Structural and functional interactions between the Ca(2+)-, ATP-, and caffeine-binding sites of skeletal muscle ryanodine receptor (RyR1)

Ryanodine receptor type 1 (RyR1) releases Ca(2+) ions from the sarcoplasmic reticulum of skeletal muscle cells to initiate muscle contraction. Multiple endogenous and exogenous effectors regulate RyR1, such as ATP, Ca(2+), caffeine (Caf), and ryanodine. Cryo-EM identified binding sites for the three coactivators Ca(2+), ATP, and Caf. However, the mechanism of coregulation and synergy between these activators remains to be determined. Here, we used [(3)H]ryanodine ligand-binding assays and molecular dynamics simulations to test the hypothesis that both the ATP- and Caf-binding sites communicate with the Ca(2+)-binding site to sensitize RyR1 to Ca(2+). We report that either phosphomethylphosphonic acid adenylate ester (AMPPCP), a nonhydrolyzable ATP analog, or Caf can activate RyR1 in the absence or the presence of Ca(2+). However, enhanced RyR1 activation occurred in the presence of Ca(2+), AMPPCP, and Caf. In the absence of Ca(2+), Na(+) inhibited [(3)H]ryanodine binding without impairing RyR1 activation by AMPPCP and Caf. Computational analysis suggested that Ca(2+)-, ATP-, and Caf-binding sites modulate RyR1 protein stability through interactions with the carboxyterminal domain and other domains in the activation core. In the presence of ATP and Caf but the absence of Ca(2+), Na(+) is predicted to inhibit RyR1 by interacting with the Ca(2+)-binding site. Our data suggested that ATP and Caf binding affected the conformation of the Ca(2+)-binding site, and conversely, Ca(2+) binding affected the conformation of the ATP- and Caf-binding sites. We conclude that Ca(2+), ATP, and Caf regulate RyR1 through a network of allosteric interactions involving the Ca(2+)-, ATP-, and Caf-binding sites.