Excitation-contraction (EC) coupling in skeletal muscle refers to the process by which depolarization of the muscle plasma membrane leads to release of calcium ions into the cytoplasm from the sarcoplasmic reticulum (SR). EC coupling is performed by an array of voltage-sensor proteins (dihydropyridine receptors (DHPRs) in the plasma membrane/transverse tubule system that physically interact with and control an array of SR- embedded calcium-release channels (ryanodine receptors (RyRs), in conjunction with numerous other proteins. This assemblage of proteins (termed a couplon) occurs at regular intervals in muscle, and elucidating its structural architecture, ultimately at the atomic level, is essential to understanding the molecular mechanism of EC coupling in healthy and diseased muscle. Current models of the couplon structure are based largely upon visual interpretation of classical electron microscopy (EM) images, and are highly schematized and qualitative.
The first aim of this proposal is to use the latest quantitative cryo-EM and tomographic reconstruction techniques to determine actual mass density distributions of the proteins and membranes comprising the couplon. Focused-ion-beam milling, a new technique for cutting sections of frozen-hydrated tissue that is being developed at our Center, will be used to prepare muscle for cryo-EM;the micrographs and tomograms obtained using this new technique will be compared to those obtained by the more standard technique of cryo-ultramicrotomy. Once optimal methodology is established, hundreds of tomograms will be obtained, and RyRs, which, being large 2.3 MDa complexes, are easily detected in tomograms, will be computationally extracted, classified, and averaged to maximize the contrast and resolution (the goal is 3-4.5 nm). The averaged RyRs are expected to resolve RyR-associated proteins, such as the RyR-DHPR complex which has never been observed.
For aim 2, in vitro assembly experiments will be done to make complexes of purified RyR with its natural ligands, such as components of the DHPR and the regulatory protein calmodulin. Cryo-EM and 3D single-particle image analysis will be applied to these complexes to determine in more detail the nature of the interactions and dynamics of RyR and its binding partners.

Public Health Relevance

Muscle diseases such as malignant hyperthermia and central-core disease result from inherited mutations of protein components of the excitation-contraction apparatus, including the ryanodine receptor and the dihydropyridine receptor. This proposal will use advanced electron microscopy technology to characterize the structural organization and interactions among these proteins. The findings will be important for understanding normal and diseased muscle function.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Research Project (R01)
Project #
Application #
Study Section
Skeletal Muscle and Exercise Physiology Study Section (SMEP)
Program Officer
Boyce, Amanda T
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Wadsworth Center
United States
Zip Code
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