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Updated: Dec 11, 2025
Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study
Published on: July 27, 2016
Structural mechanism of two gain-of-function cardiac and skeletal RyR mutations at an equivalent site by cryo-EM
Kavita A Iyer1, Yifan Hu1, Ashok R Nayak1
1Department of Physiology and Biophysics, School of Medicine, Virginia Commonwealth University, Richmond, VA, USA.
Abstract:
Mutations in ryanodine receptors (RyRs), intracellular Ca2+ channels, are associated with deadly disorders. Despite abundant functional studies, the molecular mechanism of RyR malfunction remains elusive. We studied two single-point mutations at an equivalent site in the skeletal (RyR1 R164C) and cardiac (RyR2 R176Q) isoforms using ryanodine binding, Ca2+ imaging, and cryo-electron microscopy (cryo-EM) of the full-length protein. Loss of the positive charge had greater effect on the skeletal isoform, mediated via distortion of a salt bridge network, a molecular latch inducing rotation of a cytoplasmic domain, and partial progression to open-state traits of the large cytoplasmic assembly accompanied by alteration of the Ca2+ binding site, which concur with the major "hyperactive" feature of the mutated channel. Our cryo-EM studies demonstrated the allosteric effect of a mutation situated ~85 Å away from the pore and identified an isoform-specific structural effect.
Insights
Mutations in ryanodine receptors (RyRs) cause deadly disorders. Cryo-EM revealed how these RyR mutations alter channel structure and function, explaining their hyperactivity.
Area of Science:
- Molecular biology
- Structural biology
- Biophysics
Background:
- Ryanodine receptors (RyRs) are intracellular Ca2+ channels crucial for muscle function.
- Mutations in RyRs are linked to severe human diseases, but the underlying molecular mechanisms are not fully understood.
Purpose of the Study:
- To elucidate the molecular mechanisms of RyR malfunction caused by specific mutations.
- To investigate the structural and functional consequences of single-point mutations in skeletal (RyR1 R164C) and cardiac (RyR2 R176Q) RyR isoforms.
Main Methods:
- Cryo-electron microscopy (cryo-EM) of full-length RyR proteins.
- Functional assays including ryanodine binding and Ca2+ imaging.
Main Results:
- Loss of a positive charge at equivalent sites in RyR1 and RyR2 isoforms induced distinct structural changes.
- The RyR1 R164C mutation distorted a salt bridge network, causing cytoplasmic domain rotation and partial opening, leading to channel hyperactivity.
- Cryo-EM revealed allosteric effects of mutations distant from the pore and identified isoform-specific structural impacts.
Conclusions:
- The study provides a structural basis for RyR channel hyperactivation due to specific mutations.
- Understanding these isoform-specific structural effects is critical for developing therapeutic strategies for RyR-associated disorders.
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