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

本研究旨在探讨心肌肌浆网钙信号蛋白调控的分子机制，以及甲状腺激素对原代分离的新生大鼠心肌细胞磷蛋白和Ca-ATPase基因表达水平的影响。Northern印迹分析表明，T_3降低了磷蛋白基因的表达水平，而增加了Ca-ATPase基因的表达水平。T_3增加了钙吸收的Vmax，降低了钙吸收的K_(0.5)。这些结果表明，磷蛋白对Ca-ATPase的调节有两种方式，在短时间内，通过磷酸化来降低Ca-ATPase的亲和力，在长时间内，通过改变两种蛋白之间的分子比来降低Ca-ATPase的亲和力。磷蛋白胞质1A区残基的突变导致磷蛋白对钙转运的抑制作用丧失，提示两种蛋白的功能结合所必需的区域位于磷蛋白的胞质1A区。当Ca-ATPase发生突变并与磷蛋白共表达时，只有氨基酸Lys^&lt；397&gt；突变为Val^&lt；402&gt；影响磷蛋白与Ca-ATPase的结合。这些结果表明，氨基酸Lys^&lt；397&gt；-Val^&lt；402&gt；构成了Ca-ATPase中与磷蛋白相互作用的部位，电荷残基和疏水残基的适当平衡是相互作用的重要特征。

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.