1. Structural Biology and Molecular Biophysics

The mechanism of MICU-dependent gating of the mitochondrial Ca2+ uniporter

  1. Vivek Garg  Is a corresponding author
  2. Junji Suzuki
  3. Ishan Paranjpe
  4. Tiffany Unsulangi
  5. Liron Boyman
  6. Lorin S Milescu
  7. W Jonathan Lederer
  8. Yuriy Kirichok  Is a corresponding author
  1. University of Maryland, United States
  2. University of California San Francisco, United States
  3. University of Maryland Baltimore, United States
  4. University of Maryland School of Medicine, United States
https://doi.org/10.7554/eLife.69312
Share this article
Cite this article
  1. Vivek Garg
  2. Junji Suzuki
  3. Ishan Paranjpe
  4. Tiffany Unsulangi
  5. Liron Boyman
  6. Lorin S Milescu
  7. W Jonathan Lederer
  8. Yuriy Kirichok
(2021)
The mechanism of MICU-dependent gating of the mitochondrial Ca2+ uniporter
eLife 10:e69312.
https://doi.org/10.7554/eLife.69312
  2. Download BibTeX
  3. Download .RIS

Abstract

Ca2+ entry into mitochondria is through the mitochondrial calcium uniporter complex (MCUcx), a Ca2+-selective channel composed of five subunit types. Two MCUcx subunits (MCU and EMRE) span the inner mitochondrial membrane, while three Ca2+-regulatory subunits (MICU1, MICU2 and MICU3) reside in the intermembrane space. Here we provide rigorous analysis of Ca2+ and Na+ fluxes via MCUcx in intact isolated mitochondria to understand the function of MICU subunits. We also perform direct patch clamp recordings of macroscopic and single MCUcx currents to gain further mechanistic insight. This comprehensive analysis shows that the MCUcx pore, composed of the EMRE and MCU subunits, is not occluded nor plugged by MICUs during the absence or presence of extramitochondrial Ca2+ as has been widely reported. Instead, MICUs potentiate activity of MCUcx as extramitochondrial Ca2+ is elevated. MICUs achieve this by modifying the gating properties of MCUcx allowing it to spend more time in the open state.

Data availability

Due to the size of the dataset, raw electrophysiology traces are available on request to the corresponding author. All information has been extracted from the raw electrophysiological traces and is available to download as source data files. All the codes or software used in analyzing the data and their sources are listed in the Key Resources Table.

Article and author information

Author details

  1. Vivek Garg

    Physiology, University of Maryland, Baltimore, United States
    For correspondence
    vgarg@som.umaryland.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6940-5415

  2. Junji Suzuki

    Physiology, University of California San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.

  3. Ishan Paranjpe

    Department of Physiology, University of California San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.

  4. Tiffany Unsulangi

    Department of Physiology, University of California San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.

  5. Liron Boyman

    Physiology, University of Maryland, Baltimore, United States
    Competing interests
    The authors declare that no competing interests exist.

  6. Lorin S Milescu

    Biology, University of Maryland Baltimore, College Park, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3177-7010

  7. W Jonathan Lederer

    University of Maryland School of Medicine, Baltimore, United States
    Competing interests
    The authors declare that no competing interests exist.

  8. Yuriy Kirichok

    Department of Physiology, University of California San Francisco, San Francisco, United States
    For correspondence
    yuriy.kirichok@ucsf.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7155-843X

Funding

National Institutes of Health (R01GM134536)

  • Yuriy Kirichok

National Institutes of Health (R35GM136415)

  • Yuriy Kirichok

American Heart Association (17SDG33660926)

  • Vivek Garg

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

Ethics

Animal experimentation: All animal experiments were performed according to procedures approved by the UCSF Institutional Animal Care and Use Committee (approval # AN183460-02A) and adhered to NIH standards.

Reviewing Editor

  1. Richard S Lewis, Stanford University School of Medicine, United States

Version history

  1. Preprint posted: April 5, 2020 (view preprint)
  2. Received: April 11, 2021
  3. Accepted: August 9, 2021
  4. Accepted Manuscript published: August 31, 2021 (version 1)
  5. Version of Record published: September 13, 2021 (version 2)

Copyright

© 2021, Garg 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

  • 2,559
    Page views
  • 377
    Downloads
  • 31
    Citations

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

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. Vivek Garg
  2. Junji Suzuki
  3. Ishan Paranjpe
  4. Tiffany Unsulangi
  5. Liron Boyman
  6. Lorin S Milescu
  7. W Jonathan Lederer
  8. Yuriy Kirichok
(2021)
The mechanism of MICU-dependent gating of the mitochondrial Ca2+ uniporter
eLife 10:e69312.
https://doi.org/10.7554/eLife.69312

