A pathogenic human Orai1 mutation unmasks STIM1-independent rapid inactivation of Orai1 channels

  1. Priscilla S-W Yeung
  2. Megumi Yamashita
  3. Murali Prakriya  Is a corresponding author
  1. Northwestern University, United States

Abstract

Ca2+ release-activated Ca2+ (CRAC) channels are activated by direct physical interactions between Orai1, the channel protein, and STIM1, the endoplasmic reticulum Ca2+ sensor. A hallmark of CRAC channels is fast Ca2+-dependent inactivation (CDI) which provides negative feedback to limit Ca2+ entry through CRAC channels. Although STIM1 is thought to be essential for CDI, its molecular mechanism remains largely unknown. Here, we examined a poorly understood gain-of-function (GOF) human Orai1 disease mutation, L138F, that causes tubular aggregate myopathy. Through pairwise mutational analysis, we determine that large amino acid substitutions at either L138 or the neighboring T92 locus located on the pore helix evoke highly Ca2+-selective currents in the absence of STIM1. We find that the GOF phenotype of the L138 pathogenic mutation arises due to steric clash between L138 and T92. Surprisingly, strongly activating L138 and T92 mutations showed CDI in the absence of STIM1, contradicting prevailing views that STIM1 is required for CDI. CDI of constitutively open T92W and L138F mutants showed enhanced intracellular Ca2+ sensitivity, which was normalized by re-adding STIM1 to the cells. Truncation of the Orai1 C-terminus reduced T92W CDI indicating a key role for the Orai1 C-terminus for CDI. Overall, these results identify the molecular basis of a disease phenotype with broad implications for activation and inactivation of Orai1 channels.

Data availability

Source Data files containing the numerical data used in Figures 1-8 and the associated Supplementary figures have been uploaded.

Article and author information

Author details

  1. Priscilla S-W Yeung

    Department of Pharmacology, Northwestern University, Chicago, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5400-8639
  2. Megumi Yamashita

    Department of Pharmacology, Northwestern University, Chicago, United States
    Competing interests
    No competing interests declared.
  3. Murali Prakriya

    Department of Pharmacology, Northwestern University, Chicago, United States
    For correspondence
    m-prakriya@northwestern.edu
    Competing interests
    Murali Prakriya, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0781-4480

Funding

National Institutes of Health (R01 NS057499)

  • Murali Prakriya

National Institutes of Health (R01 NS115508)

  • Murali Prakriya

National Institutes of Health (F31NS101830)

  • Priscilla S-W Yeung

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

Reviewing Editor

  1. Mohamed Trebak, University of Pittsburgh, United States

Version history

  1. Received: July 29, 2022
  2. Preprint posted: August 15, 2022 (view preprint)
  3. Accepted: February 10, 2023
  4. Accepted Manuscript published: February 20, 2023 (version 1)
  5. Version of Record published: March 7, 2023 (version 2)

Copyright

© 2023, Yeung 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

  • 793
    views
  • 158
    downloads
  • 3
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

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. Priscilla S-W Yeung
  2. Megumi Yamashita
  3. Murali Prakriya
(2023)
A pathogenic human Orai1 mutation unmasks STIM1-independent rapid inactivation of Orai1 channels
eLife 12:e82281.
https://doi.org/10.7554/eLife.82281

Share this article

https://doi.org/10.7554/eLife.82281

Further reading

    1. Biochemistry and Chemical Biology
    2. Neuroscience
    Maximilian Nagel, Marco Niestroj ... Marc Spehr
    Research Article

    In most mammals, conspecific chemosensory communication relies on semiochemical release within complex bodily secretions and subsequent stimulus detection by the vomeronasal organ (VNO). Urine, a rich source of ethologically relevant chemosignals, conveys detailed information about sex, social hierarchy, health, and reproductive state, which becomes accessible to a conspecific via vomeronasal sampling. So far, however, numerous aspects of social chemosignaling along the vomeronasal pathway remain unclear. Moreover, since virtually all research on vomeronasal physiology is based on secretions derived from inbred laboratory mice, it remains uncertain whether such stimuli provide a true representation of potentially more relevant cues found in the wild. Here, we combine a robust low-noise VNO activity assay with comparative molecular profiling of sex- and strain-specific mouse urine samples from two inbred laboratory strains as well as from wild mice. With comprehensive molecular portraits of these secretions, VNO activity analysis now enables us to (i) assess whether and, if so, how much sex/strain-selective ‘raw’ chemical information in urine is accessible via vomeronasal sampling; (ii) identify which chemicals exhibit sufficient discriminatory power to signal an animal’s sex, strain, or both; (iii) determine the extent to which wild mouse secretions are unique; and (iv) analyze whether vomeronasal response profiles differ between strains. We report both sex- and, in particular, strain-selective VNO representations of chemical information. Within the urinary ‘secretome’, both volatile compounds and proteins exhibit sufficient discriminative power to provide sex- and strain-specific molecular fingerprints. While total protein amount is substantially enriched in male urine, females secrete a larger variety at overall comparatively low concentrations. Surprisingly, the molecular spectrum of wild mouse urine does not dramatically exceed that of inbred strains. Finally, vomeronasal response profiles differ between C57BL/6 and BALB/c animals, with particularly disparate representations of female semiochemicals.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Claudia D Consalvo, Adedeji M Aderounmu ... Brenda L Bass
    Research Article

    Invertebrates use the endoribonuclease Dicer to cleave viral dsRNA during antiviral defense, while vertebrates use RIG-I-like Receptors (RLRs), which bind viral dsRNA to trigger an interferon response. While some invertebrate Dicers act alone during antiviral defense, Caenorhabditis elegans Dicer acts in a complex with a dsRNA binding protein called RDE-4, and an RLR ortholog called DRH-1. We used biochemical and structural techniques to provide mechanistic insight into how these proteins function together. We found RDE-4 is important for ATP-independent and ATP-dependent cleavage reactions, while helicase domains of both DCR-1 and DRH-1 contribute to ATP-dependent cleavage. DRH-1 plays the dominant role in ATP hydrolysis, and like mammalian RLRs, has an N-terminal domain that functions in autoinhibition. A cryo-EM structure indicates DRH-1 interacts with DCR-1’s helicase domain, suggesting this interaction relieves autoinhibition. Our study unravels the mechanistic basis of the collaboration between two helicases from typically distinct innate immune defense pathways.