Molecular basis for allosteric regulation of RyR1 channel gating: effects of a therapeutic drug and oxidation-mimicking mutation

Abstract RyR1, an enormous, approximately 2.2 MDa, tetrameric calcium-release channel in the sarcoplasmic reticulum (SR) of skeletal muscle, is essential for excitation-contraction coupling. Pathogenic mutations in RyR1 cause RyR1-related myopathies. ARM210 (S48168), a Rycal-class drug, has shown promise in treating RyR1-related myopathies in a recently concluded phase 1b clinical trial. Structural studies revealed that ARM210 binds to the peripheral RY1&2 domain of RyR1, stabilizing it in a closed state to prevent aberrant SR Ca 2+ leak. Here, we investigate ARM210’s mechanism of action at the atomistic level using extensive all-atom (~2.5 million atoms) explicit-solvent molecular dynamics simulations totaling 36.6 μs. Simulations revealed a long-range allosteric pathway in the wild-type RyR1 that propagates over 200 Å, linking the RY1&2 domain to the pore-forming S6 helices of adjacent protomers and enabling pore opening. Subsequently, we demonstrated that ARM210 binding stabilizes the RY1&2 domain in a closed conformation, rigidifying the cytosolic shell and activation core and disrupting the allosteric network necessary for pore dilation. These findings describe a plausible mode of action for how ARM210 stabilizes RyR1 in a closed state by modulating long-range allostery. Furthermore, we show that this allosteric regulatory network is perturbed by the oxidative stress-mimicking C3892D mutation near the Ca 2+ activator site, identified via mass spectrometry, live-cell fluorescence microscopy, and electrophysiology.