Structure and gating of Ryanodine receptor in lipid environment

Katrien Willegems1,2 and Rouslan G. Efremov1,2

1 Center for Structural Biology, Vlaams Instituut voor Biotechnologie, 1050 Brussels, Belgium

2 Structural Biology Brussels, Department of Bioengineering Sciences, Vrije Universiteit Brussel, 1050 Brussels, Belgium

Concentration of calcium ions in cytoplasm of the cells regulates many physiological processes including transcription, cell cycle, apoptosis and muscular contraction. Calcium signalling in muscle contraction is mediated by ryanodine receptors, RyR, large 2.2 MDa homotetrameric ion channels that are primarily gated by changes in concentration of calcium ions in the cytoplasm and are regulated by multiple factors including ions, small organic molecules as well as via interactions with partner proteins. Here we used single particle cryo-EM to investigate the conformational transition in RyR1 from closed to open state for the protein reconstituted into lipid nanodiscs. We show that RyR1 reconstituted into lipid nanodiscs is stabilized in the open conformation when bound to the plant toxin ryanodine, but not in the presence of its physiological activators, calcium and ATP. Further, using ryanodine binding assays we show that membrane mimetics influence RyR1 transition between closed and open channel conformations. We observe that all detergents, including fluorinated detergent added to nanodiscs-reconstituted RyR1, stabilize closed state of RyR1. Further biochemical experiments support structural data and suggest optimal conditions for structural studies of RyR1 gating.

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