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

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。我们将研究重点放在这两个离子通道上。一个是CIC氯通道家族成员，CIC-4，具有独特的二聚体结构。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重要作用。已知包括PSD-95在内的许多突触后密度蛋白与NMDA受体相互作用。然而，目前还不清楚NMDA受体的通道活性是否受这些蛋白的调节。在本研究中，我们研究了蛋白质-蛋白质相互作用在这两种离子通道结构和功能中的作用。为了寻找与CIC-4相互作用的蛋白质，我们利用人脑cDNA文库，以CIC-4的N-末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末端区域较多，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胞质结构域的融合蛋白，并检测了融合蛋白之间的相互作用。我们发现N-末端结构域与C-末端结构域结合，也与N-末端结构域本身结合。除了N终端域之外，C终端域还绑定C终端域本身。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，这些细胞质结构域之间的相互作用是CIC-4通道二聚体形成的原因。我们通过卵母细胞表达系统检测了PSD-95对NR1/NR2A受体通道活性的影响，并与NR1/NR2B受体的作用进行了比较。PSD-95的表达可抑制蛋白激酶C对NR1/NR2A通道和NR1/NR2B通道的增强作用。此外，我们还证明了PSD-95消除了Src对卵母细胞中表达的NR1/NR2A通道的诱导增强作用，并降低了通道对锌的敏感性。我们的结果表明，PSD-95偶联的NR1/NR2A通道没有Src诱导的增强作用，并不是因为这些通道对锌的敏感性降低。这些结果表明，PSD-95在功能上调节NR1/NR2A通道，并解释了为什么Src诱导的海马神经元NMDA受体电流的增强不是通过减少锌抑制来介导的。较少

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