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

心血管系统中浆网钙调节的新机制

• 批准号: 12835009
• 负责人: KIMURA Yoshihiro
• 金额: $ 2.3万
• 依托单位: Yamaguchi University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12835009/
• 关键词: cardiac myocytes; vascular smooth muscle; sarco (endo) plasmic reticulum; calcium ATPase; phospholamban

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.

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。

项目成果

Zvaritch, E.: "The transgenic expression of highly inhibitory monomeric forms of phospholamban heart impairs cardiac contractolity"Journal of Biological Chemistry. 275. 14985-14991 (2000)

Zvaritch, E.：“受磷蛋白心脏的高度抑制单体形式的转基因表达会损害心脏收缩力”生物化学杂志。

Kimura,Y., Inui,M.: "Reconstitution of the cytoplasmic interaction between phospholamban and Ca^<2+>-ATPasc of cardiac sarcoplasmic reticulum"Mol. Pharmacol. 61 (3). 667-673 (2002)

Kimura，Y.，Inui，M.：“心脏肌浆网的受磷蛋白和Ca^2-ATPasc之间的细胞质相互作用的重建”Mol。

Yamada,Y., Iwamoto,T., Watanabc,Y., Sobue,K. and Inui,M.: "PSD-95 eliminates src-induced potentiation of NR1/NR2A-subtype NMDA receptor channels and reduces hign-affinity zinc inhibition"J. Neurochem. (in press). (2002)

山田，Y.，岩本，T.，Watanabc，Y.，Sobue，K.

Haghighi,K., Schmidt,A.G., Hoit,B.D., Brittsan,A.G., Yatani,A., Lester,J.W., Zhai,J., Kimura,Y., Dorn,G.W.2nd, MacLennan,D.H., and Kranias,E.G.: "Superinhibition of sarcoplasmic reticulum Junction by phospholamban induces cardiac contractile failure"J Bio

Haghighi,K.、Schmidt,A.G.、Hoit,B.D.、Brittsan,A.G.、Yatani,A.、Lester,J.W.、Zhai,J.、Kimura,Y.、Dorn,G.W.2nd、MacLennan,D.H. 和 Kranias,E.G.：

Zhai,J., Schmidt,A.G., Hoit,B.D., Kimuya,Y., MacLennan,D.H., and Kranias,E.G.: "Cardiac-specific overexpression of a superinhibitory pentameric phospholamban mutant enhances inhibition of cardiac function in vivo"J Biol Chem. 275 (14). 10538-10544 (2000)

Zhai,J.、Schmidt,A.G.、Hoit,B.D.、Kimuya,Y.、MacLennan,D.H. 和 Kranias,E.G.：“超抑制性五聚磷光班突变体的心脏特异性过表达可增强体内心脏功能的抑制”J Biol Chem。