Imaging of receptor trafficking by using α-bungarotoxin-binding-site-tagged receptors
- Howard Hughes Medical Institute, Department of Neuroscience, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205
-
Contributed by Richard L. Huganir, October 18, 2004
Abstract
α-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 α-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.
Footnotes
-
↵ * To whom correspondence should be addressed. E-mail: rhuganir{at}jhmi.edu.
-
↵ † Under a licensing agreement between Upstate Group, Inc., and The Johns Hopkins University, R.L.H. is entitled to a share of royalty received by the University on sales of products described in this article. R.L.H. is a paid consultant to Upstate Group, Inc. The terms of this arrangement are being managed by The Johns Hopkins University in accordance with its conflict-of-interest policies.
-
Abbreviations: AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate; BTX, α-bungarotoxin; BBS, BTX-binding site; HEK, human embryonic kidney; HRP, horseradish peroxidase.
-
Freely available online through the PNAS open access option.
- Copyright © 2004, The National Academy of Sciences
