Activation by cleavage of the epithelial Na+ channel α and γ subunits independently coevolved with the vertebrate terrestrial migration

  1. Xue-Ping Wang
  2. Deidra M Balchak
  3. Clayton Gentilcore
  4. Nathan L Clark
  5. Ossama B Kashlan  Is a corresponding author
  1. University of Pittsburgh, United States
  2. University of Utah, United States

Abstract

Vertebrates evolved mechanisms for sodium conservation and gas exchange in conjunction with migration from aquatic to terrestrial habitats. Epithelial Na+ channel (ENaC) function is critical to systems responsible for extracellular fluid homeostasis and gas exchange. ENaC is activated by cleavage at multiple specific extracellular polybasic sites, releasing inhibitory tracts from the channel’s α and γ subunits. We found that proximal and distal polybasic tracts in ENaC subunits coevolved, consistent with the dual cleavage requirement for activation observed in mammals. Polybasic tract pairs evolved with the terrestrial migration and the appearance of lungs, coincident with the ENaC activator aldosterone, and appeared independently in the a and g subunits. In summary, sites within ENaC for protease activation developed in vertebrates when renal Na+ conservation and alveolar gas exchange was required for terrestrial survival.

Data availability

Source data for figures 1 and 2 are provided in Supplementary Data. Source data for trait evolution analysis, Figures 3 and Figure 4 are provided as image and Microsoft Excel files. Images and source data of electrophysiology traces in Figure 3 are provided at the Zenodo data repository (10.5281/zenodo.5790375).

The following data sets were generated
    1. Kashlan
    2. Ossama B
    (2021) Figure 3-source data 2
    Zenodo, doi:10.5281/zenodo.5790375.

Article and author information

Author details

  1. Xue-Ping Wang

    Department of Medicine, University of Pittsburgh, Pittsburgh, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Deidra M Balchak

    Department of Medicine, University of Pittsburgh, Pittsburgh, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Clayton Gentilcore

    Department of Medicine, University of Pittsburgh, Pittsburgh, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Nathan L Clark

    Department of Human Genetics, University of Utah, Salt Lake City, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Ossama B Kashlan

    Department of Medicine, University of Pittsburgh, Pittsburgh, United States
    For correspondence
    obk2@pitt.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0537-6720

Funding

National Institute of Diabetes and Digestive and Kidney Diseases (Grant R01 DK125439)

  • Ossama B Kashlan

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

Reviewing Editor

  1. Kenton J Swartz, National Institute of Neurological Disorders and Stroke, National Institutes of Health, United States

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols (#20037084 and #21018704) of the University of Pittsburgh. All surgery was performed following tricaine methane sulfonate anesthesia and sacrifice, and every effort was made to minimize suffering.

Version history

  1. Received: November 23, 2021
  2. Accepted: December 28, 2021
  3. Accepted Manuscript published: January 5, 2022 (version 1)
  4. Version of Record published: January 26, 2022 (version 2)

Copyright

© 2022, Wang 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

  • 862
    Page views
  • 139
    Downloads
  • 4
    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. Xue-Ping Wang
  2. Deidra M Balchak
  3. Clayton Gentilcore
  4. Nathan L Clark
  5. Ossama B Kashlan
(2022)
Activation by cleavage of the epithelial Na+ channel α and γ subunits independently coevolved with the vertebrate terrestrial migration
eLife 11:e75796.
https://doi.org/10.7554/eLife.75796

Share this article

https://doi.org/10.7554/eLife.75796

Further reading

    1. Cell Biology
    2. Evolutionary Biology
    Jonathan E Phillips, Duojia Pan
    Research Advance

    The genomes of close unicellular relatives of animals encode orthologs of many genes that regulate animal development. However, little is known about the function of such genes in unicellular organisms or the evolutionary process by which these genes came to function in multicellular development. The Hippo pathway, which regulates cell proliferation and tissue size in animals, is present in some of the closest unicellular relatives of animals, including the amoeboid organism Capsaspora owczarzaki. We previously showed that the Capsaspora ortholog of the Hippo pathway nuclear effector Yorkie/YAP/TAZ (coYki) regulates actin dynamics and the three-dimensional morphology of Capsaspora cell aggregates, but is dispensable for cell proliferation control (Phillips et al., 2022). However, the function of upstream Hippo pathway components, and whether and how they regulate coYki in Capsaspora, remained unknown. Here, we analyze the function of the upstream Hippo pathway kinases coHpo and coWts in Capsaspora by generating mutant lines for each gene. Loss of either kinase results in increased nuclear localization of coYki, indicating an ancient, premetazoan origin of this Hippo pathway regulatory mechanism. Strikingly, we find that loss of either kinase causes a contractile cell behavior and increased density of cell packing within Capsaspora aggregates. We further show that this increased cell density is not due to differences in proliferation, but rather actomyosin-dependent changes in the multicellular architecture of aggregates. Given its well-established role in cell density-regulated proliferation in animals, the increased density of cell packing in coHpo and coWts mutants suggests a shared and possibly ancient and conserved function of the Hippo pathway in cell density control. Together, these results implicate cytoskeletal regulation but not proliferation as an ancestral function of the Hippo pathway kinase cascade and uncover a novel role for Hippo signaling in regulating cell density in a proliferation-independent manner.

    1. Evolutionary Biology
    2. Immunology and Inflammation
    Zachary Paul Billman, Stephen Bela Kovacs ... Edward A Miao
    Research Article

    Gasdermins oligomerize to form pores in the cell membrane, causing regulated lytic cell death called pyroptosis. Mammals encode five gasdermins that can trigger pyroptosis: GSDMA, B, C, D, and E. Caspase and granzyme proteases cleave the linker regions of and activate GSDMB, C, D, and E, but no endogenous activation pathways are yet known for GSDMA. Here, we perform a comprehensive evolutionary analysis of the gasdermin family. A gene duplication of GSDMA in the common ancestor of caecilian amphibians, reptiles, and birds gave rise to GSDMA–D in mammals. Uniquely in our tree, amphibian, reptile, and bird GSDMA group in a separate clade than mammal GSDMA. Remarkably, GSDMA in numerous bird species contain caspase-1 cleavage sites like YVAD or FASD in the linker. We show that GSDMA from birds, amphibians, and reptiles are all cleaved by caspase-1. Thus, GSDMA was originally cleaved by the host-encoded protease caspase-1. In mammals the caspase-1 cleavage site in GSDMA is disrupted; instead, a new protein, GSDMD, is the target of caspase-1. Mammal caspase-1 uses exosite interactions with the GSDMD C-terminal domain to confer the specificity of this interaction, whereas we show that bird caspase-1 uses a stereotypical tetrapeptide sequence to confer specificity for bird GSDMA. Our results reveal an evolutionarily stable association between caspase-1 and the gasdermin family, albeit a shifting one. Caspase-1 repeatedly changes its target gasdermin over evolutionary time at speciation junctures, initially cleaving GSDME in fish, then GSDMA in amphibians/reptiles/birds, and finally GSDMD in mammals.