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Biomimetic divalent ligands for the acute disruption of synaptic AMPAR stabilization

Abstract

The interactions of the AMPA receptor (AMPAR) auxiliary subunit Stargazin with PDZ domain–containing scaffold proteins such as PSD-95 are critical for the synaptic stabilization of AMPARs. To investigate these interactions, we have developed biomimetic competing ligands that are assembled from two Stargazin-derived PSD-95/DLG/ZO-1 (PDZ) domain–binding motifs using 'click' chemistry. Characterization of the ligands in vitro and in a cellular FRET-based model revealed an enhanced affinity for the multiple PDZ domains of PSD-95 compared to monovalent peptides. In cultured neurons, the divalent ligands competed with transmembrane AMPAR regulatory protein (TARP) for the intracellular membrane-associated guanylate kinase resulting in increased lateral diffusion and endocytosis of surface AMPARs, while showing strong inhibition of synaptic AMPAR currents. This provides evidence for a model in which the TARP-containing AMPARs are stabilized at the synapse by engaging in multivalent interactions. In light of the prevalence of PDZ domain clusters, these new biomimetic chemical tools could find broad application for acutely perturbing multivalent complexes.

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Figure 1: Role of multivalency in the stabilization of AMPAR–Stargazin complex interactions with PSD-95 (working model) and implication for the development of chemical-based perturbating tools.
Figure 2: Structure of the tandem PDZ domains of PSD-95 (domains 1 and 2, PDB: 3GSL).
Figure 3: Structure of the monovalent and divalent ligands.
Figure 4: Characterization of the peptide-based ligand interactions with PDZ domains.
Figure 5: FRET-FLIM system for the characterization of the interactions between the divalent ligand and the Stargazin–PSD-95 complex in live cells.
Figure 6: TAT-[STG15]2 induces a rapid and transient increase in the surface mobility of GluA2-AMPARs.
Figure 7: Effect of intracellular [STG15]2 on AMPAR synaptic currents.

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Acknowledgements

This research was supported by the Human Frontiers Science Program (RGP0007/2006, M.S., D.C. and B.I.), the European Commission Marie Curie postdoctoral fellowship (PICK-CPP to M.S.), grants from the Agence Nationale pour la Recherche (ChemTraffic to M.S. and D.C) and the Conseil Regional d'Aquitaine. The microscopy was done in the Bordeaux Imaging Center of the Neurosciences Institute of the University of Bordeaux II. The help of P. Legros is acknowledged. We are grateful to the staff at the National Synchrotron Light Source beamline X6A for access via the General User program and especially to J. Jackonic for assistance in data collection and reduction.

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B.I. and M.S. designed the biomimetic ligands and developed their synthesis and characterization in vitro; M.S. performed the experiments. Crystallization of the PDZ domains and structural analysis was performed by M.S., N.B.O. and M.S. collected the data and solved the structure. The affinity-based isolation experiments were performed by M.S., C.T. and L.B. performed the mobility experiments. C.T. performed the electrophysiology and endocytosis experiments. C.P. and K.T. performed the FRET experiments. C.B. performed the cloning for FRET and neuronal cultures. B.I. and D.C. equally provided project management. The manuscript was written by M.S., B.I. and D.C. with input from C.T., N.B.O. and C.P.

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Correspondence to Daniel Choquet or Barbara Imperiali.

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Sainlos, M., Tigaret, C., Poujol, C. et al. Biomimetic divalent ligands for the acute disruption of synaptic AMPAR stabilization. Nat Chem Biol 7, 81–91 (2011). https://doi.org/10.1038/nchembio.498

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