Cryo-EM structures of the DCPIB-inhibited volume-regulated anion channel LRRC8A in lipid nanodiscs
- University of California, Berkeley, United States
- Memorial Sloan Kettering Cancer Center, United States
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.
-
Final map of LRRC8A-DCPIB in MSPE3D1 nanodiscs (masked constricted state)Electron Microscopy Data Bank, EMDB-0562.
-
Final map of LRRC8A-DCPIB in MSPE3D1 nanodiscs (masked expanded state)Electron Microscopy Data Bank, EMDB-0563.
-
Atomic coordinates (LRRC8A-DCPIB in MSPE3D1 nanodiscs expanded state)Protein Data Bank, 6NZZ.
-
Map of apo-LRRC8A in MSP2N2 nanodiscs in a constricted stateElectron Microscopy Data Bank, EMDB-0564.
-
Coordinates of apo-LRRC8A in MSP2N2 nanodiscs in a constricted stateProtein Data Bank, 6O00.
-
Original micrograph moviesElectron Microscopy Public Image Archive, EMPIAR-10366.
-
Original micrograph moviesElectron Microscopy Public Image Archive, EMPIAR-10367.
Article and author information
Author details
Stephen Graf Brohawn
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
© 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
-
- 5,711
- views
-
- 849
- downloads
-
- 87
- 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)
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
Further reading
-
- Neuroscience
Locomotion is controlled by spinal circuits that interact with supraspinal drives and sensory feedback from the limbs. These sensorimotor interactions are disrupted following spinal cord injury. The thoracic lateral hemisection represents an experimental model of an incomplete spinal cord injury, where connections between the brain and spinal cord are abolished on one side of the cord. To investigate the effects of such an injury on the operation of the spinal locomotor network, we used our computational model of cat locomotion recently published in eLife (Rybak et al., 2024) to investigate and predict changes in cycle and phase durations following a thoracic lateral hemisection during treadmill locomotion in tied-belt (equal left-right speeds) and split-belt (unequal left-right speeds) conditions. In our simulations, the ‘hemisection’ was always applied to the right side. Based on our model, we hypothesized that following hemisection the contralesional (‘intact’, left) side of the spinal network is mostly controlled by supraspinal drives, whereas the ipsilesional (‘hemisected’, right) side is mostly controlled by somatosensory feedback. We then compared the simulated results with those obtained during experiments in adult cats before and after a mid-thoracic lateral hemisection on the right side in the same locomotor conditions. Our experimental results confirmed many effects of hemisection on cat locomotion predicted by our simulations. We show that having the ipsilesional hindlimb step on the slow belt, but not the fast belt, during split-belt locomotion substantially reduces the effects of lateral hemisection. The model provides explanations for changes in temporal characteristics of hindlimb locomotion following hemisection based on altered interactions between spinal circuits, supraspinal drives, and somatosensory feedback.
-
- Neuroscience
A dysfunctional signaling pathway in the hippocampus has been linked to chronic pain-related memory impairment in mice.
