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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37AR018687-37
Application #
8287699
Study Section
Skeletal Muscle and Exercise Physiology Study Section (SMEP)
Program Officer
Boyce, Amanda T
Project Start
1976-05-01
Project End
2015-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
37
Fiscal Year
2012
Total Cost
$394,521
Indirect Cost
$127,157
Name
University of North Carolina Chapel Hill
Department
Biochemistry
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
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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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Petrotchenko, Evgeniy V; Yamaguchi, Naohiro; Pasek, Daniel A et al. (2011) Mass spectrometric analysis and mutagenesis predict involvement of multiple cysteines in redox regulation of the skeletal muscle ryanodine receptor ion channel complex. Res Rep Biol 2011:13-21
Yamaguchi, Naohiro; Prosser, Benjamin L; Ghassemi, Farshid et al. (2011) Modulation of sarcoplasmic reticulum Ca2+ release in skeletal muscle expressing ryanodine receptor impaired in regulation by calmodulin and S100A1. Am J Physiol Cell Physiol 300:C998-C1012
Kovacs, Erika; Xu, Le; Pasek, Daniel A et al. (2010) Regulation of ryanodine receptors by sphingosylphosphorylcholine: involvement of both calmodulin-dependent and -independent mechanisms. Biochem Biophys Res Commun 401:281-6
Zhou, Haiyan; Lillis, Suzanne; Loy, Ryan E et al. (2010) Multi-minicore disease and atypical periodic paralysis associated with novel mutations in the skeletal muscle ryanodine receptor (RYR1) gene. Neuromuscul Disord 20:166-73

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