Document Detail

Identification of target domains of the cardiac ryanodine receptor to correct channel disorder in failing hearts.
MedLine Citation:
PMID:  18227387     Owner:  NLM     Status:  MEDLINE    
BACKGROUND: We previously demonstrated that defective interdomain interaction between N-terminal (0 to 600) and central regions (2000 to 2500) of ryanodine receptor 2 (RyR2) induces Ca2+ leak in failing hearts and that K201 (JTV519) inhibits the Ca2+ leak by correcting the defective interdomain interaction. In the present report, we identified the K201-binding domain and characterized the role of this novel domain in the regulation of the RyR2 channel. METHODS AND RESULTS: An assay using a quartz-crystal microbalance technique (a very sensitive mass-measuring technique) revealed that K201 specifically bound to recombinant RyR2 fragments 1741 to 2270 and 1981 to 2520 but not to other RyR2 fragments from the 1 to 2750 region (1 to 610, 494 to 1000, 741 to 1260, 985 to 1503, 1245 to 1768, 2234 to 2750). By further analysis of the fragment(1741-2270), K201 was found to specifically bind to its subfragment(2114-2149). With the use of the peptide matching this subfragment (DP(2114-2149)) as a carrier, the RyR2 was fluorescently labeled with methylcoumarin acetate (MCA) in a site-directed manner. After tryptic digestion, the major MCA-labeled fragment of RyR2 (155 kDa) was detected by an antibody raised against the central region (Ab(2132)). Moreover, of several recombinant RyR2 fragments, only fragment(2234-2750) was specifically MCA labeled; this suggests that the K201-binding domain(2114-2149) binds with domain(2234-2750). Addition of DP(2114-2149) to the MCA-labeled sarcoplasmic reticulum interfered with the interaction between domain(2114-2149) and domain(2234-2750), causing domain unzipping, as evidenced by an increased accessibility of the bound MCA to a large-size fluorescence quencher. In failing cardiomyocytes, the frequency of spontaneous Ca2+ spark was markedly increased compared with normal cardiomyocytes, whereas incorporation of DP(2114-2149) markedly decreased the frequency of spontaneous Ca2+ spark. CONCLUSIONS: We first identified the K201-binding site as domain(2114-2149) of RyR2. Interruption of the interdomain interaction between the domain(2114-2149) and central domain(2234-2750) seems to mediate stabilization of RyR2 in failing hearts, which may lead to a novel therapeutic strategy against heart failure and perhaps lethal arrhythmia.
Takeshi Yamamoto; Masafumi Yano; XiaoJuan Xu; Hitoshi Uchinoumi; Hiroki Tateishi; Mamoru Mochizuki; Tetsuro Oda; Shigeki Kobayashi; Noriaki Ikemoto; Masunori Matsuzaki
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2008-01-28
Journal Detail:
Title:  Circulation     Volume:  117     ISSN:  1524-4539     ISO Abbreviation:  Circulation     Publication Date:  2008 Feb 
Date Detail:
Created Date:  2008-02-12     Completed Date:  2008-02-21     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0147763     Medline TA:  Circulation     Country:  United States    
Other Details:
Languages:  eng     Pagination:  762-72     Citation Subset:  AIM; IM    
Department of Medicine and Clinical Science, Division of Cardiology, Yamaguchi University Graduate School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi, 755-8505, Japan.
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MeSH Terms
Amino Acid Sequence
Annexin A5 / chemistry,  metabolism
Binding Sites
Calcium / metabolism*
Disease Models, Animal
Heart Failure / metabolism
Linear Models
Molecular Sequence Data
Myocytes, Cardiac / metabolism*
Ryanodine Receptor Calcium Release Channel / chemistry,  metabolism*
Sarcoplasmic Reticulum
Sequence Homology, Amino Acid
Thiazepines / metabolism
Grant Support
Reg. No./Substance:
0/Annexin A5; 0/K201 compound; 0/Ryanodine Receptor Calcium Release Channel; 0/Thiazepines; 7440-70-2/Calcium

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