Ultrastructural Analysis of Self-Associated RyR2s

Abstract

In heart, type-2 ryanodine receptor (RyR2) forms discrete supramolecular clusters in the sarcoplasmic reticulum known as calcium release units (CRUs), which are responsible for most of the Ca(2+) released for muscle contraction. To learn about the substructure of the CRU, we sought to determine whether RyR2s have the ability to self-associate in the absence of other factors and if so, whether they do it in a specific manner. Purified RyR2 was negatively stained and imaged on the transmission electron microscope, and RyR2 particles closely associated were further analyzed using bias-free multivariate statistical analysis and classification. The resulting two-dimensional averages show that RyR2s can interact in two rigid, reproducible configurations: "adjoining", with two RyR2s alongside each other, and "oblique", with two partially overlapped RyR2s forming an angle of 12°. The two configurations are nearly identical under two extreme physiological Ca(2+) concentrations. Pseudo-atomic models for these two interactions indicate that the adjoining interaction involves contacts between the P1, SPRY1 and the helical domains. The oblique interaction is mediated by extensive contacts between the SPRY1 domains (domains 9) and P1 domains (domains 10) of both RyR2s and not through domain 6 as previously thought; in addition its asymmetric interface imposes steric constrains that inhibit the growth of RyR2 as a checkerboard, which is the configuration usually assumed, and generates new configurations, i.e., "branched" and "interlocked". This first, to our knowledge, structural detailed analysis of the inter-RyR2 interactions helps to understand important morphological and functional aspects of the CRU in the context of cardiac EC coupling.

Size Matters: Ryanodine Receptor Cluster Size Affects Arrhythmogenic Sarcoplasmic Reticulum Calcium Release

“…Seminal quantitative electron microscopy by Franzini‐Armstrong et al4 inferred a typical RyR cluster in rat ventricular myocyte junctions to be 267 in circular arrays, requiring a fully packed checkerboard array of 553 nm diameter (for 30‐nm center‐center RyR distance) 41, 42. The value of 267 RyRs per cluster may be at the high end of normal for 3 reasons.…”

Junctophilin-2 in the nanoscale organisation and functional signalling of ryanodine receptor clusters in cardiomyocytes

“…the model by Walker et al (2015)]. Increased RyR channel distances within a cluster might also secondarily reduce channel excitability by decreasing the likelihood of inter-channel interactions, such as those assumed in quasi-crystalline arrays, in which each channel can physically interact with up to four neighbouring channels (Yin et al, 2005;Cabra et al, 2016). This arrangement has been suggested to contribute to co-operative channel opening through coupled gating (Marx et al, 2001), which, if normally physiologically present, could potentially be reduced in JPH2-OE myocytes.…”

Ryanodine Receptor Structure and Function in Health and Disease

“…RyRs are known to form paracrystalline arrays in checkerboard patterns (Franzini-Armstrong et al 1999; Cabra et al 2016). This feature provides a mechanism by which opening of a single channel can mechanically trigger neighboring channels and greatly amplify Ca 2+ release via coupled gating (Marx et al 1998).…”