[Quantification of AMPA-type glutamate receptors trafficking by ligand-directed two-step labeling]

Glutamate receptors mediate excitatory neurotransmission in the central nervous system, which have essential roles in our learning and memory. Recent studies have revealed that the trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA)-type glutamate receptors (AMPA receptors) are dynamically regulated during synaptic plasticity, the cellular basis of learning and memory. Conventionally, biochemical methods such as surface-biotin labeling or genetic incorporation of fluorescent proteins have been utilized to analyze the AMPA receptors dynamics. However, conflicting findings have been reported because of serious issues in these conventional methods. As the alternative, we have developed a new method for labeling AMPA receptors endogenously expressed in neurons by chemical approaches. This is based on a covalent chemical labeling strategy driven by selective ligand-protein recognition to tether small fluorophores to the target receptors, termed ligand-directed acyl imidazole chemistry. This method has successfully visualized AMPA receptors endogenously expressed in neurons. However, the original method required several hours for fluorophore labeling, which hampered analyzing the dynamics of AMPA receptors in detail. As the alternative, we have recently developed an improved strategy for rapid and selective labeling of chemical probes to cell-surface AMPA receptors by combining ligand-directed chemistry and bio-orthogonal click chemistry. This method allowed to quantify their trafficking, which revealed unique features of AMPA receptors such as long lifetime and rapid recycling in neurons. Notably, this method can be expanded to other receptors. Thus, the two-step labeling method would be a useful tool for understanding the physiological or pathophysiological roles of glutamate receptors in neurons.