1994 Fiscal Year Final Research Report Summary
Investigation for Molecular Mechanism of Regulation of Calcium Signaling Proteins in Cardiac Sarcoplasmic Reticulum and Those Biological Significance
| Project/Area Number |
04404045
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| Research Category |
Grant-in-Aid for General Scientific Research (A)
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| Allocation Type | Single-year Grants |
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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 Osaka Univ., Medical School., Professor, 医学部, 教授 (90093434)
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| Co-Investigator(Kenkyū-buntansha) |
大津 欣也 大阪大学, 医学部・付属病院, 医員
HOSHIDA Shiro Osaka Univ., Medical School., Associate Prof., 医学部, 助手 (80238732)
KUZUYA Tsunehiko Osaka Univ., Medical School., Associate Prof., 医学部, 助教授 (80150340)
OTSU Kinya Osaka Univ., Medical School., Resident
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| Project Period (FY) |
1992 – 1994
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| Keywords | sarcoplasmic reticulum / Calcium signaling / Molecular mechanism / Cell biology / Phospholamban / Ca ATPase / Ca release channel / Malignant hyperthermia |
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| Research Abstract |
In this project, we investigated the molecular mechanism of the regulation of calcium signaling proteins in cardiac sarcoplasmic reticulum.
We studied the effect of thyroid hormone on levels of phospholamban and Ca ATPase mRNA in primary isolated neonatal rat myocardial cells. Northern blot analysis showed that T_3 decreased phospholamban mRNA levels, whereas increased Ca ATPase mRNA levels. T_3 inceased Vmax of Ca uptake with significant reduction of K_<0.5> for Ca. These results suggested that phospholamban regulates the Ca ATPase in dual modes ; in short time range, by decreasing the affinity of the Ca ATPase by phosphorylation of phospholamban, and long time range, by changing the molecular ratio between the two proteins.
In order to identify the sites in phospholamban which inteact with Ca ATPase, a series of mutants of phospholamban were coexpressed with Ca ATPase. Mutation of residues in the cytoplasmic 1A domain of phospholamban resulted in loss of inhibitory effect of phospholamban on Ca transport, suggesting a region essential for functional association of the two proteins lies in the cytoplasmic 1A domain of phospholamban. When mutations were made in Ca ATPase and the mutants were coexpressed with phospholamban, only mutation of amino acids Lys^<397> to Val^<402> affected phospholamban association with Ca ATPase. These results demonstrated that amino acids Lys^<397>-Val^<402> comprise the interaction site with phospholamban in the Ca ATPase and that the appropriate balance of charged and hydrophobic residues is an important feature of the interaction.
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