Quantitative Single-Molecule Analysis of Ryanodine Receptor 2 Subunit Assembly in Cardiac and Neuronal Tissues

We developed a method for ex vivo receptor
encapsulation
and single-molecule imaging techniques from neuronal and cardiac tissues,
illustrating the method’s broad applicability for measuring
membrane receptor assembly. Ryanodine receptor 2 (RyR2) is a tetrameric
Ca2+ channel governing intracellular Ca2+ dynamics,
which is critical for muscle contraction. Employing GFP-RyR2 knock-in
mice, we isolated individual receptor proteins in tissue-specific
nanovesicles and performed subunit counting analyses to yield quantitative
assessment of stoichiometric distributions across different organs.
With this method, we explored the potential heterogeneity of brain-derived
RyR2, which has been reported to form heteromeric assemblies with
other ryanodine receptor isoforms.