Ca2+ dependent calmodulin binding to cardiac ryanodine receptor (RyR2) calmodulin-binding domains

Malene Brohus, Mads Toft Søndergaard, Wayne S.R. Chen, Filip Van Petegem, Michael Toft Overgaard

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3 Citations (Scopus)
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Abstract

The Ca 2+ sensor calmodulin (CaM) regulates cardiac ryanodine receptor (RyR2)-mediated Ca 2+ release from the sarcoplasmic reticulum. CaM inhibits RyR2 in a Ca 2+ -dependent manner and aberrant CaM-dependent inhibition results in life-threatening cardiac arrhyth-mias. However, the molecular details of the CaM-RyR2 interaction remain unclear. Four CaM-binding domains (CaMBD1a, -1b, -2, and -3) in RyR2 have been proposed. Here, we investigated the Ca 2+ -dependent interactions between CaM and these CaMBDs by monitoring changes in the fluorescence anisotropy of carboxytetramethylrhodamine (TAMRA)-labeled CaMBD peptides during titration with CaM at a wide range of Ca 2+ concentrations. We showed that CaM bound to all four CaMBDs with affinities that increased with Ca 2+ concentration. CaM bound to CaMBD2 and -3 with high affinities across all Ca 2+ concentrations tested, but bound to CaMBD1a and -1b only at Ca 2+ concentrations above 0.2 mM. Binding experiments using individual CaM domains revealed that the CaM C-domain preferentially bound to CaMBD2, and the N-domain to CaMBD3. Moreover, the Ca 2+ affinity of the CaM C-domain in complex with CaMBD2 or -3 was so high that these complexes are essentially Ca 2+ saturated under resting Ca 2+ conditions. Conversely, the N-domain senses Ca 2+ exactly in the transition from resting to activating Ca 2+ when complexed to either CaMBD2 or -3. Altogether, our results support a binding model where the CaM C-domain is anchored to RyR2 CaMBD2 and saturated with Ca 2+ during Ca 2+ oscillations, while the CaM N-domain functions as a dynamic Ca 2+ sensor that can bridge noncontiguous regions of RyR2 or clamp down onto CaMBD2.

Original languageEnglish
JournalBiochemical Journal
Volume476
Issue number2
Pages (from-to)193-209
ISSN0264-6021
DOIs
Publication statusPublished - 18 Jan 2019

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Ryanodine Receptor Calcium Release Channel
Calmodulin
Fluorescence Polarization
Sensors
Clamping devices
Sarcoplasmic Reticulum
Titration

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@article{50056eaef7ba436d9d9f7c0c817c244d,
title = "Ca2+ dependent calmodulin binding to cardiac ryanodine receptor (RyR2) calmodulin-binding domains",
abstract = "The Ca 2+ sensor calmodulin (CaM) regulates cardiac ryanodine receptor (RyR2)-mediated Ca 2+ release from the sarcoplasmic reticulum. CaM inhibits RyR2 in a Ca 2+ -dependent manner and aberrant CaM-dependent inhibition results in life-threatening cardiac arrhyth-mias. However, the molecular details of the CaM-RyR2 interaction remain unclear. Four CaM-binding domains (CaMBD1a, -1b, -2, and -3) in RyR2 have been proposed. Here, we investigated the Ca 2+ -dependent interactions between CaM and these CaMBDs by monitoring changes in the fluorescence anisotropy of carboxytetramethylrhodamine (TAMRA)-labeled CaMBD peptides during titration with CaM at a wide range of Ca 2+ concentrations. We showed that CaM bound to all four CaMBDs with affinities that increased with Ca 2+ concentration. CaM bound to CaMBD2 and -3 with high affinities across all Ca 2+ concentrations tested, but bound to CaMBD1a and -1b only at Ca 2+ concentrations above 0.2 mM. Binding experiments using individual CaM domains revealed that the CaM C-domain preferentially bound to CaMBD2, and the N-domain to CaMBD3. Moreover, the Ca 2+ affinity of the CaM C-domain in complex with CaMBD2 or -3 was so high that these complexes are essentially Ca 2+ saturated under resting Ca 2+ conditions. Conversely, the N-domain senses Ca 2+ exactly in the transition from resting to activating Ca 2+ when complexed to either CaMBD2 or -3. Altogether, our results support a binding model where the CaM C-domain is anchored to RyR2 CaMBD2 and saturated with Ca 2+ during Ca 2+ oscillations, while the CaM N-domain functions as a dynamic Ca 2+ sensor that can bridge noncontiguous regions of RyR2 or clamp down onto CaMBD2.",
author = "Malene Brohus and S{\o}ndergaard, {Mads Toft} and Chen, {Wayne S.R.} and {Van Petegem}, Filip and Overgaard, {Michael Toft}",
year = "2019",
month = "1",
day = "18",
doi = "10.1042/BCJ20180545",
language = "English",
volume = "476",
pages = "193--209",
journal = "Biochemical Journal",
issn = "0264-6021",
publisher = "Portland Press Ltd.",
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Ca2+ dependent calmodulin binding to cardiac ryanodine receptor (RyR2) calmodulin-binding domains. / Brohus, Malene; Søndergaard, Mads Toft; Chen, Wayne S.R.; Van Petegem, Filip; Overgaard, Michael Toft.

