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Ligand-directed two-step labeling to quantify neuronal glutamate receptor trafficking

Kento Ojima, Kazuki Shiraiwa, Tomohiro Doura, Mikiko Takato, Kazuhiro Komatsu, Michisuke Yuzaki, Itaru Hamachi, View ORCID ProfileShigeki Kiyonaka
doi: https://doi.org/10.1101/2020.05.20.105296
Kento Ojima
1Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan
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Kazuki Shiraiwa
1Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan
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Tomohiro Doura
2Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Nagoya 464-8603, Japan
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Mikiko Takato
1Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan
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Kazuhiro Komatsu
1Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan
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Michisuke Yuzaki
3Department of Physiology, School of Medicine, Keio University, Tokyo 160-8582, Japan
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Itaru Hamachi
1Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan
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  • For correspondence: ihamachi@sbchem.kyoto-u.ac.jp kiyonaka@chembio.nagoya-u.ac.jp
Shigeki Kiyonaka
2Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Nagoya 464-8603, Japan
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  • ORCID record for Shigeki Kiyonaka
  • For correspondence: ihamachi@sbchem.kyoto-u.ac.jp kiyonaka@chembio.nagoya-u.ac.jp
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ABSTRACT

The regulation of glutamate receptor localization is critical for development and synaptic plasticity in the central nervous system. Conventional biochemical and molecular biological approaches have been widely used to analyze glutamate receptor trafficking, especially for AMPA-type glutamate receptors (AMPARs). However, conflicting findings have been reported because of a lack of useful tools for analyzing endogenous AMPARs. Here, we develop a new method for the rapid and selective labeling of chemical probes to AMPARs by combining affinity-based protein labeling and bioorthogonal click chemistry under physiological conditions. This method allowed us to quantify AMPAR distribution and trafficking, which revealed some unique features of AMPARs, such as a long lifetime and a rapid recycling in neurons. This method was also successfully expanded to selectively label NMDA-type glutamate receptors. Thus, bioorthogonal two-step labeling may be a versatile tool for investigating the physiological and pathophysiological roles of glutamate receptors in neurons.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted May 22, 2020.
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Ligand-directed two-step labeling to quantify neuronal glutamate receptor trafficking
Kento Ojima, Kazuki Shiraiwa, Tomohiro Doura, Mikiko Takato, Kazuhiro Komatsu, Michisuke Yuzaki, Itaru Hamachi, Shigeki Kiyonaka
bioRxiv 2020.05.20.105296; doi: https://doi.org/10.1101/2020.05.20.105296
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Ligand-directed two-step labeling to quantify neuronal glutamate receptor trafficking
Kento Ojima, Kazuki Shiraiwa, Tomohiro Doura, Mikiko Takato, Kazuhiro Komatsu, Michisuke Yuzaki, Itaru Hamachi, Shigeki Kiyonaka
bioRxiv 2020.05.20.105296; doi: https://doi.org/10.1101/2020.05.20.105296

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