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

Kento Ojima, Kazuki Shiraiwa, Kyohei Soga, Tomohiro Doura, Mikiko Takato, Kazuhiro Komatsu, Michisuke Yuzaki, Itaru Hamachi () and Shigeki Kiyonaka ()
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Kento Ojima: Kyoto University
Kazuki Shiraiwa: Kyoto University
Kyohei Soga: Nagoya University
Tomohiro Doura: Nagoya University
Mikiko Takato: Kyoto University
Kazuhiro Komatsu: Kyoto University
Michisuke Yuzaki: Keio University
Itaru Hamachi: Kyoto University
Shigeki Kiyonaka: Nagoya University

Nature Communications, 2021, vol. 12, issue 1, 1-14

Abstract: 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 α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate-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 method for the rapid and selective labeling of AMPARs with chemical probes, by combining affinity-based protein labeling and bioorthogonal click chemistry under physiological temperature in culture medium. This method allows us to quantify AMPAR distribution and trafficking, which reveals some unique features of AMPARs, such as a long lifetime and a rapid recycling in neurons. This method is also successfully expanded to selectively label N-methyl-D-aspartate-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.

Date: 2021
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DOI: 10.1038/s41467-021-21082-x

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