A tryptophan residue in the caffeine-binding site of the ryanodine receptor regulates Ca²⁺ sensitivity

Ryanodine receptors (RyRs) are Ca²⁺ release channels in the sarcoplasmic reticulum of skeletal and cardiac muscles and are essential for muscle contraction. Mutations in genes encoding RyRs cause various muscle and arrhythmogenic heart diseases. Although RyR channels are activated by Ca²⁺, the actual mechanism of Ca²⁺ binding remains largely unknown. Here, we report the molecular basis of Ca²⁺ binding to RyRs for channel activation and discuss its implications in disease states. RyR1 and RyR2 carrying mutations in putative Ca²⁺ and caffeine-binding sites were functionally analysed. The results were interpreted with respect to recent near-atomic resolution RyR1 structures in various ligand states. We demonstrate that a tryptophan residue in the caffeine-binding site controls the structure of the Ca²⁺-binding site to regulate the Ca²⁺ sensitivity. Our results reveal the initial step of RyR channel activation by Ca²⁺ and explain the molecular mechanism of Ca²⁺ sensitization by caffeine and disease-causing mutations.