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.

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

  • 3,671
    views
  • 607
    downloads
  • 55
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

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. 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
(2017)
A selectivity filter at the intracellular end of the acid-sensing ion channel pore
eLife 6:e24630.
https://doi.org/10.7554/eLife.24630

Share this article

https://doi.org/10.7554/eLife.24630

Further reading

    1. Immunology and Inflammation
    2. Structural Biology and Molecular Biophysics
    Douwe Schulte, Marta Šiborová ... Joost Snijder
    Research Article

    Antibodies are a major component of adaptive immunity against invading pathogens. Here, we explore possibilities for an analytical approach to characterize the antigen-specific antibody repertoire directly from the secreted proteins in convalescent serum. This approach aims to perform simultaneous antibody sequencing and epitope mapping using a combination of single particle cryo-electron microscopy (cryoEM) and bottom-up proteomics techniques based on mass spectrometry (LC-MS/MS). We evaluate the performance of the deep-learning tool ModelAngelo in determining de novo antibody sequences directly from reconstructed 3D volumes of antibody-antigen complexes. We demonstrate that while map quality is a critical bottleneck, it is possible to sequence antibody variable domains from cryoEM reconstructions with accuracies of up to 80–90%. While the rate of errors exceeds the typical levels of somatic hypermutation, we show that the ModelAngelo-derived sequences can be used to assign the used V-genes. This provides a functional guide to assemble de novo peptides from LC-MS/MS data more accurately and improves the tolerance to a background of polyclonal antibody sequences. Following this proof-of-principle, we discuss the feasibility and future directions of this approach to characterize antigen-specific antibody repertoires.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Yamato Niitani, Kohei Matsuzaki ... Michio Tomishige
    Research Article

    The two identical motor domains (heads) of dimeric kinesin-1 move in a hand-over-hand process along a microtubule, coordinating their ATPase cycles such that each ATP hydrolysis is tightly coupled to a step and enabling the motor to take many steps without dissociating. The neck linker, a structural element that connects the two heads, has been shown to be essential for head–head coordination; however, which kinetic step(s) in the chemomechanical cycle is ‘gated’ by the neck linker remains unresolved. Here, we employed pre-steady-state kinetics and single-molecule assays to investigate how the neck-linker conformation affects kinesin’s motility cycle. We show that the backward-pointing configuration of the neck linker in the front kinesin head confers higher affinity for microtubule, but does not change ATP binding and dissociation rates. In contrast, the forward-pointing configuration of the neck linker in the rear kinesin head decreases the ATP dissociation rate but has little effect on microtubule dissociation. In combination, these conformation-specific effects of the neck linker favor ATP hydrolysis and dissociation of the rear head prior to microtubule detachment of the front head, thereby providing a kinetic explanation for the coordinated walking mechanism of dimeric kinesin.