Super-resolution imaging of synaptic and extra-synaptic AMPA receptors with different-sized fluorescent probes
- University of Illinois at Urbana-Champaign, United States
- University of California, San Francisco, United States
- University of Illinois at Urbana Champaign, United States
- University of Campinas, Brazil
- University of Chicago, United States
- State University of New York, United States
- University of Texas, United States
Abstract
Previous studies tracking AMPA receptor (AMPAR) diffusion at synapses observed a large mobile extrasynaptic AMPAR pool. Using super-resolution microscopy, we examined how fluorophore size and photostability affected AMPAR trafficking outside of, and within, post-synaptic densities (PSDs) from rats. Organic fluorescent dyes (≈4 nm), quantum dots, either small (≈10 nm diameter; sQDs) or big (>20 nm; bQDs), were coupled to AMPARs via different-sized linkers. We find that >90% of AMPARs labeled with fluorescent dyes or sQDs were diffusing in confined nanodomains in PSDs, which were stable for 15 minutes or longer. Less than 10% of sQD-AMPARs were extrasynaptic and highly mobile. In contrast, 5–10% of bQD-AMPARs were in PSDs and 90-95% were extrasynaptic as previously observed. Contrary to the hypothesis that AMPAR entry is limited by the occupancy of open PSD "slots", our findings suggest that AMPARs rapidly enter stable "nanodomains" in PSDs with lifetime ≥15 minutes, and do not accumulate in extrasynaptic membranes.
Article and author information
Author details
Funding
National Institutes of Health (NIH NS090903)
- William N Green
- Paul R Selvin
National Science Foundation (PHY-1430124)
- Paul R Selvin
National Science Foundation (CBET-1264051)
- Sheldon Park
National Institutes of Health (NIH NS100019)
- William N Green
- Paul R Selvin
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: Primary hippocampal cultures were prepared from E18 rats according to UIUC guidelines. All rats were handled according to approved institutional animal care and use committee (IACUC) protocols (#15254) of UIUC.
Copyright
© 2017, Lee et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 7,292
- views
-
- 1,224
- downloads
-
- 59
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Super-resolution imaging of synaptic and extra-synaptic AMPA receptors with different-sized fluorescent probes
eLife 6:e27744.
https://doi.org/10.7554/eLife.27744
Further reading
-
- Structural Biology and Molecular Biophysics
Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor transcription factor that regulates gene expression programs in response to ligand binding. Endogenous and synthetic ligands, including covalent antagonist inhibitors GW9662 and T0070907, are thought to compete for the orthosteric pocket in the ligand-binding domain (LBD). However, we previously showed that synthetic PPARγ ligands can cooperatively cobind with and reposition a bound endogenous orthosteric ligand to an alternate site, synergistically regulating PPARγ structure and function (Shang et al., 2018). Here, we reveal the structural mechanism of cobinding between a synthetic covalent antagonist inhibitor with other synthetic ligands. Biochemical and NMR data show that covalent inhibitors weaken—but do not prevent—the binding of other ligands via an allosteric mechanism, rather than direct ligand clashing, by shifting the LBD ensemble toward a transcriptionally repressive conformation, which structurally clashes with orthosteric ligand binding. Crystal structures reveal different cobinding mechanisms including alternate site binding to unexpectedly adopting an orthosteric binding mode by altering the covalent inhibitor binding pose. Our findings highlight the significant flexibility of the PPARγ orthosteric pocket, its ability to accommodate multiple ligands, and demonstrate that GW9662 and T0070907 should not be used as chemical tools to inhibit ligand binding to PPARγ.
