| Identification of target domains of the cardiac ryanodine receptor to correct channel disorder in failing hearts. | |
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MedLine Citation:
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PMID: 18227387 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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. |
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Authors:
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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:
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Type: Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't Date: 2008-01-28 |
Journal Detail:
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Title: Circulation Volume: 117 ISSN: 1524-4539 ISO Abbreviation: Circulation Publication Date: 2008 Feb |
Date Detail:
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Created Date: 2008-02-12 Completed Date: 2008-02-21 Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 0147763 Medline TA: Circulation Country: United States |
Other Details:
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Languages: eng Pagination: 762-72 Citation Subset: AIM; IM |
Affiliation:
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Department of Medicine and Clinical Science, Division of Cardiology, Yamaguchi University Graduate School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi, 755-8505, Japan. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Amino Acid Sequence Animals Annexin A5 / chemistry, metabolism Binding Sites Calcium / metabolism* Disease Models, Animal Dogs 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 | |
ID/Acronym/Agency:
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HL072841/HL/NHLBI NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Annexin A5; 0/K201 compound; 0/Ryanodine Receptor Calcium Release Channel; 0/Thiazepines; 7440-70-2/Calcium |
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