to RyR2 in failing hearts, but data are lacking on how CaM regulates human RyR2 and how this regulation is affected by RyR2 phosphorylation. Physiological concentrations of CaM (100 nM) inhibited the diastolic activity of RyR2 isolated from failing human hearts by ~50% but had no effect on RyR2 from healthy human hearts. Using FRET between donor-FKBP12.6 and acceptor-CaM bound to RyR2, we determined that CaM binds to RyR2 from healthy human heart with a K d = 121 ± 14 nM. Ex-vivo phosphorylation/dephosphorylation experiments suggested that the divergent CaM regulation of healthy and failing human RyR2 was caused by differences in RyR2 phosphorylation by protein kinase A and Ca-CaM-dependent kinase II. Ca 2+ -spark measurements in murine cardiomyocytes harbouring RyR2 phosphomimetic or phosphoablated mutants at S2814 and S2808 suggest that phosphorylation of residues corresponding to either human RyR2-S2808 or S2814 is both necessary and sufficient for RyR2 regulation by CaM. Our results challenge the current concept that CaM universally functions as a canonical inhibitor of RyR2 across species. Rather, CaM's biological action on human RyR2 appears to be more nuanced, with inhibitory activity only on phosphorylated RyR2 channels, which occurs during exercise or in patients with heart failure.

KW - Calcium sparks

KW - Calmodulin

KW - Cardiomyopathy

KW - Lipid bilayer

KW - Phosphorylation

KW - Ryanodine receptor

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DO - 10.1016/j.yjmcc.2019.03.018

M3 - Article

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EP - 106

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JF - Journal of Molecular and Cellular Cardiology

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