Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

  1. Stephanie A Heusser
  2. Christian B Borg
  3. Janne M Colding
  4. Stephan A Pless  Is a corresponding author
  1. University of Copenhagen, Denmark

Abstract

Acid-sensing ion channels (ASICs) are trimeric proton-gated cation channels involved in fast synaptic transmission. Pharmacological inhibition of ASIC1a reduces neurotoxicity and stroke infarct volumes, with the cysteine knot toxin Psalmotoxin-1 (PcTx1) being one of the most potent and selective inhibitors. PcTx1 binds at the subunit interface in the extracellular domain (ECD), but the mechanism and conformational consequences of the interaction, as well as the number of toxin molecules required for inhibition remain unknown. Here we use voltage-clamp fluorometry and subunit concatenation to decipher the mechanism and stoichiometry of PcTx1 inhibition of ASIC1a. Besides the known inhibitory binding mode, we propose PcTx1 to have at least two additional binding modes that are decoupled from the pore. One of these modes induces a long-lived ECD conformation that reduces the activity of an endogenous neuropeptide. This long-lived conformational state is proton-dependent and can be destabilized by a mutation that decreases PcTx1 sensitivity. Lastly, the use of concatemeric channel constructs reveal that disruption of a single PcTx1 binding site is sufficient to destabilize the toxin-induced conformation, while functional inhibition is not impaired until two or more binding sites are mutated. Together, our work provides insight into the mechanism of PcTx1 inhibition of ASICs and uncovers a prolonged conformational change with possible pharmacological implications.

Data availability

Figure 1 - Source Data 1, Figure 1 - Source Data 2, Figure 1 - figure supplement 1 - Source Data 1, Figure 1 - figure supplement 1 - Source Data 2, Figure 2 - Source Data 1, Figure 2 - Source Data 2, Figure 2 - Source Data 3, Figure 3 - Source Data 1, Figure 3 - Source Data 2, Figure 3 - Source Data 3, Figure 4 - Source Data 1, Figure 4 - Source Data 2, Figure 4 - Source Data 3, Figure 5 - Source Data 1 and Figure 5 - figure supplement 1 - Source Data 1 contain the numerical data used to generate the figures.

Article and author information

Author details

  1. Stephanie A Heusser

    Department of Drug Design and Pharmacology, University of Copenhagen, Copehagen, Denmark
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3224-4547
  2. Christian B Borg

    Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark
    Competing interests
    No competing interests declared.
  3. Janne M Colding

    Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark
    Competing interests
    No competing interests declared.
  4. Stephan A Pless

    Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark
    For correspondence
    stephan.pless@sund.ku.dk
    Competing interests
    Stephan A Pless, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6654-114X

Funding

Lundbeckfonden (R303-2018-3030)

  • Stephanie A Heusser

European Union's Horizon 2020 research and innovation program under der Marie Sklodowska-Curie grant agreement (No 834274)

  • Stephanie A Heusser

Hartmann Fonden (N/A)

  • Stephanie A Heusser

Lundbeckfonden (R313-2019-571)

  • Stephan A Pless

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

Reviewing Editor

  1. Jon T Sack, University of California Davis School of Medicine, United States

Publication history

  1. Preprint posted: June 22, 2021 (view preprint)
  2. Received: August 26, 2021
  3. Accepted: February 11, 2022
  4. Accepted Manuscript published: February 14, 2022 (version 1)
  5. Version of Record published: February 24, 2022 (version 2)

Copyright

© 2022, Heusser 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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  1. Stephanie A Heusser
  2. Christian B Borg
  3. Janne M Colding
  4. Stephan A Pless
(2022)
Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition
eLife 11:e73384.
https://doi.org/10.7554/eLife.73384

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