A synthetic peptide that prevents cAMP regulation in mammalian Hyperpolarization-activated Cyclic Nucleotide-regulated (HCN) channels

  1. Andrea Saponaro
  2. Francesca Cantini
  3. Alessandro Porro
  4. Annalisa Bucchi
  5. Dario DiFrancesco
  6. Vincenzo Maione
  7. Chiara Donadoni
  8. Bianca Introini
  9. Pietro Mesirca
  10. Matteo Elia Mangoni
  11. Gerhard Thiel
  12. Lucia Banci
  13. Bina Santoro
  14. Anna Moroni  Is a corresponding author
  1. University of Milan, Italy
  2. University of Florence, Italy
  3. Université de Montpellier, CNRS, INSERM, France
  4. Technische Universität Darmstadt, Germany
  5. Columbia University, United States

Abstract

Binding of TRIP8b to the cyclic nucleotide binding domain (CNBD) of mammalian HCN channels prevents their regulation by cAMP. Since TRIP8b is expressed exclusively in the brain, we envisage that it can be used for orthogonal control of HCN channels beyond the central nervous system. To this end, we have identified by rational design a 40-aa long peptide (TRIP8bnano) that recapitulates affinity and gating effects of TRIP8b in HCN isoforms (human hHCN1, mHCn2, rbHCN4) and in the cardiac current If in rabbit and mouse sinoatrial node cardiomyocytes. Guided by a NMR-derived structural model that identifies the key molecular interactions between TRIP8bnano and HCN CNBD, we further designed a cell-penetrating peptide (TAT-TRIP8bnano) which successfully prevented b-adrenergic activation of mouse If leaving the stimulation of the L-type calcium current (ICaL) unaffected. TRIP8bnano represents a novel approach to selectively control HCN activation, which yields the promise of a more targeted pharmacology compared to pore blockers.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figure 3.

Article and author information

Author details

  1. Andrea Saponaro

    Department of Biosciences, University of Milan, Milan, Italy
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5035-5174
  2. Francesca Cantini

    Centro Risonanze Magnetiche (CERM), University of Florence, Florence, Italy
    Competing interests
    The authors declare that no competing interests exist.
  3. Alessandro Porro

    Department of Biosciences, University of Milan, Milan, Italy
    Competing interests
    The authors declare that no competing interests exist.
  4. Annalisa Bucchi

    Department of Biosciences, University of Milan, Milan, Italy
    Competing interests
    The authors declare that no competing interests exist.
  5. Dario DiFrancesco

    Department of Biosciences, University of Milan, Milan, Italy
    Competing interests
    The authors declare that no competing interests exist.
  6. Vincenzo Maione

    Centro Risonanze Magnetiche (CERM), University of Florence, Florence, Italy
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8229-6612
  7. Chiara Donadoni

    Department of Biosciences, University of Milan, Milan, Italy
    Competing interests
    The authors declare that no competing interests exist.
  8. Bianca Introini

    Department of Biosciences, University of Milan, Milan, Italy
    Competing interests
    The authors declare that no competing interests exist.
  9. Pietro Mesirca

    Institut de Génomique Fonctionnelle, Université de Montpellier, CNRS, INSERM, Montpellier, France
    Competing interests
    The authors declare that no competing interests exist.
  10. Matteo Elia Mangoni

    Institut de Génomique Fonctionnelle, Université de Montpellier, CNRS, INSERM, Montpellier, France
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8892-3373
  11. Gerhard Thiel

    Department of Biology, Technische Universität Darmstadt, Darmstadt, Germany
    Competing interests
    The authors declare that no competing interests exist.
  12. Lucia Banci

    Centro Risonanze Magnetiche (CERM), University of Florence, Florence, Italy
    Competing interests
    The authors declare that no competing interests exist.
  13. Bina Santoro

    Department of Neuroscience, Columbia University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  14. Anna Moroni

    Department of Biosciences, University of Milan, Milan, Italy
    For correspondence
    anna.moroni@unimi.it
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1860-406X

Funding

Fondazione Cariplo

  • Anna Moroni

National Institutes of Health

  • Anna Moroni

H2020 European Research Council

  • Anna Moroni

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Volker Dötsch, J.W. Goethe-University, Germany

Ethics

Animal experimentation: Experiments on rabbit SAN cells were performed using left-over cells obtained during experiments approved by the Animal Welfare Body of the University of Milan and by the Italian Ministry of Health (license n.1127/2015-PR). Animal procedures were conformed to the guidelines of the care and use of laboratory animals established by Italian and European Directives (D. Lgs no 2014/26, 2010/63/UE).Mouse primary pacemaker cells were isolated from adult C5B6/J mice as previously described (Mangoni and Nargeot, Cardiovasc Res 2001), in accordance with the Guide for the Care and Use of Laboratory Animals (eighth edition, 2011), published by the US National Institute of Health and European directives (2010/63/EU). The protocol was approved by the ethical committee of the University of Montpellier and the French Ministry of Agriculture (protocol N{degree sign}: 2017010310594939).

Version history

  1. Received: February 7, 2018
  2. Accepted: June 14, 2018
  3. Accepted Manuscript published: June 20, 2018 (version 1)
  4. Version of Record published: June 28, 2018 (version 2)

Copyright

© 2018, Saponaro 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.

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  1. Andrea Saponaro
  2. Francesca Cantini
  3. Alessandro Porro
  4. Annalisa Bucchi
  5. Dario DiFrancesco
  6. Vincenzo Maione
  7. Chiara Donadoni
  8. Bianca Introini
  9. Pietro Mesirca
  10. Matteo Elia Mangoni
  11. Gerhard Thiel
  12. Lucia Banci
  13. Bina Santoro
  14. Anna Moroni
(2018)
A synthetic peptide that prevents cAMP regulation in mammalian Hyperpolarization-activated Cyclic Nucleotide-regulated (HCN) channels
eLife 7:e35753.
https://doi.org/10.7554/eLife.35753

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https://doi.org/10.7554/eLife.35753

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