Structural Insight into the Phosphorylation Domain in Ryanodine Receptors

Abstract

Ryanodine receptors (RyRs) are large Ca2+-release channels located in the SR membrane. They play a central role in the excitation-contraction coupling of skeletal (RyR1) and cardiac (RyR2) muscle. Over 500 disease mutations have been identified in RyRs that can cause several skeletal muscle disorders and cardiac arrhythmias. RyRs are also the target for phosphorylation, most notably by CaMKII and PKA. Although a lot of controversy surrounds these events, several studies indicate that phosphorylation can upregulate RyR activity. Here we present crystal structures of a domain in all three different RyR isoforms, containing the Ser2843 (RyR1) and Ser2808/Ser2814 (RyR2) phosphorylation sites. There are 11 disease mutations located in the RyR1 domain, several of which cluster near the phosphorylation site, suggesting that phosphorylation and disease mutations may affect the same interface. Crystal structures of the disease mutants show that the mutations affect either surface properties or intradomain salt bridges. One mutation, L2867G, causes a drastic reduction of thermal stability of the domain, and results in aggregation at room temperature. In vitro phosphorylation experiments identify several novel PKA and CaMKII phosphorylation sites in the same loop region of the RyR2 phosphorylation domain, also supported by another recent in vivo study. The observation of simultaneous phosphorylation events on multiple sites indicates that RyRs may be truly ‘hyperphosphorylated'. Docking into cryo-electron microscopy maps locates the domain in the clamp region, a region that has been shown to couple allosterically to channel opening. Disease mutations and phosphorylation may therefore cause conformational changes that affect the allosteric coupling and facilitate channel opening.