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.
Subject(s)
Neurons/metabolism , Receptors, AMPA/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Staining and Labeling/methods , Animals , Cerebral Cortex/cytology , Cerebral Cortex/metabolism , Excitatory Amino Acid Antagonists/chemistry , Fluorescein/chemistry , Fluorescent Dyes/chemistry , Gene Expression , HEK293 Cells , Half-Life , Hippocampus/cytology , Hippocampus/metabolism , Humans , Ligands , Mice , Mice, Inbred ICR , Neurons/ultrastructure , Primary Cell Culture , Protein Transport , Quinoxalines/chemistry , Rats , Rats, Sprague-Dawley , Receptors, AMPA/chemistry , Receptors, AMPA/genetics , Receptors, N-Methyl-D-Aspartate/chemistry , Receptors, N-Methyl-D-Aspartate/geneticsABSTRACT
Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) can inflict damage to biomolecules under oxidative stress and also act as signaling molecules at physiological levels. Here we developed a unique chemical tool to elucidate the biological roles of ROS using both fluorescence imaging and conditional proteomics. H2O2-responsive protein labeling reagents (Hyp-L) were designed to selectively tag proteins under the oxidative conditions in living cells and tissues. The Hyp-L signal remained even after sample fixation, which was compatible with conventional immunostaining. Moreover, Hyp-L allowed proteomic profiling of the labeled proteins using a conditional proteomics workflow. The integrative analysis enabled the identification of ROS generation and/or accumulation sites with a subcellular resolution. For the first time, we characterized that autophagosomes were enriched with H2O2 in activated macrophages. Hyp-L was further applied to mouse brain tissues and clearly revealed oxidative stress within mitochondria by the conditional proteomics.