Categories and tags

Research organism

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics

    The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Ga chaperone and GEF Ric-8A

    Daniel Muñoz-Reyes, Levi J McClelland ... Maria Jose Sanchez-Barrena
    Research Article

    The Neuronal Calcium Sensor 1, an EF-hand Ca2+ binding protein, and Ric-8A coregulate synapse number and probability of neurotransmitter release. Recently, the structures of Ric-8A bound to Ga have revealed how Ric-8A phosphorylation promotes Ga recognition and activity as a chaperone and guanine nucleotide exchange factor. However, the molecular mechanism by which NCS-1 regulates Ric-8A activity and its interaction with Ga subunits is not well understood. Given the interest in the NCS-1/Ric-8A complex as a therapeutic target in nervous system disorders, it is necessary to shed light on this molecular mechanism of action at atomic level. We have reconstituted NCS-1/Ric-8A complexes to conduct a multimodal approach and determine the sequence of Ca2+ signals and phosphorylation events that promote the interaction of Ric-8A with Ga. Our data show that the binding of NCS-1 and Ga to Ric-8A are mutually exclusive. Importantly, NCS-1 induces a structural rearrangement in Ric-8A that traps the protein in a conformational state that is inaccessible to Casein Kinase II-mediated phosphorylation, demonstrating one aspect of its negative regulation of Ric-8A-mediated G-protein signaling. Functional experiments indicate a loss of Ric-8A GEF activity towards Ga when complexed with NCS-1, and restoration of nucleotide exchange activity upon increasing Ca2+ concentration. Finally, the high-resolution crystallographic data reported here define the NCS-1/Ric-8A interface and will allow the development of therapeutic synapse function regulators with improved activity and selectivity.

    1. Cell Biology
    2. Structural Biology and Molecular Biophysics

    Baited reconstruction with 2D template matching for high-resolution structure determination in vitro and in vivo without template bias

    Bronwyn A Lucas, Benjamin A Himes, Nikolaus Grigorieff
    Research Advance

    Previously we showed that 2D template matching (2DTM) can be used to localize macromolecular complexes in images recorded by cryogenic electron microscopy (cryo-EM) with high precision, even in the presence of noise and cellular background (Lucas et al., 2021; Lucas et al., 2022). Here, we show that once localized, these particles may be averaged together to generate high-resolution 3D reconstructions. However, regions included in the template may suffer from template bias, leading to inflated resolution estimates and making the interpretation of high-resolution features unreliable. We evaluate conditions that minimize template bias while retaining the benefits of high-precision localization, and we show that molecular features not present in the template can be reconstructed at high resolution from targets found by 2DTM, extending prior work at low-resolution. Moreover, we present a quantitative metric for template bias to aid the interpretation of 3D reconstructions calculated with particles localized using high-resolution templates and fine angular sampling.

    1. Plant Biology
    2. Structural Biology and Molecular Biophysics

    Vicia faba SV channel VfTPC1 is a hyperexcitable variant of plant vacuole Two Pore Channels

    Jinping Lu, Ingo Dreyer ... Rainer Hedrich
    Research Article

    To fire action-potential-like electrical signals, the vacuole membrane requires the two-pore channel TPC1, formerly called SV channel. The TPC1/SV channel functions as a depolarization-stimulated, non-selective cation channel that is inhibited by luminal Ca2+. In our search for species-dependent functional TPC1 channel variants with different luminal Ca2+ sensitivity, we found in total three acidic residues present in Ca2+ sensor sites 2 and 3 of the Ca2+-sensitive AtTPC1 channel from Arabidopsis thaliana that were neutral in its Vicia faba ortholog and also in those of many other Fabaceae. When expressed in the Arabidopsis AtTPC1-loss-of-function background, wild-type VfTPC1 was hypersensitive to vacuole depolarization and only weakly sensitive to blocking luminal Ca2+. When AtTPC1 was mutated for these VfTPC1-homologous polymorphic residues, two neutral substitutions in Ca2+ sensor site 3 alone were already sufficient for the Arabidopsis At-VfTPC1 channel mutant to gain VfTPC1-like voltage and luminal Ca2+ sensitivity that together rendered vacuoles hyperexcitable. Thus, natural TPC1 channel variants exist in plant families which may fine-tune vacuole excitability and adapt it to environmental settings of the particular ecological niche.