1. Structural Biology and Molecular Biophysics

Activation mechanism of ATP-sensitive K+ channels explored with real-time nucleotide binding

  1. Michael Puljung  Is a corresponding author
  2. Natascia Vedovato
  3. Samuel Usher
  4. Frances Ashcroft  Is a corresponding author
  1. University of Oxford, United Kingdom
https://doi.org/10.7554/eLife.41103
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  1. Michael Puljung
  2. Natascia Vedovato
  3. Samuel Usher
  4. Frances Ashcroft
(2019)
Activation mechanism of ATP-sensitive K+ channels explored with real-time nucleotide binding
eLife 8:e41103.
https://doi.org/10.7554/eLife.41103
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Abstract

The response of ATP-sensitive K+ channels (KATP) to cellular metabolism is coordinated by three classes of nucleotide binding site (NBS). We used a novel approach involving labeling of intact channels in a native, membrane environment with a non-canonical fluorescent amino acid and measurement (using FRET with fluorescent nucleotides) of steady-state and time-resolved nucleotide binding to dissect the role of NBS2 of the accessory SUR1 subunit of KATP in channel gating. Binding to NBS2 was Mg2+-independent, but Mg was required to trigger a conformational change in SUR1. Mutation of a lysine (K1384A) in NBS2 that coordinates bound nucleotides increased the EC50 for trinitrophenyl-ADP binding to NBS2, but only in the presence of Mg2+, indicating that this mutation disrupts the ligand-induced conformational change. Comparison of nucleotide-binding with ionic currents suggests a model in which each nucleotide binding event to NBS2 of SUR1 is independent and promotes KATP activation by the same amount.

Data availability

Data available from the Dryad Digital Repository: https://doi.org/10.5061/dryad.6mh0sv3

The following data sets were generated

Article and author information

Author details

  1. Michael Puljung

    Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom
    For correspondence
    michael.puljung@dpag.ox.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9335-0936

  2. Natascia Vedovato

    Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  3. Samuel Usher

    Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2487-6547

  4. Frances Ashcroft

    Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom
    For correspondence
    frances.ashcroft@dpag.ox.ac.uk
    Competing interests
    The authors declare that no competing interests exist.

Funding

Biotechnology and Biological Sciences Research Council (BB/R002517/1)

  • Michael Puljung
  • Frances Ashcroft

H2020 European Research Council (322620)

  • Michael Puljung
  • Natascia Vedovato
  • Frances Ashcroft

Wellcome Trust Oxion Graduate Program

  • Samuel Usher

John Fell Fund, University of Oxford

  • Michael Puljung

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

Reviewing Editor

  1. Kenton Jon Swartz, National Institute of Neurological Disorders and Stroke, National Institutes of Health, United States

Version history

  1. Received: August 14, 2018
  2. Accepted: February 14, 2019
  3. Accepted Manuscript published: February 21, 2019 (version 1)
  4. Version of Record published: March 5, 2019 (version 2)

Copyright

© 2019, Puljung 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. Michael Puljung
  2. Natascia Vedovato
  3. Samuel Usher
  4. Frances Ashcroft
(2019)
Activation mechanism of ATP-sensitive K+ channels explored with real-time nucleotide binding
eLife 8:e41103.
https://doi.org/10.7554/eLife.41103

Share this article

https://doi.org/10.7554/eLife.41103

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