2007 Fiscal Year Final Research Report Summary
Ryanodine receptor as a novel therapeutic target of lethal arrhythmia
| Project/Area Number |
18590777
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Circulatory organs internal medicine
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| Research Institution | Yamaguchi University |
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Principal Investigator |
YAMAMOTO Takeshi Yamaguchi University, Graduate School of Medicine, Assistant Professor (50363122)
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| Co-Investigator(Kenkyū-buntansha) |
YANO Masafumi Yamaguchi University, Hospital, Associate Professor (90294628)
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| Project Period (FY) |
2006 – 2007
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| Keywords | sarcoplasmic reticulu / calcium / rvanodine recentor / heart failure / lethal arrhythmia / point mutation |
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| Research Abstract |
We previously demonstrated that K201 (JTV5I9) inhibits the Call leak by correcting the defective inter-domain interaction between N-terminal (0-600) and central regions (2000-2500) of the ryanodine receptor (RyR2) in failing hearts. Here, we identified the K201-binding domain and characterized the role of this novel domain on RyR2 channel gating. An assay using a quartz-crystal microbalance technique revealed that K201 specifically bound to recombinant RyR2 fragment: 1741-2270, but not to other RyR2 fragments (-500 amino acid residues) from the 1-2750 region. By further analysis of the fragment^<1741-2270>, K201 was found to specifically bind to its sub-fragment^<2114-2149>. Using the peptide matching this sub-fragment (DP^<2114-2149>) as a carrier, the RyR2 was specifically labeled with methylcoumarin acetate (MCA). Moreover, of several recombinant RyR2 fragments (1-2750), 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 SR 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 (F; n=15), the frequency of spontaneous Ca^<2+> spark (CaSF: s-1.100μm-1) was markedly increased compared with normal (N; n=15) cardiomyocytes (F: 2.7±0.7, N: 0.9±0.4; p<0.01 vs N), whereas incorporation of DP^<2114-2149> markedly decreased CaSF (1.3±0.5, p<0.01 vs F), the same effect as that produced by K201. In conclusion, we first identified the K201-binding site as domain^<2114-2149> of RyR2. Interruption of the inter-domain 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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