The goal of the proposed research is to define the regulation of normal and mutant cardiac muscle Ca2+ release channels (ryanodine receptors, RyR2s) under normal conditions as well as those in ischemic and post-ischemic heart. The cardiac Ca2+ release channel is a 30S protein complex comprised of four 560 kDa RyR2 subunits and four 12.6 kDa FK506 binding protein (FKBP12.6) subunits. Multiple endogenous effector molecules and post-translational modifications regulate RyR2, including Ca2+, Mg2+, ATP, calmodulin, protein phosphorylation, and thiol oxidation/reduction and S-nitrosylation. The principal hypotheses to be tested in the proposed research are that RyR2 is differentially regulated by calmodulin and by redox activation pathways involving free thiol groups. These hypotheses will be addressed using biochemical and electrophysiological methods and by creating mutant embryonic stem (ES) cell lines and mice.
The Specific Aims are: (1) Characterize the regulation of RyR2 by calmodulin (CAM) and identify by mutagenesis the apocalmodulin (apoCaM) and Ca2+-calmodulin (CaCaM) regulatory sites in RyR2. (2) Characterize the regulation of RyR2 by redox active and NO-related molecules and identify regulatory redox-sensitive and S-nitrosylation sites by chemical analysis and mutagenesis. (3) Determine the in vivo role of the above RyR2 regulatory mechanisms by creating mutant ES cells differentiated into cardiomyocytes deficient in RyR2 calmodulin binding and S- nitrosylation sites and mice expressing RyR2s deficient in calmodulin binding and S-nitrosylation sites. The functional properties of normal and mutant RyR2s will be determined in intact cells and with isolated membranes and purified channels under normal and simulated ischemic and postischemic conditions. The functional effects of Ca2+, Mg2+, ATP, pH, calmodulin, and redox-active and NO-related molecules will be assessed in Ca2+ imaging, SR vesicle-Ca2+ flux, [3H]ryanodine binding and single channel measurements. The studies will provide new insights into the complex interaction of the cardiac RyR with its regulatory ligands and how these regulatory processes are altered in the ischemic and post-ischemic heart.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL073051-03
Application #
6877999
Study Section
Cardiovascular and Pulmonary Research A Study Section (CVA)
Program Officer
Przywara, Dennis
Project Start
2003-04-15
Project End
2007-03-31
Budget Start
2005-04-01
Budget End
2006-03-31
Support Year
3
Fiscal Year
2005
Total Cost
$425,675
Indirect Cost
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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Huang, Tai-Qin; Zou, Min-Xu; Pasek, Daniel A et al. (2015) mTOR signaling in mice with dysfunctional cardiac ryanodine receptor ion channel. J Receptor Ligand Channel Res 8:43-51
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
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Yamaguchi, Naohiro; Chakraborty, Asima; Pasek, Daniel A et al. (2011) Dysfunctional ryanodine receptor and cardiac hypertrophy: role of signaling molecules. Am J Physiol Heart Circ Physiol 300:H2187-95
Meissner, Gerhard (2010) Regulation of Ryanodine Receptor Ion Channels Through Posttranslational Modifications. Curr Top Membr 66:91-113
Meissner, Gerhard; Wang, Ying; Xu, Le et al. (2009) Silencing genes of sarcoplasmic reticulum proteins clarifies their roles in excitation-contraction coupling. J Physiol 587:3089-90

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