Ancestral acetylcholine receptor β-subunit forms homopentamers that prime before opening spontaneously

Abstract

Human adult muscle-type acetylcholine receptors are heteropentameric ion channels formed from two α-subunits, and one each of the β-, d-, and e-subunits. To form functional channels, the subunits must assemble with one another in a precise stoichiometry and arrangement. Despite being different, the four subunits share a common ancestor that is presumed to have formed homopentamers. The extent to which the properties of the modern-day receptor result from its subunit complexity is unknown. Here we discover that a reconstructed ancestral muscle-type β-subunit can form homopentameric ion channels. These homopentamers open spontaneously and display single-channel hallmarks of muscle-type acetylcholine receptor activity. Our findings attest to the homopentameric origin of the muscle-type acetylcholine receptor, and demonstrate that signature features of its function are both independent of agonist and do not necessitate the complex heteropentameric architecture of the modern-day protein.

Data availability

All data generated and analysed during this study are included in the manuscript and as source data

Article and author information

Author details

  1. Christian JG Tessier

    Center for Chemical and Synthetic Biology, University of Ottawa, Ottawa, Canada
    Competing interests
    The authors declare that no competing interests exist.
  2. R Michel Sturgeon

    Center for Chemical and Synthetic Biology, University of Ottawa, Ottawa, Canada
    Competing interests
    The authors declare that no competing interests exist.
  3. Johnathon R Emlaw

    Center for Chemical and Synthetic Biology, University of Ottawa, Ottawa, Canada
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7255-182X
  4. Gregory D McCluskey

    Center for Chemical and Synthetic Biology, University of Ottawa, Ottawa, Canada
    Competing interests
    The authors declare that no competing interests exist.
  5. F Javier Pérez-Areales

    Center for Chemical and Synthetic Biology, University of Ottawa, Ottawa, Canada
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9525-9346
  6. Corrie JB daCosta

    Center for Chemical and Synthetic Biology, University of Ottawa, Ottawa, Canada
    For correspondence
    cdacosta@uottawa.ca
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9546-5331

Funding

Natural Sciences and Engineering Research Council of Canada (Discovery Grant,RGPIN-2016-04801)

  • Corrie JB daCosta

Canadian Institutes of Health Research (Project Grant,377068)

  • Corrie JB daCosta

Ontario Council on Graduate Studies, Council of Ontario Universities (Graduate Scholarship)

  • Christian JG Tessier

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

Reviewing Editor

  1. Stephan A Pless, University of Copenhagen, Denmark

Publication history

  1. Received: December 18, 2021
  2. Preprint posted: January 13, 2022 (view preprint)
  3. Accepted: June 21, 2022
  4. Accepted Manuscript published: July 4, 2022 (version 1)

Copyright

© 2022, Tessier 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

  • 280
    Page views
  • 110
    Downloads
  • 0
    Citations

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

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. Christian JG Tessier
  2. R Michel Sturgeon
  3. Johnathon R Emlaw
  4. Gregory D McCluskey
  5. F Javier Pérez-Areales
  6. Corrie JB daCosta
(2022)
Ancestral acetylcholine receptor β-subunit forms homopentamers that prime before opening spontaneously
eLife 11:e76504.
https://doi.org/10.7554/eLife.76504

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Rajesh Sharma et al.
    Research Article

    Cyclic GMP-dependent protein kinases (PKGs) are key mediators of the nitric oxide/cGMP signaling pathway that regulates biological functions as diverse as smooth muscle contraction, cardiac function, and axon guidance. Understanding how cGMP differentially triggers mammalian PKG isoforms could lead to new therapeutics that inhibit or activate PKGs, complementing drugs that target nitric oxide synthases and cyclic nucleotide phosphodiesterases in this signaling axis. Alternate splicing of PRKG1 transcripts confers distinct leucine zippers, linkers, and auto-inhibitory pseudo-substrate sequences to PKG Iα and Iβ that result in isoform-specific activation properties, but the mechanism of enzyme auto-inhibition and its alleviation by cGMP is not well understood. Here we present a crystal structure of PKG Iβ in which the auto-inhibitory sequence and the cyclic nucleotide binding domains are bound to the catalytic domain, providing a snapshot of the auto-inhibited state. Specific contacts between the PKG Iβ auto-inhibitory sequence and the enzyme active site help explain isoform-specific activation constants and the effects of phosphorylation in the linker. We also present a crystal structure of a PKG I cyclic nucleotide binding domain with an activating mutation linked to Thoracic Aortic Aneurysms and Dissections. Similarity of this structure to wild type cGMP-bound domains and differences with the auto-inhibited enzyme provide a mechanistic basis for constitutive activation. We show that PKG Iβ auto-inhibition is mediated by contacts within each monomer of the native full-length dimeric protein, and using the available structural and biochemical data we develop a model for the regulation and cooperative activation of PKGs.

    1. Physics of Living Systems
    2. Structural Biology and Molecular Biophysics
    Enrico F Semeraro et al.
    Research Article Updated

    We report the real-time response of Escherichia coli to lactoferricin-derived antimicrobial peptides (AMPs) on length scales bridging microscopic cell sizes to nanoscopic lipid packing using millisecond time-resolved synchrotron small-angle X-ray scattering. Coupling a multiscale scattering data analysis to biophysical assays for peptide partitioning revealed that the AMPs rapidly permeabilize the cytosolic membrane within less than 3 s—much faster than previously considered. Final intracellular AMP concentrations of ∼80–100 mM suggest an efficient obstruction of physiologically important processes as the primary cause of bacterial killing. On the other hand, damage of the cell envelope and leakage occurred also at sublethal peptide concentrations, thus emerging as a collateral effect of AMP activity that does not kill the bacteria. This implies that the impairment of the membrane barrier is a necessary but not sufficient condition for microbial killing by lactoferricins. The most efficient AMP studied exceeds others in both speed of permeabilizing membranes and lowest intracellular peptide concentration needed to inhibit bacterial growth.