Old dog, new trick: Charybdotoxin as a novel ryanodine receptor blocker

Ryanodine receptors (RyRs) are Ca^{2 +}-release channels of the sarcoplasmic reticulum. Because uncontrolled Ca^{2 +}-release underlies several muscle and cardiac disorders, pharmacological inhibition of RyRs represents an attractive therapeutic strategy. Natural peptide toxins have provided valuable molecular templates for inhibitor design, with calcins being a particularly promising group. Calcins bind RyRs with high specificity, but, instead of producing the desired full channel block, they stabilize subconductance states that promote Ca^{2 +} leak. Given the mechanistic similarity between calcins' action and the well-known charybdotoxin (ChTX)-mediated K⁺ channel blockade, we examined whether ChTX showed functional cross-reactivity with RyR channels. Single-channel recordings were performed on skeletal muscle RyRs reconstituted into lipid bilayers. ChTX induced long-lasting (>0.5 s) closed events (LLCEs) in a voltage-dependent manner with an apparent K_d of 85 nm at +60 mV. Under these conditions, channels spent ∼10 s min^-1 in the LLCE state. Notably, isosteric substitution of K27, which is essential for K⁺ channel block, retained RyR inhibition, indicating a pore interaction mechanistically distinct from that described in K⁺ channels. Cardiac RyRs were similarly affected by the K27N variant. Guided by in silico docking to the RyR cryo-electron microscopy structure, we mapped the ChTX-RyR interaction surface using five additional ChTX variants, focusing on basic residues. We found that the R25Q and R34Q substitutions abolished LLCE formation, whereas others had no effect or enhanced channel block. These results describe a novel ChTX-RyR interaction mode functionally convergent with calcin peptides, and they also identify K27N ChTX as a selective RyR inhibitor and a promising scaffold for future drug design. KEY POINTS: Charybdotoxin (ChTX) blocks ryanodine receptor (RyR) single channel currents in bilayer recordings. Amino acid residues R25 and R34 are essential for the block. K27, which is important for K+ channel block, is irrelevant in RyR block; thereby, the K27N ChTX variant represents a RyR-selective blocker. ChTX shares a common binding site with calcin peptides in the channel vestibule, outside the gate.