Dataset Information

FRET-based mapping of calmodulin bound to the RyR1 Ca2+ release channel.

ABSTRACT: Calmodulin (CaM) functions as a regulatory subunit of ryanodine receptor (RyR) channels, modulating channel activity in response to changing [Ca(2+)](i). To investigate the structural basis of CaM regulation of the RyR1 isoform, we used site-directed labeling of channel regulatory subunits and fluorescence resonance energy transfer (FRET). Donor fluorophore was targeted to the RyR1 cytoplasmic assembly by preincubating sarcoplasmic reticulum membranes with a fluorescent FK506-binding protein (FKBP), and FRET was determined following incubations in the presence of fluorescent CaMs in which acceptor fluorophore was attached within the N lobe, central linker, or C lobe. Results demonstrated strong FRET to acceptors attached within CaM's N lobe, whereas substantially weaker FRET was observed when acceptor was attached within CaM's central linker or C lobe. Surprisingly, Ca(2+) evoked little change in FRET to any of the 3 CaM domains. Donor-acceptor distances derived from our FRET measurements provide insights into CaM's location and orientation within the RyR1 3D architecture and the conformational switching that underlies CaM regulation of the channel. These results establish a powerful new approach to resolving the structure and function of RyR channels.

SUBMITTER: Cornea RL

PROVIDER: S-EPMC2662960 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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FRET-based mapping of calmodulin bound to the RyR1 Ca2+ release channel.

Cornea Razvan L RL Nitu Florentin F Gruber Simon S Kohler Katherine K Satzer Michael M Thomas David D DD Fruen Bradley R BR

Proceedings of the National Academy of Sciences of the United States of America 20090330 15

Calmodulin (CaM) functions as a regulatory subunit of ryanodine receptor (RyR) channels, modulating channel activity in response to changing [Ca(2+)](i). To investigate the structural basis of CaM regulation of the RyR1 isoform, we used site-directed labeling of channel regulatory subunits and fluorescence resonance energy transfer (FRET). Donor fluorophore was targeted to the RyR1 cytoplasmic assembly by preincubating sarcoplasmic reticulum membranes with a fluorescent FK506-binding protein (FK ...[more]

PMID: 19332786

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Project description:Human gene variants affecting ion channel biophysical activity and/or membrane localization are linked to potentially fatal cardiac arrhythmias. However, the mechanism for many human arrhythmia variants remains undefined despite more than a decade of investigation. Posttranslational modulation of membrane proteins is essential for normal cardiac function. Importantly, aberrant myocyte signaling has been linked to defects in cardiac ion channel posttranslational modifications and disease. We recently identified a novel pathway for posttranslational regulation of the primary cardiac voltage-gated Na(+) channel (Na(v)1.5) by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). However, a role for this pathway in cardiac disease has not been evaluated.We evaluated the role of CaMKII-dependent phosphorylation in human genetic and acquired disease. We report an unexpected link between a short motif in the Na(v)1.5 DI-DII loop, recently shown to be critical for CaMKII-dependent phosphorylation, and Na(v)1.5 function in monogenic arrhythmia and common heart disease. Experiments in heterologous cells and primary ventricular cardiomyocytes demonstrate that the human arrhythmia susceptibility variants (A572D and Q573E) alter CaMKII-dependent regulation of Na(v)1.5, resulting in abnormal channel activity and cell excitability. In silico analysis reveals that these variants functionally mimic the phosphorylated channel, resulting in increased susceptibility to arrhythmia-triggering afterdepolarizations. Finally, we report that this same motif is aberrantly regulated in a large-animal model of acquired heart disease and in failing human myocardium.We identify the mechanism for 2 human arrhythmia variants that affect Na(v)1.5 channel activity through direct effects on channel posttranslational modification. We propose that the CaMKII phosphorylation motif in the Na(v)1.5 DI-DII cytoplasmic loop is a critical nodal point for proarrhythmic changes to Na(v)1.5 in congenital and acquired cardiac disease.

Dataset Information

FRET-based mapping of calmodulin bound to the RyR1 Ca2+ release channel.

Publications

FRET-based mapping of calmodulin bound to the RyR1 Ca2+ release channel.

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