New mechanisms of SERCA2a regulation: role of luminal calcium

项目摘要/摘要 SERCA2a钙泵在心脏功能中起着核心作用。SERCA2a从土壤中去除钙的速度 胞浆是心肌松弛速率的主要决定因素。SERCA2a还设置 肌浆网(SR)中的钙，它决定心脏收缩的强度。这并不令人惊讶， SERCA2a功能受损已被报道在许多病理情况下，包括心力衰竭 (Hf)。因此，了解SERCA的调控机制具有重要的临床意义。除了激活 在肌肉收缩中，肌浆网腔钙([Ca]SR)在肌浆网蛋白功能的调节中起着重要作用。而当 SERCA2a活性控制[Ca]SR，关于[Ca]SR的变化如何影响SERCA2a钙转运的研究较少。我们的 初步结果提示，肌浆网腔内钙在SERCA2a的调节中起重要作用。在这个项目中 我们将使用先进的结构分析，创新的分子生物学技术，新的细胞器特有的 传感器、最新的光学方法和体内基因传递来探索SERCA2a的这一新机制 监管。在目标1中，我们将检验流明钙通过增加泵的 通过减轻磷脂蛋白(PLB)的抑制作用而发挥其催化作用。分子动力学模拟将 用于选择SERCA2a腔面上参与钙调节的特定结构域。站点定向 突变将被用来鉴定形成腔内钙结合位点的特定氨基酸，并开发 鲁米那钙不敏感SERCA2a突变体。我们将评估腔内钙调节对钙转运的影响， ATPase活性和原球蛋白相互作用。然后，表达腔内钙不敏感的SERCA2a的心肌细胞 将对突变体进行研究，以确定这一新机制在心脏钙循环中的作用。我们预计， 这项工作的结果将极大地促进我们对SERCA2a功能的理解。我们期待着结果 这些研究将提供对SERCA2a调控这一新机制的详细看法，促进我们的 了解钙泵的功能。在目标2中，我们将检验流明氧化还原电位调节的假设。 SERCA2a通过稳定其腔内钙结合部位，从而改善[Ca]SR对泵的调节。分子 动态模拟将被用来预测SERCA2a鲁米二硫键在泵催化中的作用 周而复始。我们将评估腔内半胱氨酸突变是否会导致功能丧失表型 腔钙对SERCA2a的调节作用。测量管腔氧化还原电位和[Ca]SR的新方法 将被用来定义管腔氧化还原电位和SERCA2a活性之间的串扰。我们会调查的 这一新机制在心力衰竭时SERCA2a功能障碍和钙处理不当中的作用。可能的结果是 在这些研究中，有一个新概念可以解释SERCA2a结构/功能的改变是如何导致缺陷的 心衰时钙调节的变化。

PROJECT SUMMARY/ABSTRACT SERCA2a Ca pump plays a central role in heart function. The speed at which SERCA2a removes Ca from the cytosol is the main determinant of the rate of cardiac muscle relaxation. SERCA2a also sets the total amount of Ca in the sarcoplasmic reticulum (SR), which determines the strength of cardiac contraction. It is not surprising, that impaired SERCA2a function has been reported in a number of pathological conditions, including heart failure (HF). Thus, understanding mechanisms of SERCA regulation is of great clinical importance. Besides activation of muscle contraction, SR luminal Ca ([Ca]SR) plays an important role in regulation of SR protein function. While SERCA2a activity controls [Ca]SR, less is known about how changes in [Ca]SR affect SERCA2a Ca transport. Our preliminary results suggest that SR luminal Ca plays an important role in regulation of SERCA2a. In this project we will use advanced structural analyses, innovative molecular biological techniques, new organelle-specific sensors, state-of-the-art optical methods and in vivo gene delivery to explore this new mechanism of SERCA2a regulation. In Aim 1, we will test the hypothesis that luminal Ca regulates SERCA2a by increasing the pump’s catalytic efficacy and by relieving the phospholamban (PLB) inhibitory effect. Molecular dynamic simulations will be used to select specific domains on the SERCA2a luminal side that are involved in Ca regulation. Site-directed mutagenesis will be used to identify the specific amino acids that form the luminal Ca-binding sites and to develop the luminal Ca-insensitive SERCA2a mutant. We will assess effects of the luminal Ca regulation on Ca transport, the ATPase activity and the PLB interaction. Then, myocytes expressing the luminal Ca-insensitive SERCA2a mutant will be studied to define the role of this novel mechanism in cardiac Ca cycling. We expect that the outcome of this work will greatly advance our understanding of SERCA2a function. We expect that the outcome of these studies will provide a detailed view of this new mechanism of SERCA2a regulation, advancing our understanding of the Ca pump’s function. In Aim 2, we will test the hypothesis that luminal redox potential regulates SERCA2a by stabilizing its luminal Ca binding sites, thus, improving the pump’s regulation by [Ca]SR. Molecular dynamic simulations will be used to forecast the role of the SERCA2a luminal disulfide bond in the pump catalytic cycle. We will assess whether mutation of luminal cysteines leads to a loss-of-function phenotype by abolishing SERCA2a regulation by luminal Ca. Newly developed approaches to measure luminal redox potential and [Ca]SR will be used to define the cross-talk between luminal redox potential and SERCA2a activity. We will investigate the contribution of this new mechanism to SERCA2a dysfunction and Ca mishandling in HF. The likely outcome of these studies is a new concept that can explain how alterations in SERCA2a structure/function cause defects in Ca regulation in HF.