Imaging of receptor trafficking by using alpha-bungarotoxin-binding-site-tagged receptors.
alpha-Amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors mediate excitatory synaptic transmission and are dynamically regulated during synaptic plasticity in the CNS. The membrane trafficking of AMPA receptors to synapses is critical for the regulation of the efficacy of excitatory synaptic transmission. Direct imaging of AMPA receptors in various cell compartments is important to dissecting the regulation of distinct steps in receptor membrane trafficking. In this study, we have developed an approach for the imaging of receptor trafficking with subunits tagged with a 13-aa alpha-bungarotoxin (BTX)-binding site (BBS). The small polypeptide neurotoxin BTX has been used for decades to study the nicotinic acetylcholine receptor. Similar high-affinity ligands are rarely available for most receptors. Engineering the BBS tag into receptor subunits allowed the high-affinity binding of fluorescent, radioactive, and biotinylated BTX to the tagged receptor subunits. By using this approach, the total receptor expression, surface expression, internalization, and insertion of receptors into the plasma membrane could be visualized and quantified in fixed or live cells including cultured neurons. The BBS tag is a flexible approach for labeling membrane proteins and studying their dynamic trafficking.
Sekine-Aizawa
Yoko
Y
Howard Hughes Medical Institute, Department of Neuroscience, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.
Huganir
Richard L
RL
D000818:Animals; D001665:Binding Sites; D002038:Bungarotoxins; D002478:Cells, Cultured; D003952:Diagnostic Imaging / Q000379:methods; D006801:Humans; D018613:Microscopy, Confocal / Q000379:methods*; D015335:Molecular Probes; D009474:Neurons / Q000166:cytology; D021381:Protein Transport; D051381:Rats; D018091:Receptors, AMPA / Q000032:analysis / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000032:analysis / Q000378:metabolism; D014162:Transfection
D002038:Bungarotoxins; D015335:Molecular Probes; D018091:Receptors, AMPA; D011956:Receptors, Cell Surface