Molecular principles of assembly, activation, and inhibition in epithelial sodium channel

  1. Sigrid Noreng
  2. Richard Posert
  3. Arpita Bharadwaj
  4. Alexandra Houser
  5. Isabelle Baconguis  Is a corresponding author
  1. Oregon Health and Science University, United States

Abstract

The molecular bases of heteromeric assembly and link between Na+ self-inhibition and protease-sensitivity in epithelial sodium channels (ENaCs) are not fully understood. Previously, we demonstrated that ENaC subunits – α, β, and γ – assemble in a counterclockwise configuration when viewed from outside the cell with the protease-sensitive GRIP domains in the periphery (Noreng et al., 2018). Here we describe the structure of ENaC resolved by cryo-electron microscopy at 3 Å. We find that a combination of precise domain arrangement and complementary hydrogen bonding network defines the subunit arrangement. Furthermore, we determined that the α subunit has a primary functional module consisting of the finger and GRIP domains. The module is bifurcated by the α2 helix dividing two distinct regulatory sites: Na+ and the inhibitory peptide. Removal of the inhibitory peptide perturbs the Na+ site via the α2 helix highlighting the critical role of the α2 helix in regulating ENaC function.

Data availability

All cryo-EM maps have been deposited in the Electron Microscopy Data Bank under the accession code EMD-21896 for ENaC. Model coordinates have been deposited in the Protein Data Bank under the accession code 6WTH.

The following data sets were generated

Article and author information

Author details

  1. Sigrid Noreng

    Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5767-1399
  2. Richard Posert

    Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9010-2104
  3. Arpita Bharadwaj

    Vollum Institute, Oregon Health and Science University, Portland, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3867-7610
  4. Alexandra Houser

    Neuroscience Graduate Program, Oregon Health and Science University, Portland, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Isabelle Baconguis

    Vollum Institute, Oregon Health and Science University, Portland, United States
    For correspondence
    bacongui@ohsu.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5440-2289

Funding

National Institutes of Health (DP5OD017871)

  • Isabelle Baconguis

American Heart Association (19TPA34760754)

  • Isabelle Baconguis

American Heart Association (18PRE33990205)

  • Sigrid Noreng

National Science Foundation (DGE-1937961)

  • Alexandra Houser

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

Reviewing Editor

  1. Sriram Subramaniam, University of British Columbia, Canada

Publication history

  1. Received: May 26, 2020
  2. Accepted: July 29, 2020
  3. Accepted Manuscript published: July 30, 2020 (version 1)
  4. Version of Record published: August 7, 2020 (version 2)

Copyright

© 2020, Noreng 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

  • 1,816
    Page views
  • 287
    Downloads
  • 16
    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. Sigrid Noreng
  2. Richard Posert
  3. Arpita Bharadwaj
  4. Alexandra Houser
  5. Isabelle Baconguis
(2020)
Molecular principles of assembly, activation, and inhibition in epithelial sodium channel
eLife 9:e59038.
https://doi.org/10.7554/eLife.59038

Further reading

    1. Immunology and Inflammation
    2. Structural Biology and Molecular Biophysics
    Toshiyuki Oda et al.
    Research Article

    Langerhans cells are specialized antigen-presenting cells localized within the epidermis and mucosal epithelium. Upon contact with Langerhans cells, pathogens are captured by the C-type lectin langerin and internalized into a structurally unique vesicle known as a Birbeck granule. Although the immunological role of Langerhans cells and Birbeck granules have been extensively studied, the mechanism by which the characteristic zippered membrane structure of Birbeck granules is formed remains elusive. In this study, we observed isolated Birbeck granules using cryo-electron tomography and reconstructed the 3D structure of the repeating unit of the honeycomb lattice of langerin at 6.4 Å resolution. We found that the interaction between the two langerin trimers was mediated by docking the flexible loop at residues 258-263 into the secondary carbohydrate-binding cleft. Mutations within the loop inhibited Birbeck granule formation and the internalization of HIV pseudovirus. These findings suggest a molecular mechanism for membrane zippering during Birbeck granule biogenesis and provide insight into the role of langerin in the defense against viral infection.

    1. Structural Biology and Molecular Biophysics
    Huan-Huan Hu et al.
    Research Article Updated

    Phosphoribosyl pyrophosphate (PRPP) is a key intermediate in the biosynthesis of purine and pyrimidine nucleotides, histidine, tryptophan, and cofactors NAD and NADP. Abnormal regulation of PRPP synthase (PRPS) is associated with human disorders, including Arts syndrome, retinal dystrophy, and gouty arthritis. Recent studies have demonstrated that PRPS can form filamentous cytoophidia in eukaryotes. Here, we show that PRPS forms cytoophidia in prokaryotes both in vitro and in vivo. Moreover, we solve two distinct filament structures of E. coli PRPS at near-atomic resolution using Cryo-EM. The formation of the two types of filaments is controlled by the binding of different ligands. One filament type is resistant to allosteric inhibition. The structural comparison reveals conformational changes of a regulatory flexible loop, which may regulate the binding of the allosteric inhibitor and the substrate ATP. A noncanonical allosteric AMP/ADP binding site is identified to stabilize the conformation of the regulatory flexible loop. Our findings not only explore a new mechanism of PRPS regulation with structural basis, but also propose an additional layer of cell metabolism through PRPS filamentation.