Structural determinants of skeletal muscle ryanodine receptor gating

Srinivas Ramachandran, Asima Chakraborty, Le Xu, Yingwu Mei, Montserrat Samsó, Nikolay V. Dokholyan, Gerhard Meissner

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Abstract

Ryanodine receptor type 1 (RyR1) releases Ca2+ from intracellular stores upon nerve impulse to trigger skeletal muscle contraction. Effector binding at the cytoplasmic domain tightly controls gating of the pore domain of RyR1 to release Ca2+. However, the molecular mechanism that links effector binding to channel gating is unknown due to lack of structural data. Here, we used a combination of computational and electrophysiological methods and cryo-EM densities to generate structural models of the open and closed states of RyR1. Using our structural models, we identified an interface between the porelining helix (Tyr-4912-Glu-4948) and a linker helix (Val-4830- Val-4841) that lies parallel to the cytoplasmic membrane leaflet. To test the hypothesis that this interface controls RyR1 gating, we designed mutations in the linker helix to stabilize either the open (V4830W and T4840W) or closed (H4832W and G4834W) state and validated them using single channel experiments. To further confirm this interface, we designed mutations in the pore-lining helix to stabilize the closed state (Q4947N, Q4947T, and Q4947S), which we also validated using single channel experiments. The channel conductance and selectivity of the mutations that we designed in the linker and pore-lining helices were indistinguishable from those of WT RyR1, demonstrating our ability to modulate RyR1 gating without affecting ion permeation. Our integrated computational and experimental approach significantly advances the understanding of the structure and function of an unusually large ion channel.

Original languageEnglish (US)
Pages (from-to)6154-6165
Number of pages12
JournalJournal of Biological Chemistry
Volume288
Issue number9
DOIs
Publication statusPublished - Mar 1 2013

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All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Ramachandran, S., Chakraborty, A., Xu, L., Mei, Y., Samsó, M., Dokholyan, N. V., & Meissner, G. (2013). Structural determinants of skeletal muscle ryanodine receptor gating. Journal of Biological Chemistry, 288(9), 6154-6165. https://doi.org/10.1074/jbc.M112.433789