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

Abstract
Data availability
Article and author information
Metrics

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) Final map of LRRC8A-DCPIB in MSPE3D1 nanodiscs (masked constricted state)
Electron Microscopy Data Bank, EMDB-0562.

http://www.ebi.ac.uk/pdbe/emdb/ EMDB-0562
1. Kern DM
2. Oh S
3. Hite RK
4. Brohawn SG
(2019) Final map of LRRC8A-DCPIB in MSPE3D1 nanodiscs (masked expanded state)
Electron Microscopy Data Bank, EMDB-0563.

http://www.ebi.ac.uk/pdbe/emdb/ EMDB-0563
1. Kern DM
2. Oh S
3. Hite RK
4. Brohawn SG
(2019) Atomic coordinates (LRRC8A-DCPIB in MSPE3D1 nanodiscs constricted state)
Protein Data Bank, 6NZW.

http://www.rcsb.org/structure/6NZW
1. Kern DM
2. Oh S
3. Hite RK
4. Brohawn SG
(2019) Atomic coordinates (LRRC8A-DCPIB in MSPE3D1 nanodiscs expanded state)
Protein Data Bank, 6NZZ.

http://www.rcsb.org/structure/6NZZ
1. Kern DM
2. Oh S
3. Hite RK
4. Brohawn SG
(2019) Map of apo-LRRC8A in MSP2N2 nanodiscs in a constricted state
Electron Microscopy Data Bank, EMDB-0564.

http://www.ebi.ac.uk/pdbe/emdb/EMDB-0564
1. Kern DM
2. Oh S
3. Hite RK
4. Brohawn SG
(2019) Coordinates of apo-LRRC8A in MSP2N2 nanodiscs in a constricted state
Protein Data Bank, 6O00.

http://www.rcsb.org/structure/6O00
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

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.
SeCheol Oh

Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, United States

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-1685-5922
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.
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.

"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.

Copyright

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.