1. Neuroscience
  2. Structural Biology and Molecular Biophysics

Cryo-EM structures of the DCPIB-inhibited volume-regulated anion channel LRRC8A in lipid nanodiscs

  1. David M Kern
  2. SeCheol Oh
  3. Richard K Hite  Is a corresponding author
  4. Stephen Graf Brohawn  Is a corresponding author
  1. University of California, Berkeley, United States
  2. Memorial Sloan Kettering Cancer Center, United States
https://doi.org/10.7554/eLife.42636
Share this article
Cite this article
  1. David M Kern
  2. SeCheol Oh
  3. Richard K Hite
  4. Stephen Graf Brohawn
(2019)
Cryo-EM structures of the DCPIB-inhibited volume-regulated anion channel LRRC8A in lipid nanodiscs
eLife 8:e42636.
https://doi.org/10.7554/eLife.42636
  2. Download BibTeX
  3. Download .RIS

Abstract

Hypoosmotic conditions activate volume-regulated anion channels in vertebrate cells. These channels are formed by leucine-rich repeat-containing protein 8 (LRRC8) family members and contain LRRC8A in homo- or hetero-hexameric assemblies. Here we present single-particle cryo-electron microscopy structures of Mus musculus LRRC8A in complex with the inhibitor DCPIB reconstituted in lipid nanodiscs. DCPIB plugs the channel like a cork in a bottle - binding in the extracellular selectivity filter and sterically occluding ion conduction. Constricted and expanded structures reveal coupled dilation of cytoplasmic LRRs and the channel pore, suggesting a mechanism for channel gating by internal stimuli. Conformational and symmetry differences between LRRC8A structures determined in detergent micelles and lipid bilayers related to reorganization of intersubunit lipid binding sites demonstrate a critical role for the membrane in determining channel structure. These results provide insight into LRRC8 gating and inhibition and the role of lipids in the structure of an ionic-strength sensing ion channel.

Data availability

Final maps of LRRC8A-DCPIB in MSPE3D1 nanodiscs have been deposited to the Electron Microscopy Data Bank under accession codes EMDB-0562 (masked constricted state), and EMDB-0563 (masked expanded state) and atomic coordinates have been deposited in the PDB under IDs 6NZW (constricted state) and 6NZZ (expanded state). The original micrograph movies have been deposited to EMPIAR under accession codes EMPIAR-10366 and EMPIAR-10367. The map of apo-LRRC8A in MSP2N2 nanodiscs in a constricted state has been deposited with EMDB accession code EMDB-0564 and coordinates deposited in the PDB with ID 6O00.

The following data sets were generated
    1. Kern DM
    2. Oh S
    3. Hite RK
    4. Brohawn SG
    (2019) Original micrograph movies
    Electron Microscopy Public Image Archive, EMPIAR-10366.
    https://www.ebi.ac.uk/pdbe/emdb/empiar/entry/10366
    1. Kern DM
    2. Oh S
    3. Hite RK
    4. Brohawn SG
    (2019) Original micrograph movies
    Electron Microscopy Public Image Archive, EMPIAR-10367.
    https://www.ebi.ac.uk/pdbe/emdb/empiar/entry/10367

Article and author information

Author details

  1. David M Kern

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    The authors declare that no competing interests exist.

  2. SeCheol Oh

    Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1685-5922

  3. Richard K Hite

    Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, United States
    For correspondence
    hiter@mskcc.org
    Competing interests
    The authors declare that no competing interests exist.

  4. Stephen Graf Brohawn

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    For correspondence
    brohawn@berkeley.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6768-3406

Funding

New York Stem Cell Foundation (NYSCF-R-N145)

  • Stephen Graf Brohawn

National Institute of General Medical Sciences (DP2GM123496-01)

  • Stephen Graf Brohawn

Klingenstein Third Generation Foundation (na)

  • Stephen Graf Brohawn

McKnight Endowment Fund for Neuroscience (na)

  • Stephen Graf Brohawn

National Cancer Institute (PO CA008748)

  • Richard K Hite

Searle Scholars Program (na)

  • Richard K Hite

Robertson Foundation (na)

  • Richard K Hite

National Institute of General Medical Sciences (F32GM128263)

  • David M Kern

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 Jon Swartz, National Institute of Neurological Disorders and Stroke, National Institutes of Health, United States

