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

磷蛋白（Phospholamban， PLN）通过蛋白-蛋白直接相互作用可逆抑制心肌肌浆网（SERCA2）的Ca^<2+> - atp酶，在心肌细胞内Ca^<2+>的调控中起关键作用。PLN也被认为参与血管张力的调节。PLN和SERCA2之间的相互作用发生在细胞质和膜结构域的多个位点。在这项研究中，我们利用细菌表达的PLN细胞质结构域融合蛋白和SERCA2长细胞质环重构了细胞质蛋白-蛋白相互作用。我们开发了一种评估融合蛋白结合的方法，采用96孔ELISA板。PLN融合蛋白与SERCA2融合蛋白特异性结合。结合的亲和度（K_D）为0.70 μM。camp依赖性磷酸化PLN融合蛋白和使用抗PLN单克隆抗体可抑制这种关联。在Ser^<16>的PLN磷酸化位点上，它也被取代为Asp。这些结果表明，PLN可以在没有膜结构域的情况下结合SERCA2a，并且激活SERCA2a的PLN细胞质结构域的修饰平行于两种融合蛋白之间的关联破坏。我们的检测系统可以用于筛选新型的肌力药物，这些药物可以消除PLN对SERCA2a的抑制，改善衰竭心脏的舒张和收缩性，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.