2001 Fiscal Year Final Research Report Summary
Molecular mechanisms of ion channel regulation by a protein-protein interaction
| Project/Area Number |
12470019
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General pharmacology
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| Research Institution | Yamaguchi University |
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Principal Investigator |
INUI Makoto Yamaguchi University, School of Medicine, Professor, 医学部, 教授 (70223237)
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| Co-Investigator(Kenkyū-buntansha) |
KO Jie Yamaguchi University, School of Medicine, Research Associate, 医学部, 助手 (70314797)
YAMADA Yasue Yamaguchi University, School of Medicine, Research Associate, 医学部, 助手 (00166737)
KIMURA Yoshihiro Yamaguchi University, School of Medicine, Assistant Professor, 医学部, 講師 (90301308)
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| Project Period (FY) |
2000 – 2001
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| Keywords | Ion channels / Chloride channels / Protein-protein interaction / Dimer formation / PSD-95 / NMDA receptor / Protein phosphorylation / Zinc inhibition |
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| Research Abstract |
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
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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
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[Publications] Haghighi K, Schmidt A. G, Hoit B. D, Brittsan A. G, Yatani A, Lester J. W, Zhai J, Kimura Y, Dorn G. W, MacLennan D. H, and Kranias E. G: "Superinhibition of sarcoplasmic reticulum function by phospholamban induces cardiac contractile failure"Journal of Biological Chemistry. 276. 24145-24152 (2001)
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