1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics
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Atomic mutagenesis in ion channels with engineered stoichiometry

  1. John D Lueck
  2. Adam L Mackey
  3. Daniel T Infield
  4. Jason D Galpin
  5. Jing Li
  6. Benoît Roux
  7. Christopher A Ahern  Is a corresponding author
  1. University of Iowa Carver College of Medicine, United States
  2. University of Chicago, United States
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Cite this article as: eLife 2016;5:e18976 doi: 10.7554/eLife.18976

Abstract

C-type inactivation of potassium channels fine-tunes the electrical signaling in excitable cells through an internal timing mechanism that is mediated by a hydrogen bond network in the channels' selectively filter. Previously, (Pless, 2013) we used nonsense suppression to highlight the role of the conserved Trp434-Asp447 indole hydrogen bond in Shaker potassium channels with a non-hydrogen bonding homologue of tryptophan, Ind. Here, molecular dynamics simulations indicate that the Trp434Ind hydrogen bonding partner, Asp447, unexpectedly 'flips out' towards the extracellular environment, allowing water to penetrate the space behind the selectivity filter while simultaneously reducing the local negative electrostatic charge. Additionally, a protein engineering approach is presented whereby split intein sequences are flanked by endoplasmic reticulum retention/retrieval motifs (ERret) are incorporated into the N- or C- termini of Shaker monomers or within sodium channels two-domain fragments. This system enabled stoichiometric control of Shaker monomers and the encoding of multiple amino acids within a channel tetramer.

Article and author information

Author details

  1. John D Lueck

    Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Adam L Mackey

    Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Daniel T Infield

    Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Jason D Galpin

    Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Jing Li

    Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Benoît Roux

    Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5254-2712
  7. Christopher A Ahern

    Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United States
    For correspondence
    christopher-ahern@uiowa.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7975-2744

Funding

National Institute of General Medical Sciences (106569)

  • Jason D Galpin
  • Christopher A Ahern

National Institute of General Medical Sciences (87519)

  • Jason D Galpin

National Institute of General Medical Sciences (62342)

  • Jing Li
  • Benoît Roux

American Heart Association (A22180002)

  • Christopher A Ahern

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

Reviewing Editor

  1. Richard Aldrich, The University of Texas at Austin, United States

Publication history

  1. Received: June 23, 2016
  2. Accepted: October 5, 2016
  3. Accepted Manuscript published: October 6, 2016 (version 1)
  4. Version of Record published: November 2, 2016 (version 2)

Copyright

© 2016, Lueck 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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