Related Experiment Videos
Unique phosphorylation site on the cardiac ryanodine receptor regulates calcium channel activity
D R Witcher1, R J Kovacs, H Schulman
1Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis 46202.
Abstract:
Ryanodine receptors have recently been shown to be the Ca2+ release channels of sarcoplasmic reticulum in both cardiac muscle and skeletal muscle. Several regulatory sites are postulated to exist on these receptors, but to date, none have been definitively identified. In the work described here, we localize one of these sites by showing that the cardiac isoform of the ryanodine receptor is a preferred substrate for multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase). Phosphorylation by CaM kinase occurs at a single site encompassing serine 2809. Antibodies generated to this site react only with the cardiac isoform of the ryanodine receptor, and immunoprecipitate only cardiac [3H]ryanodine-binding sites. When cardiac junctional sarcoplasmic reticulum vesicles or partially purified ryanodine receptors are fused with planar bilayers, phosphorylation at this site activates the Ca2+ channel. In tissues expressing the cardiac isoform of the ryanodine receptor, such as heart and brain, phosphorylation of the Ca2+ release channel by CaM kinase may provide a unique mechanism for regulating intracellular Ca2+ release.
Insights
Researchers identified a specific phosphorylation site on cardiac ryanodine receptors, a key calcium channel. This finding reveals a novel mechanism for regulating calcium release in heart and brain tissues.
Area of Science:
- Biochemistry
- Molecular Biology
- Cardiovascular Physiology
Background:
- Ryanodine receptors (RyRs) are crucial Ca2+ release channels in muscle sarcoplasmic reticulum.
- Regulatory sites on RyRs are proposed but not definitively identified.
- Understanding RyR regulation is vital for cardiac function.
Purpose of the Study:
- To identify and characterize regulatory sites on the cardiac ryanodine receptor.
- To investigate the role of Ca2+/calmodulin-dependent protein kinase (CaM kinase) in RyR regulation.
- To determine the functional consequences of RyR phosphorylation.
Main Methods:
- Utilized biochemical assays to identify phosphorylation sites on cardiac RyRs.
- Generated antibodies specific to the identified phosphorylation site.
- Performed electrophysiological studies using planar bilayers to assess channel activity.
- Investigated RyR phosphorylation in cardiac sarcoplasmic reticulum vesicles.
Main Results:
- Identified serine 2809 as a unique phosphorylation site on the cardiac RyR isoform by CaM kinase.
- Developed antibodies that specifically recognize phosphorylated cardiac RyR.
- Demonstrated that phosphorylation at serine 2809 activates the cardiac Ca2+ release channel.
- Confirmed cardiac RyR as a preferred substrate for CaM kinase.
Conclusions:
- Phosphorylation of cardiac ryanodine receptors at serine 2809 by CaM kinase is a key regulatory mechanism.
- This phosphorylation event directly modulates Ca2+ channel activity.
- Provides a novel pathway for regulating intracellular calcium release in cardiac and neuronal tissues.