Dynamic Na+/H+ Exchanger 1 (NHE1):Calmodulin complexes of varying stoichiometry and structure regulate Ca2+-dependent NHE1 activation

Abstract

Calmodulin (CaM) engages in Ca2+-dependent interactions with numerous proteins, including a still incompletely understood physical and functional interaction with the human Na+/H+-exchanger NHE1. Using nuclear magnetic resonance (NMR) spectroscopy, isothermal titration calorimetry, and fibroblasts stably expressing wildtype and mutant NHE1, we discovered multiple accessible states of this functionally important complex existing in different NHE1:CaM stoichiometries and structures. We determined the NMR solution structure of a ternary complex in which CaM links two NHE1 cytosolic tails. In vitro, stoichiometries and affinities could be tuned by variations in NHE1:CaM ratio and calcium ([Ca2+]) and by phosphorylation of S648 in the first CaM-binding a-helix. In cells, Ca2+-CaM-induced NHE1 activity was reduced by mimicking S648 phosphorylation and by mutation of the first CaM-binding a-helix, whereas it was unaffected by inhibition of Akt, one of several kinases phosphorylating S648. Our results demonstrate a diversity of NHE1:CaM interaction modes and suggest that CaM may contribute to NHE1 dimerization and thereby augment NHE1 regulation. We propose that a similar structural diversity is of relevance to many other CaM complexes.

Data availability

Source data files are provided for Figure 1, 3, 5 and 6. Resonance assignments of the ternary complex of CaM and two H1 have been deposited in the Biological Magnetic Resonance Bank (BMRB) under ID code 34521. The atomic coordinates for the ternary complex of CaM and two H1 have been deposited in the Protein Data Bank under the ID code 6zbi.

The following data sets were generated

Article and author information

Author details

  1. Lise M Sjøgaard-Frich

    Section for Cell Biology and Physiology, Department of Biology, University of Copenhagen, Copenhagen, Denmark
    Competing interests
    The authors declare that no competing interests exist.
  2. Andreas Prestel

    Structural Biology and NMR laboratory, Department of Biology, University of Copenhagen, Copenhagen, Denmark
    Competing interests
    The authors declare that no competing interests exist.
  3. Emilie S Pedersen

    Structural Biology and NMR laboratory, Department of Biology, University of Copenhagen, Copenhagen, Denmark
    Competing interests
    The authors declare that no competing interests exist.
  4. Marc Severin

    Section for Cell Biology and Physiology, Department of Biology, University of Copenhagen, Copenhagen, Denmark
    Competing interests
    The authors declare that no competing interests exist.
  5. Kristian Kølby Kristensen

    Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark
    Competing interests
    The authors declare that no competing interests exist.
  6. Johan G Olsen

    Structural Biology and NMR laboratory, Department of Biology, University of Copenhagen, Copenhagen, Denmark
    Competing interests
    The authors declare that no competing interests exist.
  7. Birthe B Kragelund

    Department of Biology, University of Copenhagen, Copenhagen, Denmark
    For correspondence
    bbk@bio.ku.dk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7454-1761
  8. Stine Falsig Pedersen

    Section for Cell Biology and Physiology, Department of Biology, University of Copenhagen, Copenhagen, Denmark
    For correspondence
    sfpedersen@bio.ku.dk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3044-7714

Funding

Danish Research Councils (4181-00344)

  • Birthe B Kragelund

Novo Nordisk Fonden (NNF15OC0016670)

  • Birthe B Kragelund

Novo Nordisk Fonden (NNF18OC0034070)

  • Stine Falsig Pedersen

Novo Nordisk Fonden (NNF19OC0057241)

  • Stine Falsig Pedersen

Novo Nordisk Fonden (NNF18OC0032996)

  • Birthe B Kragelund

Villum Fonden

  • Birthe B Kragelund

Carlsbergfondet (CF20-0491)

  • Stine Falsig Pedersen

Novo Nordisk

  • Lise M Sjøgaard-Frich

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

Reviewing Editor

  1. Lewis E Kay, University of Toronto, Canada

Version history

  1. Received: July 9, 2020
  2. Accepted: March 1, 2021
  3. Accepted Manuscript published: March 3, 2021 (version 1)
  4. Version of Record published: March 30, 2021 (version 2)

