The goal of the proposed research is to obtain new insights into the mechanism underlying regulation of the skeletal muscle Ca2+ release channel (type 1 ryanodine receptor, RyR1) by calmodulin (CaM), a cytoplasmic Ca2+ binding protein. RyR1 is the dominant RyR isoform in skeletal muscle and is responsible for the release of Ca2+ from the sarcoplasmic reticulum, an intracellular Ca2+ store, during muscle action potential. RyR1 is a homotetramer of ~550 kDa polypeptides and is regulated by various physiological molecules and proteins such as ATP, Ca2+, protein kinases and CaM. Involvement of domain interactions in regulation of RyR1 has been demonstrated. Although CaM binding site is conserved among all three mammalian RyR isoforms, isoform- dependent regulation is observed. RyR1 is activated, whereas RyR2 (cardiac muscle isoform) is inhibited by CaM at submicromolar Ca2+ concentrations. Corresponding point mutations in the conserved CaM binding domain differentially affect CaM binding and CaM regulation of each RyR isoform. This suggests that isoform- specific domains other than the CaM binding domain are involved in CaM regulation. A RyR1 domain (CaM- like domain;CaMLD), which was predicted to resemble the structure of CaM, was suggested to interact with the CaM binding site of RyR1. The hypothesis to be tested in the proposed research is that inter-domain interactions between the CaM binding domain and CaMLD is included in RyR1-specific CaM activation at submicromolar Ca2+ concentrations. Functional significance of CaMLD in isoform-specific CaM regulation of RyRs will be assessed by two approaches;(1) expression of recombinant RyR1/RyR2 chimera channels and mutant RyRs carrying point mutations in CaMLD, and (2) use of synthetic peptides corresponding with RyR CaM binding domain and CaMLD. We will perform functional analysis of chimera and mutant RyRs by [3H]ryanodine binding measurements and single channel recordings. Exogenous synthetic peptides will be used to disrupt inter-domain interactions, which is expected to result in isoform-dependent changes in RyR activity. We will also measure direct interactions between the CaM binding domain and CaMLD using surface plasmon resonance. In addition, we will screen an additional domain of RyR1 which interacts with CaMLD using protein overlays and surface plasmon resonance. The research will advance understanding of the domain complex involved in CaM regulation of RyR1.

Public Health Relevance

The proposed research will define the regulation mechanism of skeletal muscle Ca2+ release channel (type 1 ryanodine receptor (RyR1)) by calmodulin. Dysfunction in RyR1, thereby dysregulation of intracellular Ca2+ handling, is implicated in skeletal myopathies such as muscular dystrophy. By providing new insights in RyR1 function, our studies should help development of new treatment for muscle diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Small Research Grants (R03)
Project #
5R03AR061030-03
Application #
8490169
Study Section
Special Emphasis Panel (ZAR1-EHB (M1))
Program Officer
Boyce, Amanda T
Project Start
2011-07-01
Project End
2014-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
3
Fiscal Year
2013
Total Cost
$70,063
Indirect Cost
$22,563
Name
Medical University of South Carolina
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
183710748
City
Charleston
State
SC
Country
United States
Zip Code
29425
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Gomez, Angela C; Holford, Timothy W; Yamaguchi, Naohiro (2016) Malignant hyperthermia-associated mutations in the S2-S3 cytoplasmic loop of type 1 ryanodine receptor calcium channel impair calcium-dependent inactivation. Am J Physiol Cell Physiol 311:C749-C757
Chakraborty, Asima; Pasek, Daniel A; Huang, Tai-Qin et al. (2014) Inhibition of CaMKII does not attenuate cardiac hypertrophy in mice with dysfunctional ryanodine receptor. PLoS One 9:e104338
Gomez, Angela C; Yamaguchi, Naohiro (2014) Two regions of the ryanodine receptor calcium channel are involved in Ca(2+)-dependent inactivation. Biochemistry 53:1373-9
Arnáiz-Cot, Juan José; Damon, Brooke James; Zhang, Xiao-Hua et al. (2013) Cardiac calcium signalling pathologies associated with defective calmodulin regulation of type 2 ryanodine receptor. J Physiol 591:4287-99
Rosa, Angelo O; Yamaguchi, Naohiro; Morad, Martin (2013) Mechanical regulation of native and the recombinant calcium channel. Cell Calcium 53:264-74