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Investigation for Molecular Mechanism of Regulation of Calcium Signaling Proteins in Cardiac Sarcoplasmic Reticulum and Those Biological Significance

心肌肌浆网钙信号蛋白调控的分子机制及其生物学意义研究

基本信息

批准号：
04404045
负责人：
TADA Michihiko
金额：
$ 19.2万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (A)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1994
项目状态：
已结题

项目摘要

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.

在本项目中，我们研究了心肌肌浆网钙信号蛋白调控的分子机制。我们研究了甲状腺激素对原代离体新生大鼠心肌细胞磷蛋白和Ca - atp酶mRNA水平的影响。Northern blot分析显示，T_3降低了磷蛋白mRNA水平，而增加了Ca - atp酶mRNA水平。T_3增加了Ca摄取的Vmax，显著降低了Ca的K_<0.5>，表明磷蛋白对Ca atp酶的调节是双模式的；在短时间范围内，通过磷酸化磷蛋白降低Ca - atp酶的亲和力；在长时间范围内，通过改变两种蛋白之间的分子比例。为了确定磷蛋白中与Ca - atp酶相互作用的位点，研究了一系列与Ca - atp酶共表达的磷蛋白突变体。磷酸化蛋白胞质1A结构域残基突变导致磷酸化蛋白对钙转运的抑制作用丧失，提示磷酸化蛋白胞质1A结构域是两种蛋白功能结合所必需的区域。当Ca ATPase发生突变并与磷蛋白共表达时，只有氨基酸Lys^<397>至Val^<402>的突变影响磷蛋白与Ca ATPase的结合。这些结果表明，氨基酸Lys^<397>-Val^<402>构成了Ca atp酶与磷蛋白的相互作用位点，并且电荷和疏水残基的适当平衡是相互作用的重要特征。