description): Ryanodine receptors (RyRs) are intracellular calcium-releasing channels found in many cell types. They play a key role in regulating cytoplasmic [Ca2+], which controls such basic processes as contraction, secretion, mitosis, and neuroplasticity. RyRs are enriched in striated muscle where they play a central role in excitation-contraction (e-c) coupling, the process by which neuronal depolarization of the muscle membrane leads to release of Ca2+ from the lumen of the sarcoplasmic reticulum into the cytoplasm. A dozen or more mutations have been discovered in skeletal RyRs that together are responsible for two diseases, malignant hyperthermia and central core disease. The potentially important roles of RyRs in heart and brain function (and pathology) are also of current interest. RyRs are tetrameric complexes of net molecular masses greater than 2 million Da, making them the largest ion channels. Further, they interact with other proteins to form complex signaling assemblies, particularly in muscle. High-resolution electron microscopy of purified, frozen RyRs, combined with computerized three-dimensional image reconstruction techniques, offers the methodology for determining the structures of large, multicomponent complexes. This technology will be applied to the following problems: (1) How is the amino acid sequence of the RyR subunit distributed among the various structural domains (at least 11) that have been detected in 3D reconstructions. To accomplish this goal, structures will be determined of RyR:antibody (or Fab) complexes at moderate resolution (2-3nm) using antibodies that are specific to epitopes containing known amino acids within the overall RyR sequence (about 5,000 residues). In collaboration with another laboratory, an alternative molecular-biological approach will be pursued in which recombinant receptors will be prepared that contain epitope tags inserted into surface-exposed regions.(2) Where do natural protein ligands bind on the surface of the RyR? For example, the interaction between the RyR and the dihydropyridine receptor will be investigated, which is thought to be crucial to the mechanism in e-c coupling. (3) How do the three known genetic isoforms of the RyR differ in structure? Each isoform has slightly different properties and physiological roles. For example, only isoform 1 will mediate normal e-c coupling in skeletal muscle. Comparisons of the isoforms in defined functional states (e.g., open, closed, inactivated)in increasing detail will aid in understanding the molecular basis of their different properties and illuminate the structural basis of channel gating.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR040615-10
Application #
6374925
Study Section
Cardiovascular and Pulmonary Research A Study Section (CVA)
Program Officer
Lymn, Richard W
Project Start
1991-04-10
Project End
2005-07-31
Budget Start
2001-08-01
Budget End
2002-07-31
Support Year
10
Fiscal Year
2001
Total Cost
$301,520
Indirect Cost
Name
Wadsworth Center
Department
Type
DUNS #
110521739
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 25:4823
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
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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