Chemical labelling for visualizing native AMPA receptors in live neurons

Abstract

The location and number of neurotransmitter receptors are dynamically regulated at postsynaptic sites. However, currently available methods for visualizing receptor trafficking require the introduction of genetically engineered receptors into neurons, which can disrupt the normal functioning and processing of the original receptor. Here we report a powerful method for visualizing native α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) which are essential for cognitive functions without any genetic manipulation. This is based on a covalent chemical labelling strategy driven by selective ligand-protein recognition to tether small fluorophores to AMPARs using chemical AMPAR modification (CAM) reagents. The high penetrability of CAM reagents enables visualization of native AMPARs deep in brain tissues without affecting receptor function. Moreover, CAM reagents are used to characterize the diffusion dynamics of endogenous AMPARs in both cultured neurons and hippocampal slices. This method will help clarify the involvement of AMPAR trafficking in various neuropsychiatric and neurodevelopmental disorders.

Simultaneous Live Imaging of Multiple Endogenous Proteins Reveals a Mechanism for Alzheimer’s-Related Plasticity Impairment

“…Excitatory synapse function of cultured neurons is also altered by overexpression of CaMKIIα (Barcomb et al, 2014; Thiagarajan et al, 2002). Several methods for live imaging of endogenous protein (i.e., without overexpression) have been developed (Campagnola et al, 2002; Mikuni et al, 2016; Panza et al, 2015; Wakayama et al, 2017; Xue et al, 2015). Intrabodies that bind to specific proteins can be expressed in cells and imaged by fusion with a fluorescent protein.…”

Considerations on Probe Design for Affinity‐Guided Protein Conjugation

“…Affinity‐guided protein labeling has been utilized extensively for creation of fluorescent protein conjugates. However, the strategies differ by either allowing for direct introduction of the fluorescent label or requiring prior modification with a reactive handle that is subsequently modified with a fluorophore (tag‐and‐modify) …”

“…Due to low reactivity of the reactive moieties under dilute conditions, affinity‐guided conjugation is possible in live cells, and it is the only protein modification technique that allows for selective protein conjugation in nongenetically modified cells. The probes are often used for labeling of extracellular membrane receptors, however, some probes have been utilized for labeling intracellular proteins .…”

“…For this reason, affinity‐guided probes have also been developed for transfer of a biotin handle to the protein. In these cases, the biotin handle was used for blotting of the target protein that had been labeled in living systems …”

“…The imidazole carbamate reactive moiety has been extensively used to study the biology of proteins (Figure C), especially membrane bound receptors . During the conjugation reaction, the imidazole acts as a leaving group transferring the label containing alkyl formate to the protein.…”