Ryanodine receptors (RyRs) are intracellular calcium channels that are particularly important in skeletal and cardiac muscle where they play a key role in excitation-contraction (E-C) coupling, the process by which neuron-induced depolarization of the plasma membrane (sarcolemma) causes release of calcium ions from the sarcoplasmic reticulum (via RyRs). Mutated and malfunctioning RyRs have been implicated in certain diseases of skeletal (malignant hyperthermia, central-core disease) and, quite recently, cardiac muscle (two forms of sudden cardiac death, heart failure). In cardiac muscle RyRs also play important roles in pacemaker activity and in the generation of arrhythmogenesis. By characterizing the structure of RyRs and their interactions with associated proteins we expect to advance our understanding of RyRs' roles in E-C coupling mechanisms and in heart and skeletal muscle diseases. Purified RyRs from either natural sources (mammalian skeletal & cardiac muscle) or from transfected cultured cells that express mutated receptors will be characterized by cryo-electron microscopy and single-particle image processing to generate three- dimensional reconstructions (3D-cryoEM) at 10-30 Angstrom resolution.
The specific aims of this proposal are to: (1) Determine the location of surface-exposed amino acids and regulatory sites by tagging them with green fluorescent protein or other macromolecular ligands, and (2) Characterize dynamical aspects of RyR function at increasing resolution by 3D cryo-EM and time-resolved cryo-EM. Specifically, the mechanisms by which the numerous domains that comprise the structure interact with each other to regulate channel gating by means of very long-range (>100 Angstroms) communication will be determined. Additionally, a hypothesized disruption of interdomain interactions by mutations that cause skeletal and sudden cardiac death diseases will be characterized, as well as the structural transitions that occur upon RyR regulation by calmodulin.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
2R01AR040615-14
Application #
6967923
Study Section
Electrical Signaling, Ion Transport, and Arrhythmias Study Section (ESTA)
Program Officer
Nuckolls, Glen H
Project Start
1991-04-10
Project End
2010-07-31
Budget Start
2005-09-05
Budget End
2006-07-31
Support Year
14
Fiscal Year
2005
Total Cost
$435,234
Indirect Cost
Name
Wadsworth Center
Department
Type
DUNS #
153695478
City
Menands
State
NY
Country
United States
Zip Code
12204
Hu, Hongli; Meng, Xing (2016) Observation of Network Dynamics of Ryanodine Receptors on Skeletal Muscle Sarcoplasmic Reticulum Membranes. Eur J Transl Myol 26:5805
Wagenknecht, Terence; Hsieh, Chyongere; Marko, Michael (2015) Skeletal muscle triad junction ultrastructure by Focused-Ion-Beam milling of muscle and Cryo-Electron Tomography. Eur J Transl Myol 25:49-56
Wagenknecht, Terence; Hsieh, Chyongere; Marko, Michael (2015) Skeletal Muscle Triad Junction Ultrastructure by Focused-Ion-Beam Milling of Muscle and Cryo-Electron Tomography. Eur J Transl Myol 25:4823
Hsieh, Chyongere; Schmelzer, Thomas; Kishchenko, Gregory et al. (2014) Practical workflow for cryo focused-ion-beam milling of tissues and cells for cryo-TEM tomography. J Struct Biol 185:32-41
Tian, Xixi; Liu, Yingjie; Liu, Ying et al. (2013) Ligand-dependent conformational changes in the clamp region of the cardiac ryanodine receptor. J Biol Chem 288:4066-75
Strauss, Joshua D; Wagenknecht, Terence (2013) Structure of glutaraldehyde cross-linked ryanodine receptor. J Struct Biol 181:300-6
Huang, Xiaojun; Liu, Ying; Wang, Ruiwu et al. (2013) Two potential calmodulin-binding sequences in the ryanodine receptor contribute to a mobile, intra-subunit calmodulin-binding domain. J Cell Sci 126:4527-35
Zhong, Xiaowei; Liu, Ying; Zhu, Li et al. (2013) Conformational dynamics inside amino-terminal disease hotspot of ryanodine receptor. Structure 21:2051-60
Liu, Ying; Meng, Xing; Liu, Zheng (2013) Deformed grids for single-particle cryo-electron microscopy of specimens exhibiting a preferred orientation. J Struct Biol 182:255-8
Huang, Xiaojun; Fruen, Bradley; Farrington, Dinah T et al. (2012) Calmodulin-binding locations on the skeletal and cardiac ryanodine receptors. J Biol Chem 287:30328-35

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