Nucleotide inhibition of the pancreatic ATP-sensitive K+ channel explored with patch-clamp fluorometry

  1. Samuel G Usher
  2. Frances M Ashcroft  Is a corresponding author
  3. Michael C Puljung  Is a corresponding author
  1. University of Oxford, United Kingdom

Abstract

Pancreatic ATP-sensitive K+ channels (KATP) comprise four inward rectifier subunits (Kir6.2), each associated with a sulphonylurea receptor (SUR1). ATP/ADP binding to Kir6.2 shuts KATP. Mg-nucleotide binding to SUR1 stimulates KATP. In the absence of Mg2+, SUR1 increases the apparent affinity for nucleotide inhibition at Kir6.2 by an unknown mechanism. We simultaneously measured channel currents and nucleotide binding to Kir6.2. Fits to combined data sets suggest that KATP closes with only one nucleotide molecule bound. A Kir6.2 mutation (C166S) that increases channel activity did not affect nucleotide binding, but greatly perturbed the ability of bound nucleotide to inhibit KATP. Mutations at position K205 in SUR1 affected both nucleotide affinity and the ability of bound nucleotide to inhibit KATP. This suggests a dual role for SUR1 in KATP inhibition, both in directly contributing to nucleotide binding and in stabilising the nucleotide-bound closed state.

Data availability

All data and associated code are available on github (https://github.com/smusher/KATP_paper_2019) and have also been uploaded to Dryad (https://doi.org/10.5061/dryad.0vt4b8gtv).

The following data sets were generated

Article and author information

Author details

  1. Samuel G 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
  2. Frances M 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.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6970-1767
  3. Michael C 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

Funding

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

  • Frances M Ashcroft
  • Michael C Puljung

John Fell Fund, University of Oxford

  • Michael C Puljung

Wellcome (203731/Z/16/A)

  • Samuel G Usher

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

Reviewing Editor

  1. Gary Yellen, Harvard Medical School, United States

Publication history

  1. Received: October 16, 2019
  2. Accepted: January 7, 2020
  3. Accepted Manuscript published: January 7, 2020 (version 1)
  4. Version of Record published: February 6, 2020 (version 2)
  5. Version of Record updated: February 27, 2020 (version 3)

Copyright

© 2020, Usher 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. Samuel G Usher
  2. Frances M Ashcroft
  3. Michael C Puljung
(2020)
Nucleotide inhibition of the pancreatic ATP-sensitive K+ channel explored with patch-clamp fluorometry
eLife 9:e52775.
https://doi.org/10.7554/eLife.52775
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