Effect of Calcium in the Cardiac Ryanodine Receptor Inter-Molecular Contacts

Abstract

The cardiac ryanodine receptor (RyR2) is the major calcium (Ca2+) release channel on the sarcoplasmic reticulum (SR) in cardiomyocytes. The RYR2 gene encodes a large ∼565 kDa protein that forms homotetramers with molecular weight ∼2.26 MDa. They are organized in a large squared cytoplasmic domain of 290 x 290 x 100 A connected to a smaller, square tapering prism shaped transmembrane domain of 120 x 120 x 60 A. The corners of the cytoplasmic domain are named the clamp domains and the flat, slab-shaped sides define the handle domains.The dyads are structural elements formed by the close apposition of the plasmalemma and the junctional sarcoplasmic reticulum (jSR), while a couplon is the functional element within the dyad formed by juxtaposition of cardiac L-type voltage-gated Ca2+ channels (Cav 1.2) in the plasmalemma with RyR2 in the adjacent jSR. The clustering of RyR2 into functional Ca2+ release units is central to current models for cardiac excitation-contraction (E-C) coupling.Classical studies done in dyads suggested RyR2 inter-molecular contacts through their clamp domains, but more recent studies propose multiple and complex RyR2 arrangements, which can be modulated by diverse local factors like Mg2+ concentration, phosphorylation and redox state.In the present study the effect of the Ca2+ in RyR2-RyR2 contacts was investigated. RyR2s purified from pig cardiac muscle were incubated in 100μM Ca2+ or in 2 mM EGTA, negatively stained, imaged on the electron microscope and image-processed, yielding well-resolved images of RyR2 dimers. In addition to the clamp-clamp domain interactions, we find clamp-handle domain contacts in multiple configurations, and that their relative proportions depend on the presence or absence of Ca2+. These different RyR2-RyR2 interactions could have a vital role in various physiological and pathological conditions.