The central domain of cardiac ryanodine receptor governs channel activation, regulation, and stability

Structural analyses identified the central domain of ryanodine receptor (RyR) as a transducer converting conformational changes in the cytoplasmic platform to the RyR gate. The central domain is also a regulatory hub encompassing the Ca²⁺-, ATP-, and caffeine-binding sites. However, the role of the central domain in RyR activation and regulation has yet to be defined. Here, we mutated five residues that form the Ca²⁺ activation site and 10 residues with negatively charged or oxygen-containing side chains near the Ca²⁺ activation site. We also generated eight disease-associated mutations within the central domain of RyR2. We determined the effect of these mutations on Ca²⁺, ATP, and caffeine activation and Mg²⁺ inhibition of RyR2. Mutating the Ca²⁺ activation site markedly reduced the sensitivity of RyR2 to Ca²⁺ and caffeine activation. Unexpectedly, Ca²⁺ activation site mutation E3848A substantially enhanced the Ca²⁺-independent basal activity of RyR2, suggesting that E3848A may also affect the stability of the closed state of RyR2. Mutations in the Ca²⁺ activation site also abolished the effect of ATP/caffeine on the Ca²⁺-independent basal activity, suggesting that the Ca²⁺ activation site is also a critical determinant of ATP/caffeine action. Mutating residues with negatively charged or oxygen-containing side chains near the Ca²⁺ activation site significantly altered Ca²⁺ and caffeine activation and reduced Mg²⁺ inhibition. Furthermore, disease-associated RyR2 mutations within the central domain significantly enhanced Ca²⁺ and caffeine activation and reduced Mg²⁺ inhibition. Our data demonstrate that the central domain plays an important role in channel activation, channel regulation, and closed state stability.