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.

Metrics

  • 1,483
    Page views
  • 210
    Downloads
  • 11
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  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

Further reading

    1. Structural Biology and Molecular Biophysics
    Daan B Boltje, Jacob P Hoogenboom ... Sander den Hoedt
    Tools and Resources Updated

    Cryogenic electron tomography (cryo-ET) combined with subtomogram averaging, allows in situ visualization and structure determination of macromolecular complexes at subnanometre resolution. Cryogenic focused ion beam (cryo-FIB) micromachining is used to prepare a thin lamella-shaped sample out of a frozen-hydrated cell for cryo-ET imaging, but standard cryo-FIB fabrication is blind to the precise location of the structure or proteins of interest. Fluorescence-guided focused ion beam (FIB) milling at target locations requires multiple sample transfers prone to contamination, and relocation and registration accuracy is often insufficient for 3D targeting. Here, we present in situ fluorescence microscopy-guided FIB fabrication of a frozen-hydrated lamella to address this problem: we built a coincident three-beam cryogenic correlative microscope by retrofitting a compact cryogenic microcooler, custom positioning stage, and an inverted widefield fluorescence microscope (FM) on an existing FIB scanning electron microscope. We show FM controlled targeting at every milling step in the lamella fabrication process, validated with transmission electron microscope tomogram reconstructions of the target regions. The ability to check the lamella during and after the milling process results in a higher success rate in the fabrication process and will increase the throughput of fabrication for lamellae suitable for high-resolution imaging.

    1. Cell Biology
    2. Structural Biology and Molecular Biophysics
    Johannes Elferich, Giulia Schiroli ... Nikolaus Grigorieff
    Tools and Resources Updated

    A major goal of biological imaging is localization of biomolecules inside a cell. Fluorescence microscopy can localize biomolecules inside whole cells and tissues, but its ability to count biomolecules and accuracy of the spatial coordinates is limited by the wavelength of visible light. Cryo-electron microscopy (cryo-EM) provides highly accurate position and orientation information of biomolecules but is often confined to small fields of view inside a cell, limiting biological context. In this study, we use a new data-acquisition scheme called Defocus-Corrected Large-Area cryo-EM (DeCo-LACE) to collect high-resolution images of entire sections (100- to 250-nm-thick lamellae) of neutrophil-like mouse cells, representing 1–2% of the total cellular volume. We use 2D template matching (2DTM) to determine localization and orientation of the large ribosomal subunit in these sections. These data provide maps of ribosomes across entire sections of mammalian cells. This high-throughput cryo-EM data collection approach together with 2DTM will advance visual proteomics and provide biological insight that cannot be obtained by other methods.