The long-term goal of the proposed research is to identify the key regions within the primary sequence of theCa2+ release channel (ryanodine receptor;RyR1) of mammalian skeletal muscle sarcoplasmic reticulum (SR) which determine: 1. Sensitivity to physiologic activating and inhibiting cations, and 2. The ability of the channel to sense physiologic changes in local redox potential. Hypothesis I: Structural determinants of the cation activator and inhibitor sites are coordinated by spatially separated amino acids within the RyR1 linear sequence. Conserved negative charges within one or more of five cytoplasmic domains in the C-terminal -1000 amino acids of RyR1 coordinate cation binding and activation, whereas it appears that interactions between the C-terminal and central domains of RyR1, especially sequences encompassing difference regions D1 &D3, are needed to engage proper cation inhibition of RyRI.
Specific Aim 1. Define the structural determinants in RyR1 that influence its binding and sensitivity to Ca2+ and Mg2+ using site-specific mutations of C-terminal truncated channels and full length RyR1 along with RyR1/RyR3 chimera.
Specific Aim 2. Define the changes in cation binding constants and the activation energy (Ea) associated with channel activation for RyR1s with mutations within their cation binding motifs.
Specific Aim 3. To define how altered cation regulation of RyR1 impacts excitation-contraction coupling and excitation-coupled Ca2+ entry. Hypothesis II: Hyper-reactive sulfhydryls in the primary structure of RyR1 constitute a trans- SR redox gradient sensor.
Specific Aim 1. To mutate the six hyper-reactive thiols that we have discovered in RyR1 and define their contribution to trans SR redox sensing behavior.
Specific Aim 2. To establish mechanistic links between redox state and the ability of cations to regulate RyR1 activity. The proposed studies will provide new information concerning the molecular mechanisms by which microsomal calcium channels residing within the SR/ER membrane are regulated by two important physiological parameters, cations (Ca2+and Mg2+) and cellular redox state. It is likely that these mechanisms are conserved among the multi-member family of Ca2+ release channels and may be significant in identifying new clinical targets for the prevention and treatment of injury resulting from oxidative stress.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR043140-14
Application #
7825361
Study Section
Skeletal Muscle and Exercise Physiology Study Section (SMEP)
Program Officer
Boyce, Amanda T
Project Start
1996-04-14
Project End
2012-03-31
Budget Start
2010-04-01
Budget End
2012-03-31
Support Year
14
Fiscal Year
2010
Total Cost
$469,416
Indirect Cost
Name
Brigham and Women's Hospital
Department
Type
DUNS #
030811269
City
Boston
State
MA
Country
United States
Zip Code
02115
Henríquez-Olguín, Carlos; Altamirano, Francisco; Valladares, Denisse et al. (2015) Altered ROS production, NF-?B activation and interleukin-6 gene expression induced by electrical stimulation in dystrophic mdx skeletal muscle cells. Biochim Biophys Acta 1852:1410-9
Mijares, Alfredo; Altamirano, Francisco; Kolster, Juan et al. (2014) Age-dependent changes in diastolic Ca(2+) and Na(+) concentrations in dystrophic cardiomyopathy: Role of Ca(2+) entry and IP3. Biochem Biophys Res Commun 452:1054-9
Altamirano, Francisco; Eltit, José M; Robin, Gaëlle et al. (2014) Ca2+ influx via the Na+/Ca2+ exchanger is enhanced in malignant hyperthermia skeletal muscle. J Biol Chem 289:19180-90
Altamirano, Francisco; Perez, Claudio F; Liu, Min et al. (2014) Whole body periodic acceleration is an effective therapy to ameliorate muscular dystrophy in mdx mice. PLoS One 9:e106590
Manno, Carlo; Sztretye, Monika; Figueroa, Lourdes et al. (2013) Dynamic measurement of the calcium buffering properties of the sarcoplasmic reticulum in mouse skeletal muscle. J Physiol 591:423-42
Altamirano, Francisco; Valladares, Denisse; Henríquez-Olguín, Carlos et al. (2013) Nifedipine treatment reduces resting calcium concentration, oxidative and apoptotic gene expression, and improves muscle function in dystrophic mdx mice. PLoS One 8:e81222
Barrientos, Genaro C; Feng, Wei; Truong, Kim et al. (2012) Gene dose influences cellular and calcium channel dysregulation in heterozygous and homozygous T4826I-RYR1 malignant hyperthermia-susceptible muscle. J Biol Chem 287:2863-76
Altamirano, Francisco; López, Jose R; Henríquez, Carlos et al. (2012) Increased resting intracellular calcium modulates NF-?B-dependent inducible nitric-oxide synthase gene expression in dystrophic mdx skeletal myotubes. J Biol Chem 287:20876-87
Yuen, Benjamin; Boncompagni, Simona; Feng, Wei et al. (2012) Mice expressing T4826I-RYR1 are viable but exhibit sex- and genotype-dependent susceptibility to malignant hyperthermia and muscle damage. FASEB J 26:1311-22
Eltit, Jose Miguel; Li, Hongli; Ward, Christopher W et al. (2011) Orthograde dihydropyridine receptor signal regulates ryanodine receptor passive leak. Proc Natl Acad Sci U S A 108:7046-51

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