Copyright

© 2021, Sjøgaard-Frich 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

  • 1,449
    Page views
  • 225
    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. Lise M Sjøgaard-Frich
  2. Andreas Prestel
  3. Emilie S Pedersen
  4. Marc Severin
  5. Kristian Kølby Kristensen
  6. Johan G Olsen
  7. Birthe B Kragelund
  8. Stine Falsig Pedersen
(2021)
Dynamic Na+/H+ Exchanger 1 (NHE1):Calmodulin complexes of varying stoichiometry and structure regulate Ca2+-dependent NHE1 activation
eLife 10:e60889.
https://doi.org/10.7554/eLife.60889

Further reading

    1. Cell Biology
    2. Microbiology and Infectious Disease
    Juan Xiang, Chaoyang Fan ... Pei Xu
    Research Article Updated

    The relative positions of viral DNA genomes to the host intranuclear environment play critical roles in determining virus fate. Recent advances in the application of chromosome conformation capture-based sequencing analysis (3 C technologies) have revealed valuable aspects of the spatiotemporal interplay of viral genomes with host chromosomes. However, to elucidate the causal relationship between the subnuclear localization of viral genomes and the pathogenic outcome of an infection, manipulative tools are needed. Rapid repositioning of viral DNAs to specific subnuclear compartments amid infection is a powerful approach to synchronize and interrogate this dynamically changing process in space and time. Herein, we report an inducible CRISPR-based two-component platform that relocates extrachromosomal DNA pieces (5 kb to 170 kb) to the nuclear periphery in minutes (CRISPR-nuPin). Based on this strategy, investigations of herpes simplex virus 1 (HSV-1), a prototypical member of the human herpesvirus family, revealed unprecedently reported insights into the early intranuclear life of the pathogen: (I) Viral genomes tethered to the nuclear periphery upon entry, compared with those freely infecting the nucleus, were wrapped around histones with increased suppressive modifications and subjected to stronger transcriptional silencing and prominent growth inhibition. (II) Relocating HSV-1 genomes at 1 hr post infection significantly promoted the transcription of viral genes, termed an ‘Escaping’ effect. (III) Early accumulation of ICP0 was a sufficient but not necessary condition for ‘Escaping’. (IV) Subnuclear localization was only critical during early infection. Importantly, the CRISPR-nuPin tactic, in principle, is applicable to many other DNA viruses.

    1. Cell Biology
    Enrico Radaelli, Charles-Antoine Assenmacher ... Marco Spinazzi
    Research Article Updated

    Impaired spermatogenesis and male infertility are common manifestations associated with mitochondrial diseases, yet the underlying mechanisms linking these conditions remain elusive. In this study, we demonstrate that mice deficient for the mitochondrial intra-membrane rhomboid protease PARL, a recently reported model of the mitochondrial encephalopathy Leigh syndrome, develop early testicular atrophy caused by a complete arrest of spermatogenesis during meiotic prophase I, followed by degeneration and death of arrested spermatocytes. This process is independent of neurodegeneration. Interestingly, genetic modifications of PINK1, PGAM5, and TTC19 – three major substrates of PARL with important roles in mitochondrial homeostasis – fail to reproduce or modify this severe phenotype, indicating that the spermatogenic arrest arises from distinct molecular pathways. We further observed severe abnormalities in mitochondrial ultrastructure in PARL-deficient spermatocytes, along with prominent electron transfer chain defects, disrupted coenzyme Q (CoQ) biosynthesis, and metabolic rewiring. These mitochondrial defects are associated with a germ cell-specific decrease in GPX4 expression leading arrested spermatocytes to ferroptosis – a regulated cell death modality characterized by uncontrolled lipid peroxidation. Our results suggest that mitochondrial defects induced by PARL depletion act as an initiating trigger for ferroptosis in primary spermatocytes through simultaneous effects on GPX4 and CoQ – two major inhibitors of ferroptosis. These findings shed new light on the potential role of ferroptosis in the pathogenesis of mitochondrial diseases and male infertility warranting further investigation.