The goal of the proposed research is to uncover the molecular basis of channel opening and Ca2+ permeability of the skeletal muscle Ca2+ release channel (ryanodine receptor, RyR1). RyR1 is a 2,200 kDa ion channel that releases Ca2+ ions in response to an action potential from the sarcoplasmic reticulum (SR), an intracellular Ca2+-storing compartment in skeletal muscle. The release channel has a high conductance for monovalent (~800 pS with 250 mM K+ as conducting ion) and divalent cations (~150 pS with 50 mM Ca2+) yet is selective for Ca2+ (PCa/PK ~7). However, the molecular basis of the unique ion permeability properties is poorly understood. The principal hypothesis to be tested in the proposed research is that a combined experimental and computational approach will substantially increase our knowledge of the molecular determinants responsible for channel opening and the high ion transport rates of RyR1.
Three specific aims are to (i) to apply a novel computational methodology of finding a protein conformational ensemble consistent with cryo-electron microscopy and sequence mapping data to build a structural model comprised of the 6 predicted transmembrane segments of RyR1, (ii) apply all-atom umbrella-sampling simulations to calculate the free energy profile of ion translocation through the pore of wild type, engineered and disease-associated RyR1 mutants, and (iii) develop multiscale modeling tools, which will involve the incorporation of atomistic details of ion-pore interactions into long time-scale discrete molecular dynamics, to directly quantify ionic currents in the channel. Pore mutants already available will provide the basis for modeling channel structure and the flow of ions. These include mutants linked to central core and multi minicore diseases. Computational data in turn will be critically tested by generating additional RyR1 mutants and determining their ion permeability properties in single channel recordings using the planar lipid bilayer method. As our studies progress we expect to gain new insights in the complex mechanism of RyR1 opening, ion conductance and selectivity and how these processes are altered by mutations in RyR1 linked to core myopathies.

Public Health Relevance

The skeletal muscle Ca2+ release channel (type 1 ryanodine receptor) plays a key role in skeletal muscle by releasing Ca2+ ions required for muscle to contract. The proposed research will use experimental and computational approaches to advance our understanding of the molecular mechanisms responsible for channel opening and high ion flux rates of RyR1. Study of disease-associated mutants will directly contribute to a better understanding of mechanisms underlying an aberrant function of the release channel in humans.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Method to Extend Research in Time (MERIT) Award (R37)
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Skeletal Muscle and Exercise Physiology Study Section (SMEP)
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Boyce, Amanda T
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University of North Carolina Chapel Hill
Schools of Medicine
Chapel Hill
United States
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Xu, Le; Mowrey, David D; Chirasani, Venkat R et al. (2018) G4941K substitution in the pore-lining S6 helix of the skeletal muscle ryanodine receptor increases RyR1 sensitivity to cytosolic and luminal Ca2. J Biol Chem 293:2015-2028
Mowrey, David D; Xu, Le; Mei, Yingwu et al. (2017) Ion-pulling simulations provide insights into the mechanisms of channel opening of the skeletal muscle ryanodine receptor. J Biol Chem 292:12947-12958
Meissner, Gerhard (2017) The structural basis of ryanodine receptor ion channel function. J Gen Physiol 149:1065-1089
Xu, Le; Gomez, Angela C; Pasek, Daniel A et al. (2017) Two EF-hand motifs in ryanodine receptor calcium release channels contribute to isoform-specific regulation by calmodulin. Cell Calcium 66:62-70
Mei, Yingwu; Xu, Le; Mowrey, David D et al. (2015) Channel Gating Dependence on Pore Lining Helix Glycine Residues in Skeletal Muscle Ryanodine Receptor. J Biol Chem 290:17535-45
Shirvanyants, David; Ramachandran, Srinivas; Mei, Yingwu et al. (2014) Pore dynamics and conductance of RyR1 transmembrane domain. Biophys J 106:2375-84
Gillespie, Dirk; Xu, Le; Meissner, Gerhard (2014) Selecting ions by size in a calcium channel: the ryanodine receptor case study. Biophys J 107:2263-73
Ramachandran, Srinivas; Chakraborty, Asima; Xu, Le et al. (2013) Structural determinants of skeletal muscle ryanodine receptor gating. J Biol Chem 288:6154-65
Manno, Carlo; Figueroa, Lourdes; Royer, Leandro et al. (2013) Altered Ca2+ concentration, permeability and buffering in the myofibre Ca2+ store of a mouse model of malignant hyperthermia. J Physiol 591:4439-57
Mei, Yingwu; Xu, Le; Kramer, Henning F et al. (2013) Stabilization of the skeletal muscle ryanodine receptor ion channel-FKBP12 complex by the 1,4-benzothiazepine derivative S107. PLoS One 8:e54208

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