PHOSPHORYLATION OF RyR2 SIMULTANEOUSLY EXPANDS THE DYAD AND REARRANGES THE TETRAMERS

Area of Science:

• Cardiovascular Physiology
• Molecular Cell Biology
• Biophysics

Background:

• Ryanodine receptors (RyR2) are crucial for excitation-contraction coupling in cardiomyocytes.
• Previous studies suggested RyR2 tetramer rearrangement upon phosphorylation, but the direct role of RyR2 phosphorylation remained unclear.
• Clinically relevant mutations in RyR2 can impact cardiac function.

Approach:

• Utilized isoproterenol (ISO) stimulation in mice with specific RyR2 phosphorylation site mutations (S2030A, S2808A, S2814A, S2814D).
• Employed transmission electron microscopy (TEM) and dual-tilt electron tomography to measure dyad length and visualize RyR2 distribution.
• Correlated structural findings with existing functional data from the same mutants.

Key Points:

• The S2814D mutation alone expanded dyads and reorganized RyR2 tetramers, indicating a direct link between phosphorylation state and microarchitecture.
• ISO-induced dyad expansion occurred in wild-type, S2808A, and S2814A mice, but not S2030A mice.
• S2030 and S2808 phosphorylation sites were essential for a full β-adrenergic response, while S2814 was not.
• Each mutation uniquely affected RyR2 tetramer array organization.
• Tetramer-tetramer contacts are functionally significant.

Conclusions:

• RyR2 phosphorylation state directly influences dyad microarchitecture and RyR2 tetramer organization.
• The arrangement of RyR2 tetramers is dynamically regulated by β-adrenergic receptor agonists.
• Structural changes in the dyad and RyR2 arrangement are intrinsically linked to channel function.