Version history

  1. Received: October 7, 2018
  2. Accepted: February 14, 2019
  3. Accepted Manuscript published: February 18, 2019 (version 1)
  4. Version of Record published: February 28, 2019 (version 2)
  5. Version of Record updated: March 16, 2020 (version 3)

Copyright

© 2019, Kern 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

  • 5,256
    Page views
  • 805
    Downloads
  • 68
    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. David M Kern
  2. SeCheol Oh
  3. Richard K Hite
  4. Stephen Graf Brohawn
(2019)
Cryo-EM structures of the DCPIB-inhibited volume-regulated anion channel LRRC8A in lipid nanodiscs
eLife 8:e42636.
https://doi.org/10.7554/eLife.42636

Categories and tags

Research organism

Further reading

    1. Neuroscience

    Modeling apical and basal tree contribution to orientation selectivity in a mouse primary visual cortex layer 2/3 pyramidal cell

    Konstantinos-Evangelos Petousakis, Ji Young Park ... Panayiota Poirazi
    Research Article

    Pyramidal neurons, a mainstay of cortical regions, receive a plethora of inputs from various areas onto their morphologically distinct apical and basal trees. Both trees differentially contribute to the somatic response, defining distinct anatomical and possibly functional sub-units. To elucidate the contribution of each tree to the encoding of visual stimuli at the somatic level, we modeled the response pattern of a mouse L2/3 V1 pyramidal neuron to orientation tuned synaptic input. Towards this goal, we used a morphologically detailed computational model of a single cell that replicates electrophysiological and two-photon imaging data. Our simulations predict a synergistic effect of apical and basal trees on somatic action potential generation: basal tree activity, in the form of either depolarization or dendritic spiking, is necessary for producing somatic activity, despite the fact that most somatic spikes are heavily driven by apical dendritic spikes. This model provides evidence for synergistic computations taking place in the basal and apical trees of the L2/3 V1 neuron along with mechanistic explanations for tree-specific contributions and emphasizes the potential role of predictive and attentional feedback input in these cells.

    1. Neuroscience
    2. Structural Biology and Molecular Biophysics

    Dynamic landscape of the intracellular termini of acid-sensing ion channel 1a

    Megan M Cullinan, Robert C Klipp ... John R Bankston
    Research Article

    Acid-sensing ion channels (ASICs) are trimeric proton-gated sodium channels. Recent work has shown that these channels play a role in necroptosis following prolonged acidic exposure like occurs in stroke. The C-terminus of ASIC1a is thought to mediate necroptotic cell death through interaction with receptor interacting serine threonine kinase 1 (RIPK1). This interaction is hypothesized to be inhibited at rest via an interaction between the C- and N-termini which blocks the RIPK1 binding site. Here, we use two transition metal ion FRET methods to investigate the conformational dynamics of the termini at neutral and acidic pH. We do not find evidence that the termini are close enough to be bound while the channel is at rest and find that the termini may modestly move closer together during acidification. At rest, the N-terminus adopts a conformation parallel to the membrane about 10 Å away. The distal end of the C-terminus may also spend time close to the membrane at rest. After acidification, the proximal portion of the N-terminus moves marginally closer to the membrane whereas the distal portion of the C-terminus swings away from the membrane. Together these data suggest that a new hypothesis for RIPK1 binding during stroke is needed.

    1. Neuroscience

    Adaptive biasing of action-selective cortical build-up activity by stimulus history

    Anke Braun, Tobias H Donner
    Research Article

    Decisions under uncertainty are often biased by the history of preceding sensory input, behavioral choices, or received outcomes. Behavioral studies of perceptual decisions suggest that such history-dependent biases affect the accumulation of evidence and can be adapted to the correlation structure of the sensory environment. Here, we systematically varied this correlation structure while human participants performed a canonical perceptual choice task. We tracked the trial-by-trial variations of history biases via behavioral modeling and of a neural signature of decision formation via magnetoencephalography (MEG). The history bias was flexibly adapted to the environment and exerted a selective effect on the build-up (not baseline level) of action-selective motor cortical activity during decision formation. This effect added to the impact of the current stimulus. We conclude that the build-up of action plans in human motor cortical circuits is shaped by dynamic prior expectations that result from an adaptive interaction with the environment.