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Dynamic, inter-subunit interactions between the N-terminal and central mutation regions of cardiac ryanodine receptor

Zheng Liu1, Ruiwu Wang, Xixi Tian

  • 1Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA. liuz@wadsworth.org

Journal of Cell Science
|April 30, 2010
PubMed

Insights

Mutations in cardiac ryanodine receptor (RyR2) cause arrhythmias. Researchers developed a FRET sensor to study RyR2 dynamics, revealing inter-subunit interactions and potential drug targets for stabilizing RyR2.

Area of Science:

  • Cardiovascular Biology
  • Molecular Biophysics
  • Genetics

Background:

  • Naturally occurring mutations in the cardiac ryanodine receptor (RyR2) are associated with cardiac arrhythmias and sudden death.
  • Two key RyR2 mutation hotspots in the N-terminal and central regions are predicted to interact, forming a critical channel regulator switch.

Purpose of the Study:

  • To monitor the conformational dynamics of RyR2 involving N-terminal and central regions.
  • To develop a Förster Resonance Energy Transfer (FRET) based assay for RyR2 channel gating.
  • To investigate the potential of FRET analysis for identifying RyR2-stabilizing drugs.

Main Methods:

  • Constructed a dual fluorescent protein-labeled RyR2 (RyR2(S437-YFP/S2367-CFP)) by inserting yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) at specific RyR2 residues.
  • Transfected HEK293 cells with the labeled RyR2 constructs.
  • Examined FRET signals in live cells using confocal microscopy and assessed their modulation by RyR2 ligands and disease-mimicking peptides.

Main Results:

  • Successfully generated and validated a FRET pair within RyR2 (RyR2(S437-YFP/S2367-CFP)).
  • Demonstrated that FRET signals are sensitive to RyR2 modulators, disease-mimicking peptides, and drugs.
  • Confirmed that the N-terminal and central RyR2 regions interact via an inter-subunit mechanism, as evidenced by FRET signals in co-transfected cells.

Conclusions:

  • FRET analysis of the labeled RyR2 provides a powerful tool for investigating RyR2 conformational dynamics and channel gating.
  • This FRET-based approach can be utilized to screen for and identify novel drugs capable of stabilizing RyR2 conformations, potentially preventing arrhythmias.

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