Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, 615-8510, Japan.
Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Nagoya, 464-8603, Japan.
Nat Commun. 2021 Feb 5;12(1):831. doi: 10.1038/s41467-021-21082-x.
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.
谷氨酸受体定位的调节对于中枢神经系统的发育和突触可塑性至关重要。传统的生化和分子生物学方法已被广泛用于分析谷氨酸受体的运输，特别是对于α-氨基-3-羟基-5-甲基-4-异恶唑丙酸型谷氨酸受体（AMPARs）。然而，由于缺乏用于分析内源性 AMPAR 的有用工具，因此报道了相互矛盾的发现。在这里，我们通过在生理温度下在培养基中结合基于亲和力的蛋白质标记和生物正交点击化学，开发了一种快速且选择性地用化学探针标记 AMPAR 的方法。该方法使我们能够定量 AMPAR 的分布和运输，从而揭示了 AMPAR 的一些独特特征，例如在神经元中的长寿命和快速循环。该方法还成功扩展到选择性标记 N-甲基-D-天冬氨酸型谷氨酸受体。因此，生物正交两步标记可能是研究谷氨酸受体在神经元中的生理和病理生理作用的通用工具。
