title: Structural determinants of skeletal muscle ryanodine receptor gating.
abstract: Ryanodine receptor type 1 (RyR1) releases Ca(2+) 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 Ca(2+). 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 pore-lining 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.
authors: Department of Biochemistry and Biophysics, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7260, USA.
authors: Srinivas
authors: S
authors: Ramachandran
authors: Asima
authors: A
authors: Chakraborty
authors: Le
authors: L
authors: Xu
authors: Yingwu
authors: Y
authors: Mei
authors: Montserrat
authors: M
authors: Samsó
authors: Nikolay V
authors: NV
authors: Dokholyan
authors: Gerhard
authors: G
authors: Meissner
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000378:metabolism; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D019837:Ryanodine Receptor Calcium Release Channel