| Project/Area Number |
10557069
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
Circulatory organs internal medicine
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| Research Institution | Osaka University |
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Principal Investigator |
TADA Michihiko Graduate School of Medicine, Osaka University, Professor, 医学系研究科, 教授 (90093434)
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| Co-Investigator(Kenkyū-buntansha) |
OTSU Kinya Graduate School of Medicine, Osaka University, Assistant Professor, 医学系研究科, 助手 (20294051)
NISHIDA Masashi Graduate School of Medicine, Osaka University, Assistant Professor, 医学系研究科, 助手 (40283783)
KUZUYA Tsunehiko Graduate School of Medicine, Osaka University, Associate Professor, 医学系研究科, 助教授 (80150340)
TOYOFUKU Toshihiko Graduate School of Medicine, Osaka University, Assistant Professor, 医学系研究科, 助手
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥13,500,000 (Direct Cost: ¥13,500,000)
Fiscal Year 1999: ¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 1998: ¥10,000,000 (Direct Cost: ¥10,000,000)
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| Keywords | Gap junction / Connexin / Cardiac myocytes / Cai. aging system / patch-clamp methods / ZO-1 / Gap junctional Protein / パッチクランプ法 / gap junctional protein / connexin-43 / 部位特異的変異導入法 / パッチ・クランプシステム |
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| Research Abstract |
In excitable cells, intracellular Ca is released via the ryanodine receptor from the intracellular Ca storing structure, the sarcoplasmic reticulum. To determine whether this released Ca propagates through gap junctions to neighboring cells and thereby constitutes a long-range signaling network, we developed a cell system in which cells expressing both connexin43 and ryanodine receptor are surrounded by cells expressing only connexin43. Propagation of Ca to neighboring cells was observed with a Ca imaging system using fura-2/AM. We next evaluated the functional roles of cysteine residues in the extracellular loops of connexin43 in gap junctional communication. Mutations of cysteine residues showed that two pairs of intramolecular disulfide bonds are formed. Thus, the extracellular disulfide bonds of connexin43 are crucial for this process. Gap junctional protein connexin43, localized to the intercalated discs of cardiac myocytes, plays a critical role in the synchronization of their contractions. Co-immunoprecipitation experiments using co-expressed epitope-tagged connexin43 and ZO-1 in transfected HEK293 cells indicated that ZO-1 links connexin43. Affinity binding assay using deleted ZO-1 and deleted connexin43 fusion proteins showed that the C-terminal region of connexin43 binds to the N-terminal region of ZO-1. Overexpression of the N-terminal domain of ZO-1 in connexin43-expressing cells resulted in the redistribution of connexin43 from the cell-cell interfaces to the cytoplasmic structures, in accordance with the loss of electrical coupling. We therefore conclude that the linkage of connexin43 with ZO-1 may serve to localize the connexin43 at the intercalated discs, thereby generating the functional gap junction in cardiac myoctyes.
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