Polyunsaturated fatty acids inhibit a pentameric ligand-gated ion channel through one of two binding sites

  1. Noah M Dietzen
  2. Mark J Arcario
  3. Lawrence J Chen
  4. John T Petroff
  5. Trent K Moreland
  6. Kathiresan Krishnan
  7. Grace Brannigan
  8. Douglas F Covey
  9. Wayland WL Cheng  Is a corresponding author
  1. Washington University in St. Louis, United States
  2. Rutgers University, United States

Abstract

Polyunsaturated fatty acids (PUFAs) inhibit pentameric ligand-gated ion channels (pLGICs) but the mechanism of inhibition is not well understood. The PUFA, docosahexaenoic acid (DHA), inhibits agonist responses of the pLGIC, ELIC, more effectively than palmitic acid, similar to the effects observed in the GABAA receptor and nicotinic acetylcholine receptor. Using photo-affinity labeling and coarse-grained molecular dynamics simulations, we identified two fatty acid binding sites in the outer transmembrane domain (TMD) of ELIC. Fatty acid binding to the photolabeled sites is selective for DHA over palmitic acid, and specific for an agonist-bound state. Hexadecyl-methanethiosulfonate modification of one of the two fatty acid binding sites in the outer TMD recapitulates the inhibitory effect of PUFAs in ELIC. The results demonstrate that DHA selectively binds to multiple sites in the outer TMD of ELIC, but that state-dependent binding to a single intrasubunit site mediates DHA inhibition of ELIC.

Data availability

Figure 4- source data 1 contains the numerical data used to generate Figure 4A and 4B. Figure 4- source data 2 contains the numerical data used to generate Figure 4C and Figure 4- figure supplement 5. Figure 4- source data 3 contains the statistical analysis (linear mixed effects model) for Figure 4- figure supplement 5.

Article and author information

Author details

  1. Noah M Dietzen

    Department of Anesthesiology, Washington University in St. Louis, St Louis, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Mark J Arcario

    Department of Anesthesiology, Washington University in St. Louis, St Louis, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5017-1519
  3. Lawrence J Chen

    Department of Anesthesiology, Washington University in St. Louis, St Louis, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. John T Petroff

    Department of Anesthesiology, Washington University in St. Louis, St Louis, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1323-0273
  5. Trent K Moreland

    Department of Anesthesiology, Washington University in St. Louis, St Louis, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Kathiresan Krishnan

    Department of Developmental Biology, Washington University in St. Louis, St Louis, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Grace Brannigan

    Center for the Computational and Integrative Biology, Rutgers University, Camden, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Douglas F Covey

    Department of Developmental Biology, Washington University in St. Louis, St Louis, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Wayland WL Cheng

    Department of Anesthesiology, Washington University in St. Louis, St Louis, United States
    For correspondence
    wayland.cheng@wustl.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9529-9820

Funding

National Institutes of Health (R35GM137957)

  • Wayland WL Cheng

National Institutes of Health (F32GM139351)

  • John T Petroff

National Institutes of Health (R01HL067773)

  • Douglas F Covey

National Institutes of Health (R01GM108799)

  • Douglas F Covey

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

Reviewing Editor

  1. Marcel P Goldschen-Ohm, University of Texas at Austin, United States

Publication history

  1. Received: September 29, 2021
  2. Preprint posted: October 8, 2021 (view preprint)
  3. Accepted: December 31, 2021
  4. Accepted Manuscript published: January 4, 2022 (version 1)
  5. Accepted Manuscript updated: January 7, 2022 (version 2)
  6. Version of Record published: January 24, 2022 (version 3)

Copyright

© 2022, Dietzen 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

  • 1,035
    Page views
  • 211
    Downloads
  • 2
    Citations

Article citation count generated by polling the highest count across the following sources: PubMed Central, Crossref, 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. Noah M Dietzen
  2. Mark J Arcario
  3. Lawrence J Chen
  4. John T Petroff
  5. Trent K Moreland
  6. Kathiresan Krishnan
  7. Grace Brannigan
  8. Douglas F Covey
  9. Wayland WL Cheng
(2022)
Polyunsaturated fatty acids inhibit a pentameric ligand-gated ion channel through one of two binding sites
eLife 11:e74306.
https://doi.org/10.7554/eLife.74306

Further reading

    1. Biochemistry and Chemical Biology
    Meiling Wu, Anda Zhao ... Dongyun Shi
    Research Article

    Antioxidant intervention is considered to inhibit reactive oxygen species (ROS) and alleviates hyperglycemia. Paradoxically, moderate exercise can produce ROS to improve diabetes. The exact redox mechanism of these two different approaches remains largely unclear. Here, by comparing exercise and antioxidants intervention on type 2 diabetic rats, we found moderate exercise upregulated compensatory antioxidant capability and reached a higher level of redox balance in the liver. In contrast, antioxidant intervention achieved a low-level redox balance by inhibiting oxidative stress. Both of these two interventions could promote glucose catabolism and inhibit gluconeogenesis through activation of hepatic AMPK signaling, therefore ameliorating diabetes. During exercise, different levels of ROS generated by exercise have differential regulations on the activity and expression of hepatic AMPK. Moderate exercise-derived ROS promoted hepatic AMPK glutathionylation activation. However, excessive exercise increased oxidative damage and inhibited the activity and expression of AMPK. Overall, our results illustrate that both exercise and antioxidant intervention improve blood glucose in diabetes by promoting redox balance, despite different levels of redox balance. These results indicate that the AMPK signaling activation, combined with oxidative damage markers, could act as a sensitive biomarker, reflecting the threshold of redox balance defining effective treatment in diabetes. These findings provide theoretical evidence for the precise treatment of diabetes by antioxidants and exercise.

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Edmundo G Vides, Ayan Adhikari ... Suzanne R Pfeffer
    Research Advance

    Activating mutations in the Leucine Rich Repeat Kinase 2 (LRRK2) cause Parkinson's disease and previously we showed that activated LRRK2 phosphorylates a subset of Rab GTPases (Steger et al., 2017). Moreover, Golgi-associated Rab29 can recruit LRRK2 to the surface of the Golgi and activate it there for both auto- and Rab substrate phosphorylation. Here we define the precise Rab29 binding region of the LRRK2 Armadillo domain between residues 360-450 and show that this domain, termed 'Site #1', can also bind additional LRRK2 substrates, Rab8A and Rab10. Moreover, we identify a distinct, N-terminal, higher affinity interaction interface between LRRK2 phosphorylated Rab8 and Rab10 termed 'Site #2', that can retain LRRK2 on membranes in cells to catalyze multiple, subsequent phosphorylation events. Kinase inhibitor washout experiments demonstrate that rapid recovery of kinase activity in cells depends on the ability of LRRK2 to associate with phosphorylated Rab proteins, and phosphorylated Rab8A stimulates LRRK2 phosphorylation of Rab10 in vitro. Reconstitution of purified LRRK2 recruitment onto planar lipid bilayers decorated with Rab10 protein demonstrates cooperative association of only active LRRK2 with phospho-Rab10-containing membrane surfaces. These experiments reveal a feed-forward pathway that provides spatial control and membrane activation of LRRK2 kinase activity.