Structural basis for Ca ²⁺ -dependent allosteric modulation of cardiac ryanodine receptor by Ryanozole

The cardiac ryanodine receptor (RyR2) is a Ca ²⁺ release channel essential for excitation-contraction coupling. RyR2 mutations cause severe arrhythmogenic disorders, including catecholaminergic polymorphic ventricular tachycardia (CPVT), through gain-of-function (GOF) effects leading to aberrant Ca ²⁺ release. We have recently developed Ryanozole, a potential therapeutic compound for CPVT, which selectively stabilizes RyR2 in a Ca ²⁺ -dependent manner. Here, we define the mechanism of action of Ryanozole by combining high-resolution cryo-electron microscopy, targeted mutagenesis and functional assays. Ryanozole binds to the interface between the Ca ²⁺ -binding site and the pore-forming S6 helix, interfering with conformational changes required for Ca ²⁺ -induced channel opening at low Ca ²⁺ . We identified key residues for the binding and isoform-specific modulation of Ryanozole. Notably, Ryanozole-bound RyR2 retains its ability to open at high Ca ²⁺ via unique conformational changes. These findings provide a structural basis for CPVT-targeted therapy and redefine the paradigm of small molecule-based regulation of large ion channels.