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Gephyrin-binding peptides visualize postsynaptic sites and modulate neurotransmission

Nature Chemical Biology volume 13, pages 153–160 (2017)Cite this article

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Abstract

γ-Aminobutyric acid type A and glycine receptors are the major mediators of fast synaptic inhibition in the human central nervous system and are established drug targets. However, all drugs targeting these receptors bind to the extracellular ligand-binding domain of the receptors, which inherently is associated with perturbation of the basic physiological action. Here we pursue a fundamentally different approach, by instead targeting the intracellular receptor–gephyrin interaction. First, we defined the gephyrin peptide-binding consensus sequence, which facilitated the development of gephyrin super-binding peptides and later effective affinity probes for the isolation of native gephyrin. Next, we demonstrated that fluorescent super-binding peptides could be used to directly visualize inhibitory postsynaptic sites for the first time in conventional and super-resolution microscopy. Finally, we demonstrate that the gephyrin super-binding peptides act as acute intracellular modulators of fast synaptic inhibition by modulating receptor clustering, thus being conceptually novel modulators of inhibitory neurotransmission.

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Figure 1: Principle of action of gephyrin SBPs.
Figure 2: Peptide-array-based profiling of gephyrin.
Figure 3: Design and evaluation of gephyrin super-binding peptides.
Figure 4: Peptide-based visualization of gephyrin in HEK cells and neurons.
Figure 5: SBPs modulate fast synaptic inhibition in living neurons.

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Acknowledgements

We thank P. Breiden for her excellent technical assistance. We thank C. Geis and C. Sommer (Hans–Berger Department of Neurology, Jena University Hospital, Jena, Germany) for providing primary cultures of mouse hippocampal neurons. H.M.M. was supported by the Lundbeck Foundation (R118-A11469). S.D. and M.S. acknowledge funding by the German Research Foundation (SFB/TRR166).

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Authors and Affiliations

  1. Department of Drug Design and Pharmacology, Center for Biopharmaceuticals, University of Copenhagen, Copenhagen, Denmark

    Hans Michael Maric, Nils Ole Dalby & Kristian Strømgaard

  2. Department of Molecular Neurogenetics, Center for Molecular Neurobiology, ZMNH, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    Torben Johann Hausrat & Matthias Kneussel

  3. Department of Biotechnology and Biophysics, Biocenter, University of Würzburg, Würzburg, Germany

    Franziska Neubert, Sören Doose & Markus Sauer

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Contributions

H.M.M. and K.S. conceived the project. H.M.M., T.J.H., M.K., S.D., M.S. and K.S. designed the research. Microarrays were designed by H.M.M. and synthesized by the Intavis AG. H.M.M. carried out the μSPOT experiments, peptide synthesis, purification and analysis, the ITC measurements, competition experiments and the protein expression and purification. T.J.H. and H.M.M. carried out the preparation of brain lysates, the HEK cell and neuronal confocal microscopy experiments and their quantification, the pull-down experiments and the western blotting. T.J.H. carried out the toxicological assays as well as the immunohistochemical analysis of the SBP exposed neurons. Electrophysiological experiments were conducted and analyzed by N.O.D. Super resolution imaging and their quantification was carried out by F.N. and S.D. The manuscript was written by H.M.M. and K.S. with input from all authors.

Corresponding authors

Correspondence to Hans Michael Maric or Kristian Strømgaard.

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Competing interests

The University of Copenhagen has filed a provisional patent application (PA 2015 70783) on peptides described herein with H.M.M. and K.S. as inventors.

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Maric, H., Hausrat, T., Neubert, F. et al. Gephyrin-binding peptides visualize postsynaptic sites and modulate neurotransmission. Nat Chem Biol 13, 153–160 (2017). https://doi.org/10.1038/nchembio.2246

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