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Cardiac-specific phosphorylation site for multifunctional Ca2+/calmodulin-dependent protein kinase is conserved in
D R Witcher1, B A Strifler, L R Jones
1Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis 46202-4800.
Insights
Mammalian brain expresses the cardiac ryanodine receptor isoform, confirmed by specific antibodies. This cardiac ryanodine receptor in the brain shares a conserved Ca2+/calmodulin-dependent protein kinase phosphorylation site with the heart.
Area of Science:
- Molecular Biology
- Neuroscience
- Cardiology
Background:
- The ryanodine receptor (RyR) is a critical calcium channel involved in excitation-contraction coupling in muscle and neurotransmission in the brain.
- Different RyR isoforms exist, with the cardiac isoform known to be regulated by Ca2+/calmodulin-dependent protein kinase (CaM kinase) phosphorylation.
- The presence and function of the cardiac RyR isoform in the mammalian brain were previously unclear.
Purpose of the Study:
- To identify and characterize the ryanodine receptor isoform present in the mammalian brain.
- To investigate whether the brain ryanodine receptor is a substrate for CaM kinase.
- To determine if the CaM kinase phosphorylation site is conserved between cardiac and brain ryanodine receptors.
Main Methods:
- Generation of a cardiac isoform-specific antiserum against a unique region of the cardiac ryanodine receptor.
- Immunoprecipitation of solubilized guinea pig brain membranes using the cardiac-specific antiserum.
- Analysis of immunoprecipitated brain receptors by SDS-PAGE and comparison with cardiac receptors.
- In vitro phosphorylation assays using CaM kinase on both brain and cardiac receptors.
- Affinity purification of site-specific antibodies to block phosphorylation.
- Two-dimensional peptide mapping of phosphorylated and iodinated receptors from both tissues.
Main Results:
- Cardiac-specific antiserum immunoprecipitated over 90% of [3H]ryanodine binding sites from guinea pig brain membranes.
- The immunoprecipitated brain receptor displayed cardiac-type mobility on SDS-PAGE.
- Both brain and cardiac ryanodine receptors were phosphorylated by CaM kinase.
- Site-specific antibodies blocked phosphorylation of both receptors, and peptide mapping revealed identical major 32P-labeled peptides.
- 125I-labeled receptors also yielded identical peptide maps.
Conclusions:
- Mammalian brain expresses the cardiac isoform of the ryanodine receptor.
- The CaM kinase phosphorylation site, crucial for regulating Ca2+ channel activity, is conserved in the brain's cardiac-type ryanodine receptor.
- These findings suggest a conserved regulatory mechanism for ryanodine receptors in both cardiac and neural tissues.
Abstract:
An antiserum raised against the region of the cardiac ryanodine receptor (residues 2805-2819) containing the phosphorylation site for multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) was used to identify the brain ryanodine receptor. This antiserum, which is cardiac isoform-specific, immunoprecipitated greater than 90% of the [3H]ryanodine receptor binding sites solubilized from guinea pig brain membranes. The immunoprecipitated brain receptor exhibited the characteristic cardiac-type mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The brain ryanodine receptor, like the cardiac ryanodine receptor, was a substrate for CaM kinase. Affinity-purified, site-specific antibodies completely blocked phosphorylation of both brain and cardiac receptors by CaM kinase, and two-dimensional peptide mapping identified the same major 32P-labeled peptide in receptors from both tissues. 125I-Labeled receptors also gave the same peptide maps. These results confirm that mammalian brain expresses the cardiac isoform of the ryanodine receptor. Furthermore, the unique CaM kinase phosphorylation site, which has been shown to regulate Ca2+ channel activity, is conserved.