Abstract 2237 Cryo-EM analysis of scorpion toxin binding to Ryanodine Receptors reveals sub-conductance that is abolished by PKA phosphorylation

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We recently elucidated the interaction between scorpion venom-derived calcins and intracellular Ryanodine Receptors (RyRs). The study primarily focuses on uncovering the structural and functional dynamics of this interaction, leveraging cryo-electron microscopy (cryo-EM) for detailed insights. We analyzed the binding mechanism of calcins, small peptides from scorpion venom, to RyRs using a combination of single particle cryo-EM and electrophysiology. These channels, located within the endoplasmic and sarcoplasmic reticulum, play a crucial role in calcium ion flow. Our approach includes structural mapping of toxin-channel interaction, assessment of conductivity changes, and exploring the impact of post-translational modifications on toxin functionality. Results indicate that calcins bind within the RyR's cytosolic shell, near the transmembrane region, inducing a sub-conductance state. This binding elongates the ion conduction pathway, reducing the channel's conductivity. Notably, this study is the first to demonstrate the asymmetric conformational changes in RyRs upon toxin binding, which significantly affects subunit interactions. Additionally, we discovered that PKA phosphorylation of calcins prevents their binding to RyRs, highlighting a unique interplay between the toxin and intracellular enzymes. The conclusions drawn from this study are multi-fold: First, the observed binding mechanism of calcins offers a structural template for designing potent RyR blockers, potentially beneficial in treating disorders like heart failure and Alzheimer's disease. Second, the asymmetric conformational changes induced by calcins provide novel insights into the structural dynamics of RyRs. Lastly, the modulation of calcin function through PKA phosphorylation unveils a critical regulatory mechanism, suggesting a balance between kinase and phosphatase activities determines the functional outcome in cellular contexts. This work was funded by Canadian Institutes of Health Research grant PJT-159601 (F.V.P.), Canadian Institutes of Health Research fellowship MFE-381863 (O.H.-G.), Michael Smith Foundation for Health Research fellowship (O.H.-G.), Fonds de Recherche du Québec–Santé fellowship BF7-310636 (Y.S.C.), and National Institutes of Health grant R01-HL055438.