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.