Molecular mechanisms of ion channel regulation by a protein-protein interaction

蛋白质-蛋白质相互作用调节离子通道的分子机制

• 批准号: 12470019
• 负责人: INUI Makoto
• 金额: $ 8.96万
• 依托单位: Yamaguchi University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 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/en/grant/KAKENHI-PROJECT-12470019/
• 关键词: Ion channels; Chloride channels; Protein-protein interaction; Dimer formation; PSD-95; NMDA receptor; Protein phosphorylation; Zinc inhibition; チャネル; NMDA受容体チャネル; チロシン燐酸化; Cl^-チャネル; ClC4; 酵母two hybrid法; 融合蛋白質; 2量体; 容積依存性Cl^-チャネル

The aim of this study is to elucidate the molecular mechanisms by which the ion channels are regulated by a protein-protein interaction. We focused our study on the two ion channels. One is a member of CIC chloride channel family, CIC-4, which has unique dimeric structure. The channels are thought to be regulated by the cell volume. The other is the NMDA receptor which plays important roles in synaptic plasticity underlying memory and learning. It has been known that a number of post synaptic density proteins including PSD-95 interact with the NMDA receptor. However, it is unknown whether the channel activity of NMDA receptor is regulated by those proteins. In this study, we investigated the role of a protein-protein interaction in the structure and function of these two ion channels.To find out a protein which interacts with CIC-4, we performed two-hybrid screening with the N-terminal domain of CIC-4 as a bait using human brain cDNA library. We obtained 10 clones. One of them was the … More C-terminal region of CIC-4, indicating the intra-molecular interaction or the protein-protein interaction between CIC-4s. We constructed fusion proteins of the cytoplasmic domains of CIC-4 and examined the interaction among fusion proteins. We found that the N-terminal domain binds to the C-terminal domain and to the N-terminal domain itself. The C-terminal domain binds the C-terminal domain itself in addition to the N-terminal domain. Since the interaction between the N- and C-terminal domains occurs between a fusion protein and the full length CIC-4, these interaction between the cytoplasmic domains is responsible for the dimer formation of CIC-4 channels.We examined the effects of PSD-95 on the channel activity of the NR1/NR2A receptor with the oocyte expression system, comparing with those of the NR1/NR2B receptor. The expression of PSD-95 inhibited the protein kinase C-mediated potentiation of NR1/NR2A channels as well as NR1/NR2B channels. In addition, we demonstrated that PSD-95 eliminates the Src-induced potentiation of NR1/NR2A channels expressed in oocytes and reduces the sensitivity of the channels to Zn2+. Our results revealed that the absence of Src-induced potentiation of PSD-95-coupled NR1/NR2A channels is not to due to the reduced sensitivity of these channels to Zn^<2+>. These results indicate that PSD-95 functionally modulates NR1/NR2A channels and explain why Src-induced potentiation of NMDA receptor currents in hippocampus neurons is not mediated by a reduction in Zn^<2+> inhibition. Less

本研究的目的是阐明蛋白质-蛋白质相互作用调节离子通道的分子机制。我们的研究重点是两个离子通道。一种是CIC氯离子通道家族的成员CIC-4，具有独特的二聚体结构。这些通道被认为是由细胞体积调节的。另一个是 NMDA 受体，它在记忆和学习的突触可塑性中发挥着重要作用。众所周知，包括 PSD-95 在内的许多突触后密度蛋白与 NMDA 受体相互作用。然而，NMDA 受体通道活性是否受这些蛋白调节尚不清楚。在本研究中，我们研究了蛋白质-蛋白质相互作用在这两个离子通道的结构和功能中的作用。为了找出与CIC-4相互作用的蛋白质，我们利用人脑cDNA文库以CIC-4的N端结构域为诱饵进行了双杂交筛选。我们获得了10个克隆。其中之一是 CIC-4 的 C 末端区域，表明 CIC-4 之间存在分子内相互作用或蛋白质-蛋白质相互作用。我们构建了 CIC-4 细胞质结构域的融合蛋白并检查了融合蛋白之间的相互作用。我们发现 N 端结构域与 C 端结构域以及 N 端结构域本身结合。除了N端结构域之外，C端结构域还结合C端结构域本身。由于 N 端和 C 端结构域之间的相互作用发生在融合蛋白和全长 CIC-4 之间，因此细胞质结构域之间的这些相互作用负责 CIC-4 通道二聚体的形成。我们通过卵母细胞表达系统检查了 PSD-95 对 NR1/NR2A 受体通道活性的影响，并与 NR1/NR2B 受体的通道活性进行比较。 PSD-95 的表达抑制蛋白激酶 C 介导的 NR1/NR2A 通道以及 NR1/NR2B 通道的增强。此外，我们还证明 PSD-95 消除了 Src 诱导的卵母细胞中表达的 NR1/NR2A 通道的增强作用，并降低了通道对 Zn2+ 的敏感性。我们的结果表明，PSD-95耦合的NR1/NR2A通道的Src诱导增强的缺失并不是由于这些通道对Zn^2+的敏感性降低。这些结果表明PSD-95功能性调节NR1/NR2A通道并解释了为什么Src诱导的海马神经元中NMDA受体电流的增强不是由Zn 2+ 抑制的减少介导的。较少的

项目成果

J.Zhai: "Cardiac-specific overexpression of a superinhibitory pentameric phospholamban mutant enhances inhibition of cardiac function in vivo"Journal of Biological Chemistry. 275. 10538-10544 (2000)

J.Zhai：“超抑制性五聚受磷蛋白突变体的心脏特异性过度表达增强了体内心脏功能的抑制”《生物化学杂志》。

Yasue Yamada: "PSID-95 eliminates src-induced potentiation of NR1/NR2A-subtype NMDA receptor channels and reduces high-affinity zinc inhibition"Journal of Neurochemistry. (in press). (2002)

Yasue Yamada：“PSID-95 消除 src 诱导的 NR1/NR2A 亚型 NMDA 受体通道增强，并减少高亲和力锌抑制”《神经化学杂志》。

E.Zvaritch: "The transgenic expression of highly inhibitory monomeric forms of phospholamban in mouse heart impairs cardiac contractility"Journal of Biological Chemistry. 275. 14985-14989 (2000)

E.Zvaritch：“小鼠心脏中高抑制性单体形式的受磷蛋白的转基因表达会损害心脏收缩力”《生物化学杂志》。

Yoshihiro Kimura: "Reconstitution of the cytoplasmic interaction between phospholamban and Ca2+-ATPase of cardiac sarcoplasmic reticulum"Molecular Pharmacology. 61 (3). 667-673 (2002)

Yoshihiro Kimura：“心脏肌浆网受磷蛋白和 Ca2-ATP 酶之间细胞质相互作用的重建”分子药理学。

Yasue Yamada: "PSD-95 eliminates src0indeuced potentiation of NR1/NR2A-subtype NMDA receptor channels and reduces high-affinity zinc inhibition"Journal of Neurochemistry. (In press). (2002)

Yasue Yamada：“PSD-95 消除了 src0 诱导的 NR1/NR2A 亚型 NMDA 受体通道的增强，并减少了高亲和力锌抑制”《神经化学杂志》。