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Regulation of AMPA receptor extrasynaptic insertion by 4.1N, phosphorylation and palmitoylation

Nature Neuroscience volume 12, pages 879–887 (2009)Cite this article

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Abstract

The insertion of AMPA receptors (AMPARs) into the plasma membrane is an important step in the synaptic delivery of AMPARs during the expression of synaptic plasticity. However, the molecular mechanisms regulating AMPAR insertion remain elusive. By directly visualizing individual insertion events of the AMPAR subunit GluR1 in rodents, we found that the protein 4.1N was required for activity-dependent GluR1 insertion. Protein kinase C (PKC) phosphorylation of the serine 816 (S816) and S818 residues of GluR1 enhanced 4.1N binding to GluR1 and facilitated GluR1 insertion. In addition, palmitoylation of GluR1 C811 residue modulated PKC phosphorylation and GluR1 insertion. Finally, disrupting 4.1N-dependent GluR1 insertion decreased surface expression of GluR1 and the expression of long-term potentiation. Our study uncovers a previously unknown mechanism that governs activity-dependent GluR1 trafficking, reveals an interaction between AMPAR palmitoylation and phosphorylation, and underscores the functional importance of 4.1N in AMPAR trafficking and synaptic plasticity.

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Figure 1: Direct observation of GluR1 insertion events at extrasynaptic sites.
Figure 2: Activity-dependent GluR1 insertion.
Figure 3: GluR1 MPR is required for GluR1 insertion.
Figure 4: 4.1N is required for GluR1 insertion.
Figure 5: Phosphorylation and depalmitoylation regulate GluR1 insertion.
Figure 6: Regulation of the 4.1N and GluR1 interaction.
Figure 7: Reduction in GluR1 insertion results in reduced surface expression.
Figure 8: 4.1N is required for LTP expression in acute hippocampal slices of adult mice.

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  • 14 June 2009

    In the version of this article initially published online, two words were omitted. In the last paragraph of the first section under Results, the sentence “Acute suppression of excitatory neuronal activity by applying a cocktail of tetrodotoxin (TTX, 1 μM), 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX, 20 mM) and DL(–)-2-amino-5-phosphonovaleric acid (AP5, 200 μM) substantially the insertion frequency of R1pH (Fig. 2b)” should read “Acute suppression of excitatory neuronal activity by applying a cocktail of tetrodotoxin (TTX, 1 μM), 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX, 20 μM) and DL(–)-2-amino-5-phosphonovaleric acid (AP5, 200 μM) substantially reduced the insertion frequency of R1pH (Fig. 2b).” In the last paragraph of the fourth section under Results, the sentence “Following PKC activation, we detected greater phosphorylation of S818 in GluR1C811S compared with of GluR1 (Fig. 6e,f)” should read “Following PKC activation, we detected greater phosphorylation of S818 in GluR1C811S compared with that of GluR1 (Fig. 6e,f).” These errors have been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

We would like to thank M. Dai in the Monoclonal Antibody Core Facility of the Johns Hopkins University School of Medicine Department of Neuroscience for generating the monoclonal antibody to GluR1 N terminus, M. Coulter, R. Johnson and Y. Yu for their outstanding technical assistance and B. Bowman and R.M.E. Chalmers-Redman from the Carl Zeiss Micro-Imaging Group for their excellent technical support. This work is funded by US National Institutes of Health grant R01NS036715 and by the Howard Hughes Medical Institute.

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  1. Kogo Takamiya

    Present address: Present address: Department of Integrative Physiology, University of Miyazaki Faculty of Medicine, Miyazaki, Japan.,

Authors and Affiliations

  1. Department of Neuroscience and Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Da-Ting Lin, Yuichi Makino, Kamal Sharma, Takashi Hayashi, Kogo Takamiya & Richard L Huganir

  2. Massachusetts Institute of Technology, The Picower Institute, Cambridge, Massachusetts, USA

    Rachael Neve

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Contributions

D.-T.L. conceived the project, set up the TIR-FM imaging system, designed and executed majority of experiments, analyzed the experimental results and wrote the manuscript. Y.M. carried out in vivo virus injections, electrophysiological recordings and some biochemical experiments, and analyzed the experimental results. K.S. performed immunocytochemistry studies and analyzed the results. T.H. carried out 3H-palmitate labeling experiments. R.N. provided the HSV expression system. K.T. established the HSV and lentivirus systems and GluR1 knockout mice. R.L.H. conceived the project, designed the experiments, provided funding and guidance for the project and wrote the manuscript.

Corresponding author

Correspondence to Richard L Huganir.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 (PDF 9281 kb)

Supplementary Movie 1

Individual insertion events of pHluorin-tagged GluR1 can be observed using TIR-FM. (AVI 1635 kb)

Supplementary Movie 2

Generation of y-t maximum intensity projection images to view individual insertion events of R1pH. (AVI 102 kb)

Supplementary Movie 3

Individual insertion events of R1pH can be observed on dendrites. Dendritic spines were clearly visible when R1pH insertion was imaged under epi-fluorescent illumination mode, but insertion on spines was not observed. (AVI 280 kb)

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Lin, DT., Makino, Y., Sharma, K. et al. Regulation of AMPA receptor extrasynaptic insertion by 4.1N, phosphorylation and palmitoylation. Nat Neurosci 12, 879–887 (2009). https://doi.org/10.1038/nn.2351

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