1. Structural Biology and Molecular Biophysics
  2. Neuroscience
Download icon

A selectivity filter at the intracellular end of the acid-sensing ion channel pore

  1. Timothy Lynagh
  2. Emelie Flood
  3. Céline Boiteux
  4. Matthias Wulf
  5. Vitaly V Komnatnyy
  6. Janne M Colding
  7. Toby W Allen
  8. Stephan A Pless  Is a corresponding author
  1. University of Copenhagen, Denmark
  2. RMIT University, Australia
Research Article
  • Cited 24
  • Views 3,116
  • Annotations
Cite this article as: eLife 2017;6:e24630 doi: 10.7554/eLife.24630

Abstract

Increased extracellular proton concentrations during neurotransmission are converted to excitatory sodium influx by acid-sensing ion channels (ASICs). 10-fold sodium/potassium selectivity in ASICs has long been attributed to a central constriction in the channel pore, but experimental verification is lacking due to the sensitivity of this structure to conventional manipulations. Here, we explored the basis for ion selectivity by incorporating unnatural amino acids into the channel, engineering channel stoichiometry and performing free energy simulations. We observed no preference for sodium at the 'GAS belt' in the central constriction. Instead, we identified a band of glutamate and aspartate side chains at the lower end of the pore that enables preferential sodium conduction.

Article and author information

Author details

  1. Timothy Lynagh

    Center for Biopharmaceuticals, University of Copenhagen, Copenhagen, Denmark
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4888-4098
  2. Emelie Flood

    School of Science, RMIT University, Melbourne, Australia
    Competing interests
    The authors declare that no competing interests exist.
  3. Céline Boiteux

    School of Science, RMIT University, Melbourne, Australia
    Competing interests
    The authors declare that no competing interests exist.
  4. Matthias Wulf

    Center for Biopharmaceuticals, University of Copenhagen, Copenhagen, Denmark
    Competing interests
    The authors declare that no competing interests exist.
  5. Vitaly V Komnatnyy

    Center for Biopharmaceuticals, University of Copenhagen, Copenhagen, Denmark
    Competing interests
    The authors declare that no competing interests exist.
  6. Janne M Colding

    Center for Biopharmaceuticals, University of Copenhagen, Copenhagen, Denmark
    Competing interests
    The authors declare that no competing interests exist.
  7. Toby W Allen

    School of Science, RMIT University, Melbourne, Australia
    Competing interests
    The authors declare that no competing interests exist.
  8. Stephan A Pless

    Center for Biopharmaceuticals, University of Copenhagen, Copenhagen, Denmark
    For correspondence
    stephan.pless@sund.ku.dk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6654-114X

Funding

Lundbeckfonden (Lundbeck Foundation Fellowship R139-2012-12390)

  • Stephan A Pless

Carlsbergfondet (Equipment Grant 2013_01_0439)

  • Stephan A Pless

Det Frie Forskningsråd (Postdoctoral Fellowship 4092-00348B)

  • Timothy Lynagh

Australian Research Council (Project Grant DP170101732)

  • Toby W Allen

Novo Nordisk Foundation (Project Grant)

  • Stephan A Pless

National Health and Medical Research Council (Project Grant APP1104259)

  • Toby W Allen

National Institutes of Health (Project Grant U01-11567710)

  • Toby W Allen

Lundbeckfonden (Postdoctoral Fellowship R171-2014-558)

  • Timothy Lynagh

National Cancer Institute (dd7)

  • Toby W Allen

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

Ethics

Animal experimentation: This study was performed in accordance with the recommendations by the by the Danish Veterinary and Food Administration and approved under license 2014−15−0201−00031. Surgery was performed on Xenopus laevis frogs anaesthetized in 0.3% tricaine.

Reviewing Editor

  1. Baron Chanda, University of Wisconsin-Madison, United States

Publication history

  1. Received: December 23, 2016
  2. Accepted: May 11, 2017
  3. Accepted Manuscript published: May 12, 2017 (version 1)
  4. Version of Record published: May 30, 2017 (version 2)

Copyright

© 2017, Lynagh 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

  • 3,116
    Page views
  • 552
    Downloads
  • 24
    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)

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

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

  1. Further reading

Further reading

    1. Structural Biology and Molecular Biophysics
    Xiaochen Chen et al.
    Research Article Updated

    Human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor that maintains Ca2+ homeostasis in serum. Here, we present the cryo-electron microscopy structures of the CaSR in the inactive and agonist+PAM bound states. Complemented with previously reported structures of CaSR, we show that in addition to the full inactive and active states, there are multiple intermediate states during the activation of CaSR. We used a negative allosteric nanobody to stabilize the CaSR in the fully inactive state and found a new binding site for Ca2+ ion that acts as a composite agonist with L-amino acid to stabilize the closure of active Venus flytraps. Our data show that agonist binding leads to compaction of the dimer, proximity of the cysteine-rich domains, large-scale transitions of seven-transmembrane domains, and inter- and intrasubunit conformational changes of seven-transmembrane domains to accommodate downstream transducers. Our results reveal the structural basis for activation mechanisms of CaSR and clarify the mode of action of Ca2+ ions and L-amino acid leading to the activation of the receptor.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Quentin M Smith et al.
    Research Article

    Regulated thin filaments (RTFs) tightly control striated muscle contraction through calcium binding to troponin, which enables tropomyosin to expose myosin-binding sites on actin. Myosin binding holds tropomyosin in an open position, exposing more myosin-binding sites on actin, leading to cooperative activation. At lower calcium levels, troponin and tropomyosin turn off the thin filament; however, this is antagonised by the high local concentration of myosin, questioning how the thin filament relaxes. To provide molecular details of deactivation, we used single-molecule imaging of green fluorescent protein (GFP)-tagged myosin-S1 (S1-GFP) to follow the activation of RTF tightropes. In sub-maximal activation conditions, RTFs are not fully active, enabling direct observation of deactivation in real time. We observed that myosin binding occurs in a stochastic step-wise fashion; however, an unexpectedly large probability of multiple contemporaneous detachments is observed. This suggests that deactivation of the thin filament is a coordinated active process.