Cryo-EM structure of the ATP-sensitive potassium channel illuminates mechanisms of assembly and gating

  1. Gregory M Martin
  2. Craig Yoshioka
  3. Emily A Rex
  4. Jonathan F Fay
  5. Qing Xie
  6. Matthew R Whorton
  7. James Z Chen  Is a corresponding author
  8. Show-Ling Shyng  Is a corresponding author
  1. Oregon Health and Science University, United States

Abstract

KATP channels are metabolic sensors that couple cell energetics to membrane excitability. In pancreatic β-cells, channels formed by SUR1 and Kir6.2 regulate insulin secretion and are the targets of antidiabetic sulfonylureas. Here, we used cryo-EM to elucidate structural basis of channel assembly and gating. The structure, determined in the presence of ATP and the sulfonylurea glibenclamide, at ~6Å resolution reveals a closed Kir6.2 tetrameric core with four peripheral SUR1s each anchored to a Kir6.2 by its N-terminal transmembrane domain (TMD0). Intricate interactions between TMD0, the loop following TMD0, and Kir6.2 near the proposed PIP2 binding site, and where ATP density is observed, suggest SUR1 may contribute to ATP and PIP2 binding to enhance Kir6.2 sensitivity to both. The SUR1-ABC core is found in an unusual inward-facing conformation whereby the two nucleotide binding domains are misaligned along a two-fold symmetry axis, revealing a possible mechanism by which glibenclamide inhibits channel activity.

Article and author information

Author details

  1. Gregory M Martin

    Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Craig Yoshioka

    Department of Biomedical Engineering, Oregon Health and Science University, Portland, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Emily A Rex

    Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Jonathan F Fay

    Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Qing Xie

    Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Matthew R Whorton

    Vollum Institute, Oregon Health and Science University, Portland, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. James Z Chen

    Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, United States
    For correspondence
    chezh@ohsu.edu
    Competing interests
    The authors declare that no competing interests exist.
  8. Show-Ling Shyng

    Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, United States
    For correspondence
    shyngs@ohsu.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8230-8820

Funding

National Institute of Diabetes and Digestive and Kidney Diseases (R01DK066485)

  • Show-Ling Shyng

National Institute of Diabetes and Digestive and Kidney Diseases (F31DK105800)

  • Gregory M Martin

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

Reviewing Editor

  1. Werner Kühlbrandt, Max Planck Institute of Biophysics, Germany

Publication history

  1. Received: December 12, 2016
  2. Accepted: January 11, 2017
  3. Accepted Manuscript published: January 16, 2017 (version 1)
  4. Version of Record published: March 9, 2017 (version 2)

Copyright

© 2017, Martin 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

  • 5,883
    Page views
  • 1,353
    Downloads
  • 112
    Citations

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

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. Gregory M Martin
  2. Craig Yoshioka
  3. Emily A Rex
  4. Jonathan F Fay
  5. Qing Xie
  6. Matthew R Whorton
  7. James Z Chen
  8. Show-Ling Shyng
(2017)
Cryo-EM structure of the ATP-sensitive potassium channel illuminates mechanisms of assembly and gating
eLife 6:e24149.
https://doi.org/10.7554/eLife.24149
  1. Further reading

Further reading

    1. Structural Biology and Molecular Biophysics
    Metehan Ilter, Ramazan Kaşmer ... Ozge Sensoy
    Research Article

    Undruggability of RAS proteins has necessitated alternative strategies for the development of effective inhibitors. In this respect, phosphorylation has recently come into prominence as this reversible post-translational modification attenuates sensitivity of RAS towards RAF. As such, in this study, we set out to unveil the impact of phosphorylation on dynamics of HRASWT and aim to invoke similar behavior in HRASG12D mutant by means of small therapeutic molecules. To this end, we performed molecular dynamics (MD) simulations using phosphorylated HRAS and showed that phosphorylation of Y32 distorted Switch I, hence the RAS/RAF interface. Consequently, we targeted Switch I in HRASG12D by means of approved therapeutic molecules and showed that the ligands enabled detachment of Switch I from the nucleotide-binding pocket. Moreover, we demonstrated that displacement of Switch I from the nucleotide-binding pocket was energetically more favorable in the presence of the ligand. Importantly, we verified computational findings in vitro where HRASG12D/RAF interaction was prevented by the ligand in HEK293T cells that expressed HRASG12D mutant protein. Therefore, these findings suggest that targeting Switch I, hence making Y32 accessible might open up new avenues in future drug discovery strategies that target mutant RAS proteins.

    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.