Study on chronic heart failure using mutant phospholamban transgenic mice

突变型受磷蛋白转基因小鼠对慢性心力衰竭的研究

• 批准号: 10670662
• 负责人: KIMURA Yoshihiro
• 金额: $ 2.11万
• 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: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670662/
• 关键词: cardiac sarcoplasmic reticulum; calcium ATPase; phospholamban; heart failure; transgenic mice; トランスジェニックマウス

Phospholamban (PLN), an integral membrane protein of cardiac sarcoplasmic reticulum, reversibly inhibits the CaィイD12+ィエD1-ATPase (SERCA) to play a pivotal role in the regulation of cardiac contractility and relaxation. We examined the molecular mechanism of SERCA inhibition by PLN and we have created transgenic mice in which the dominant, highly inhibitory PLN mutant proteins are overexpressed in collaboration with Dr. D. H. MacLennan of the University of Toronto. Our results are following :1. PLN interacts with SERCA through at least two, possibly three sites ; cytoplasmic domain (domain Ia), transmembrane helix (domain II), and cytoplasmic-membrane boundary (domain Ib). Intramembrane interactions are inhibitory on SERCA activity, while the cytoplasmic interactions are not inhibitory itself although it is essential for the physiological regulatory effect. We propose that PLN and SERCA interact via a four base circuit through which long range inhibitory interactions night be propagated among a series of cytoplasmic and transmembrane interaction sites.2. PLN transmembrane domain interacts with the transmembrane sequence (M6) of SERCA. The interaction site consists of four residues which form the hydrophobic face of the proposed helix on the opposite face to the CaィイD12+ィエD1 binding site. As M& is located in a shallow groove with the key residues which form the proposed interaction site facing the lipid bilayer, it is conceivable that the groove may provide the PLN transmembrane helix with access to the interaction site.3. Transgenic mice overexpressing monomeric, dominant-acting, highly inhibitory PLN mutants were generated. They exhibited impaired cardiac contractility and relaxation.

磷蛋白是心肌肌浆网的一种完整的膜蛋白，它可逆地抑制CaィイD12+ィエD1-ATPase，在调节心肌收缩和松弛中发挥关键作用。我们研究了PLN抑制SERCA的分子机制，并与多伦多大学的D.H.MacLennan博士合作，创造了转基因小鼠，在其中，显性的、高度抑制的PLN突变蛋白过度表达。我们的研究结果如下：1.PLN与SERCA的相互作用至少通过两个或三个区域：细胞质结构域(结构域Ia)、跨膜螺旋结构域(结构域II)和细胞质-膜边界(结构域Ib)。膜内相互作用对SERCA活性有抑制作用，而细胞质相互作用本身并不抑制SERCA活性，尽管它对生理调节作用是必不可少的。我们认为PLN和SERCA通过一个四个碱基的环路相互作用，通过这个环路可以在一系列细胞质和跨膜相互作用部位之间传播长范围的抑制作用。PLN跨膜区与SERCA的跨膜序列(M6)相互作用。相互作用部位由四个残基组成，它们形成了所建议螺旋的疏水面，位于CaィイD12+ィエD1结合部位的对面。由于M&位于一个浅槽中，关键残基形成了面向脂质双层的结合位点，因此可以想象该槽可能为PLN跨膜螺旋提供了进入作用位点的途径。获得了高表达单体、显性作用、高度抑制的PLN突变体的转基因小鼠。他们表现出心脏收缩和松弛能力受损。

项目成果

Asahi M.: "Transmembrane Helix M6 in Sarco(endo)plasmic Reticulum Ca2+ ATPase Forms a Functional Interaction Site with Phospholamban"J. Biol. Chem.. 274. 32855-32862 (1999)

Asahi M.：“肌（内）质网 Ca2 ATP 酶中的跨膜螺旋 M6 与 Phospholamban 形成功能性相互作用位点”J。

Yamada,Y.: "Modulation of the channel activity of the ε2/ζ1-subtype N-methyl Daspartate receptor by PSD-95"J.Biol.Chem.. 274. 6647-6652 (1999)

Yamada, Y.：“PSD-95 对 ε2/ζ1 亚型 N-甲基 Daspartate 受体的通道活性的调节”J.Biol.Chem.. 274. 6647-6652 (1999)

MacLennan,D.H.: "Sites of regulatory interaction between calcium ATPases and phospholamban"Ann.New York Acad.Sci.. 853. 31-42 (1998)

MacLennan，D.H.：“钙 ATP 酶和受磷蛋白之间的调节相互作用位点”Ann.New York Acad.Sci. 853. 31-42 (1998)

Kimura,Y.: "Phospholamban domain I/cytochrome b5 trandsmembrane sequence chimeras do not inhibit SERCA2a"FEBS Lett.. 425. 509-512 (1998)

Kimura,Y.：“磷酸兰班结构域 I/细胞色素 b5 跨膜序列嵌合体不抑制 SERCA2a”FEBS Lett.. 425. 509-512 (1998)

Asahi, M.: "Transmembrane helix M6 in sarco (endo) plasmic reticulum CaィイD12+-ィエD1ATPase forms a functional interaction site with phosphokamban. Evidence for physical interaction at other sites."J. Biol. Chem.. 274. 32855-32862 (1999)

Asahi, M.：“肌（内）质网 CaiD12+-D1ATP 酶中的跨膜螺旋 M6 与磷酸卡班形成功能性相互作用位点。其他位点的物理相互作用的证据。”J. Biol. 274. 32855-32862 (1999) ）