A novel mechanism of serco (endo) plasmic reticulum calcium regulation in the cardiovascular system

受磷蛋白（Phospholamban，PLN）通过直接的蛋白质-蛋白质相互作用可逆地抑制心肌肌浆网Ca^2+-ATP酶（SERCA 2），在心肌细胞内Ca^2+的调节中起关键作用。PLN也被认为参与血管紧张的调节。PLN和SERCA 2之间的相互作用发生在细胞质和膜结构域内的多个位点。在这项研究中，我们已经重建了细胞质蛋白质的相互作用，使用细菌表达的融合蛋白质的胞质结构域的PLN和长胞质环的SERCA 2。我们已经开发了一种方法来评估的融合蛋白的结合，采用96孔ELISA板。PLN融合蛋白与SERCA 2融合蛋白特异性结合。结合亲和力（K_D）为0.70 μM。该协会被抑制cAMP依赖磷酸化的PLN融合蛋白和使用抗PLN单克隆抗体。在PLN的磷酸化位点将Ser^替换为Asp也降低了它<16>。这些结果表明，PLN可以结合SERCA 2a的膜结构域的情况下，和PLN的胞质结构域的修饰，激活SERCA 2a平行的破坏之间的关联的两个融合蛋白。我们的测定系统可应用于筛选新的变力剂，其消除PLN对SERCA 2a的抑制，改善衰竭心脏的舒张性和收缩性，并降低血管阻力。

Phospholamban (PLN) reversibly inhibits the Ca^<2+> -ATPase of cardiac sarcoplasmic reticulum (SERCA2) through a direct protein-protein interaction, playing a pivotal role in the regulation of intracellular Ca^<2+> in heart muscle cells. PLN is also thought to be involved in the regulation of vascular tonus. The interaction between PLN and SERCA2 occurs at multiple sites within the cytoplasmic and membrane domains. In this study, we have reconstituted the cytoplasmic protein-protein interaction using bacterially expressed fusion proteins of the cytoplasmic domain of PLN and the long cytoplasmic loop of SERCA2. We have developed a method to evaluate the binding of the fusion proteins employing a 96-well ELISA plate. The PLN fusion protein bound specifically to the SERCA2 fusion protein. The affinity of the binding (K_D) was 0.70 μM. The association was inhibited by cAMP-dependent phosphorylation of the PLN fusion protein and by usage of anti-PLN monoclonal antibody. It was also diminished by substitution at the phosphorylation site of PLN of Ser^<16> to Asp. These results suggest that PLN can bind SERCA2a in the absence of the membrane domains and that the modifications of the cytoplasmic domain of PLN that activate SERCA2a parallel the disruption of the association between the two fusion proteins. Our assay system can be applied to the screening of novel inotropic agents which remove the inhibition of SERCA2a by PLN, improving the relaxation as well as the contractility of the failing heart and reducing vascular resistance.