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Allosteric activation of membrane-bound glutamate receptors using coordination chemistry within living cells

Nature Chemistry volume 8, pages 958967 (2016) | Download Citation

Abstract

The controlled activation of proteins in living cells is an important goal in protein-design research, but to introduce an artificial activation switch into membrane proteins through rational design is a significant challenge because of the structural and functional complexity of such proteins. Here we report the allosteric activation of two types of membrane-bound neurotransmitter receptors, the ion-channel type and the G-protein-coupled glutamate receptors, using coordination chemistry in living cells. The high programmability of coordination chemistry enabled two His mutations, which act as an artificial allosteric site, to be semirationally incorporated in the vicinity of the ligand-binding pockets. Binding of Pd(2,2′-bipyridine) at the allosteric site enabled the active conformations of the glutamate receptors to be stabilized. Using this approach, we were able to activate selectively a mutant glutamate receptor in live neurons, which initiated a subsequent signal-transduction pathway.

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Acknowledgements

This work was funded by the Japan Science and Technology Agency CREST of Molecular Technologies and JSPS KAKENHI (JP15H01637) to I.H., Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (B) to S.K. (25282238), Grants-in-Aid for Research Activity Start-up to R.K. (15H06318) and by the Ministry of Education, Culture, Sports, Science and Technology in Japan. We thank Y. Mori for providing the pEGFP-F plasmid and E. Gouaux for giving the plasmid of S1S2J. We also acknowledge the help of O. Hanpanich for constructing the plasmids of mGluR1.

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Author notes

    • Shigeki Kiyonaka
    •  & Ryou Kubota

    These authors contributed equally to this work

Affiliations

  1. Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan

    • Shigeki Kiyonaka
    • , Ryou Kubota
    • , Yukiko Michibata
    •  & Itaru Hamachi
  2. Department of Technology and Ecology, Hall of Global Environmental Studies, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan

    • Shigeki Kiyonaka
  3. Graduate School of Medical Life Science, Yokohama City University, 1-7-29 Suehirocho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan

    • Masayoshi Sakakura
    •  & Hideo Takahashi
  4. Department of Physiology, School of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan

    • Tomohiro Numata
    •  & Ryuji Inoue
  5. Department of Physiology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan

    • Michisuke Yuzaki
  6. Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, 5 Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan

    • Michisuke Yuzaki
    •  & Itaru Hamachi

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Contributions

I.H. and S.K. conceived the project. R.K. and M.Y. conducted the construction of GluA2 plasmids. R.K. performed Ca2+ imaging in HEK293T and cultured cortical neurons, western blot analyses in cultured cortical neurons and the immunostaining experiment in HEK293T cells. R.K. conducted the construction of the plasmid of S1S2J(KR) and prepared the protein sample of S1S2J (WT and mutants) for 1H–15N HMQC NMR spectroscopy and fluorescence measurements. H.T. and M.S. performed and analysed 1H–15N HMQC NMR spectroscopy. S.K., R.K. and Y.M. performed the fluorescence measurements. Y.M. and M.Y. conducted the construction of the mGluR1 plasmids. Y.M. performed the fluorescent Ca2+ imaging in HEK293 cells. S.K. conducted the p-CREB assay in rat cortical neurons. T.N., R.I. and M.Y. contributed to the analysis and interpretation of Ca2+-imaging data. T.N. and R.I. conducted the electrophysiological measurements of cell capacitance and membrane potential. R.K., S.K. and I.H. wrote the manuscript. All the authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Shigeki Kiyonaka or Itaru Hamachi.

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DOI

https://doi.org/10.1038/nchem.2554