The long-term goal of this proposal is to determine the three-dimensional architecture and composition of the triad junction in striated muscle by cryo-electron microscopy and computer image processing, including three- dimensional reconstruction. Triad junctions are specialized regions in muscle where invaginations of the plasmalemma known as t-tubules and the intracellular sarcoplasmic reticulum (SR) membrane system are in close apposition. Excitation-contraction (E-C) coupling refers to the process by which neuronally induced depolarization of the plasmalemma/t-tubules causes release of Ca2+ (the immediate stimulus of contraction) from the internal Ca2+ stores of the SR. The signal transducing events associated with E-C coupling occur at the triad junction by mechanism(s) that are incompletely understood. Knowledge of the structure of the triad is essential to fully understanding E-C coupling and the for the rational search for therapies to diseases of skeletal muscle and heart. In the main strategy that will be taken, for which substantial progress has already been made, the purified solubilized ryanodine receptor/calcium release channel (RyR), the major component (by mass) of the triad junction, will be studied in isolation and complexed with site-specific probes (e.g. amino-acid-sequence specific antibodies) and macromolecular ligands known or suspected to be constituents (e.g. FK-506 binding protein) or modulators (e.g. calmodulin) of the triad junction. Comparative studies of the rabbit skeletal muscle RyR with that from heart will be done to determine whether structural differences can be correlated with the apparently different mechanisms of E-C coupling in the two muscle types. We will refine the methods we have developed to reconstitute the solubilized RyR into lipid bilayers for structural studies; structural characterization of defined structural states of the RyR (e.g. open and closed) should then be feasible. We will explore a second strategy for determining the structure of the triad in which electron tomographic reconstruction techniques will be applied to tilt-series images of isolated (fixed, sectioned, and embedded) triad junctions. Isolated triads will also be imaged in the frozen- hydrated state to maintain structural integrity, and gold cluster- containing ligands will be used to identify and map components of the triad junction with much higher precision than has been possible by conventional immunoelectron microscopy.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR040615-08
Application #
2899860
Study Section
Physiology Study Section (PHY)
Program Officer
Lymn, Richard W
Project Start
1991-04-10
Project End
2000-08-10
Budget Start
1999-04-01
Budget End
2000-08-10
Support Year
8
Fiscal Year
1999
Total Cost
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

Showing the most recent 10 out of 46 publications