In: Biochemical Journal, Vol. 476, No. 2, 18.01.2019, p. 193-209.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Ca2+ dependent calmodulin binding to cardiac ryanodine receptor (RyR2) calmodulin-binding domains

AU - Brohus, Malene

AU - Søndergaard, Mads Toft

AU - Chen, Wayne S.R.

AU - Van Petegem, Filip

AU - Overgaard, Michael Toft

PY - 2019/1/18

Y1 - 2019/1/18

N2 - The Ca 2+ sensor calmodulin (CaM) regulates cardiac ryanodine receptor (RyR2)-mediated Ca 2+ release from the sarcoplasmic reticulum. CaM inhibits RyR2 in a Ca 2+ -dependent manner and aberrant CaM-dependent inhibition results in life-threatening cardiac arrhyth-mias. However, the molecular details of the CaM-RyR2 interaction remain unclear. Four CaM-binding domains (CaMBD1a, -1b, -2, and -3) in RyR2 have been proposed. Here, we investigated the Ca 2+ -dependent interactions between CaM and these CaMBDs by monitoring changes in the fluorescence anisotropy of carboxytetramethylrhodamine (TAMRA)-labeled CaMBD peptides during titration with CaM at a wide range of Ca 2+ concentrations. We showed that CaM bound to all four CaMBDs with affinities that increased with Ca 2+ concentration. CaM bound to CaMBD2 and -3 with high affinities across all Ca 2+ concentrations tested, but bound to CaMBD1a and -1b only at Ca 2+ concentrations above 0.2 mM. Binding experiments using individual CaM domains revealed that the CaM C-domain preferentially bound to CaMBD2, and the N-domain to CaMBD3. Moreover, the Ca 2+ affinity of the CaM C-domain in complex with CaMBD2 or -3 was so high that these complexes are essentially Ca 2+ saturated under resting Ca 2+ conditions. Conversely, the N-domain senses Ca 2+ exactly in the transition from resting to activating Ca 2+ when complexed to either CaMBD2 or -3. Altogether, our results support a binding model where the CaM C-domain is anchored to RyR2 CaMBD2 and saturated with Ca 2+ during Ca 2+ oscillations, while the CaM N-domain functions as a dynamic Ca 2+ sensor that can bridge noncontiguous regions of RyR2 or clamp down onto CaMBD2.

AB - The Ca 2+ sensor calmodulin (CaM) regulates cardiac ryanodine receptor (RyR2)-mediated Ca 2+ release from the sarcoplasmic reticulum. CaM inhibits RyR2 in a Ca 2+ -dependent manner and aberrant CaM-dependent inhibition results in life-threatening cardiac arrhyth-mias. However, the molecular details of the CaM-RyR2 interaction remain unclear. Four CaM-binding domains (CaMBD1a, -1b, -2, and -3) in RyR2 have been proposed. Here, we investigated the Ca 2+ -dependent interactions between CaM and these CaMBDs by monitoring changes in the fluorescence anisotropy of carboxytetramethylrhodamine (TAMRA)-labeled CaMBD peptides during titration with CaM at a wide range of Ca 2+ concentrations. We showed that CaM bound to all four CaMBDs with affinities that increased with Ca 2+ concentration. CaM bound to CaMBD2 and -3 with high affinities across all Ca 2+ concentrations tested, but bound to CaMBD1a and -1b only at Ca 2+ concentrations above 0.2 mM. Binding experiments using individual CaM domains revealed that the CaM C-domain preferentially bound to CaMBD2, and the N-domain to CaMBD3. Moreover, the Ca 2+ affinity of the CaM C-domain in complex with CaMBD2 or -3 was so high that these complexes are essentially Ca 2+ saturated under resting Ca 2+ conditions. Conversely, the N-domain senses Ca 2+ exactly in the transition from resting to activating Ca 2+ when complexed to either CaMBD2 or -3. Altogether, our results support a binding model where the CaM C-domain is anchored to RyR2 CaMBD2 and saturated with Ca 2+ during Ca 2+ oscillations, while the CaM N-domain functions as a dynamic Ca 2+ sensor that can bridge noncontiguous regions of RyR2 or clamp down onto CaMBD2.

U2 - 10.1042/BCJ20180545

DO - 10.1042/BCJ20180545

M3 - Journal article

VL - 476

SP - 193

EP - 209

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

IS - 2

ER -