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Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study
12:43

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Published on: July 27, 2016

Regulation of the cardiac ryanodine receptor by protein kinase-dependent phosphorylation

T Takasago1, T Imagawa, K Furukawa

  • 1Department of Molecular Physiology, National Cardiovascular Center Research Institute, Osaka.

Journal of Biochemistry
|January 1, 1991
PubMed

Insights

Protein kinases like PKA and PKC, along with calmodulin, regulate the cardiac ryanodine receptor. Phosphorylation by these enzymes impacts the Ca2+ release channel

Area of Science:

  • Cardiovascular Physiology
  • Molecular Biology
  • Biochemistry

Background:

  • The cardiac ryanodine receptor (RyR2) is a critical Ca2+ release channel in the sarcoplasmic reticulum.
  • Its function is tightly regulated by post-translational modifications, including phosphorylation.
  • Understanding these regulatory mechanisms is key to comprehending cardiac excitation-contraction coupling.

Purpose of the Study:

  • To investigate the role of various protein kinases and calmodulin in the phosphorylation of the cardiac ryanodine receptor.
  • To determine how these modifications affect the function and Ca2+ binding properties of the ryanodine receptor.

Main Methods:

  • Incubation of canine cardiac microsomes with [gamma-32P]ATP and catalytic subunits of PKA, PKG, PKC, or CaM.
  • Measurement of [3H]ryanodine binding to assess receptor activity.
  • Phosphopeptide mapping and phosphoamino acid analysis to identify phosphorylation sites.
  • Photoaffinity labeling with 125I-labeled CaM to confirm CaM binding to RyR2.

Main Results:

  • PKA, PKG, and PKC rapidly phosphorylated the cardiac ryanodine receptor, increasing [3H]ryanodine binding.
  • CaM also phosphorylated the receptor, but incubation with CaM alone or with ATP decreased [3H]ryanodine binding.
  • PKA, PKG, and PKC predominantly phosphorylated serine residues in peptide 1, while CaM-kinase acted on peptide 4.
  • CaM was shown to bind directly to the cardiac ryanodine receptor.

Conclusions:

  • Protein kinase-dependent phosphorylation and calmodulin play significant regulatory roles in cardiac sarcoplasmic reticulum Ca2+ release channel function.
  • These findings highlight complex signaling pathways modulating cardiac contractility.
  • Further research into these pathways could offer therapeutic targets for cardiac dysfunction.

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