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
-
- 5,790
- views
-
- 854
- downloads
-
- 93
- 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
Detecting causal relations structures our perception of events in the world. Here, we determined for visual interactions whether generalized (i.e. feature-invariant) or specialized (i.e. feature-selective) visual routines underlie the perception of causality. To this end, we applied a visual adaptation protocol to assess the adaptability of specific features in classical launching events of simple geometric shapes. We asked observers to report whether they observed a launch or a pass in ambiguous test events (i.e. the overlap between two discs varied from trial to trial). After prolonged exposure to causal launch events (the adaptor) defined by a particular set of features (i.e. a particular motion direction, motion speed, or feature conjunction), observers were less likely to see causal launches in subsequent ambiguous test events than before adaptation. Crucially, adaptation was contingent on the causal impression in launches as demonstrated by a lack of adaptation in non-causal control events. We assessed whether this negative aftereffect transfers to test events with a new set of feature values that were not presented during adaptation. Processing in specialized (as opposed to generalized) visual routines predicts that the transfer of visual adaptation depends on the feature similarity of the adaptor and the test event. We show that the negative aftereffects do not transfer to unadapted launch directions but do transfer to launch events of different speeds. Finally, we used colored discs to assign distinct feature-based identities to the launching and the launched stimulus. We found that the adaptation transferred across colors if the test event had the same motion direction as the adaptor. In summary, visual adaptation allowed us to carve out a visual feature space underlying the perception of causality and revealed specialized visual routines that are tuned to a launch’s motion direction.
-
- Neuroscience
The classical diagnosis of Parkinsonism is based on motor symptoms that are the consequence of nigrostriatal pathway dysfunction and reduced dopaminergic output. However, a decade prior to the emergence of motor issues, patients frequently experience non-motor symptoms, such as a reduced sense of smell (hyposmia). The cellular and molecular bases for these early defects remain enigmatic. To explore this, we developed a new collection of five fruit fly models of familial Parkinsonism and conducted single-cell RNA sequencing on young brains of these models. Interestingly, cholinergic projection neurons are the most vulnerable cells, and genes associated with presynaptic function are the most deregulated. Additional single nucleus sequencing of three specific brain regions of Parkinson’s disease patients confirms these findings. Indeed, the disturbances lead to early synaptic dysfunction, notably affecting cholinergic olfactory projection neurons crucial for olfactory function in flies. Correcting these defects specifically in olfactory cholinergic interneurons in flies or inducing cholinergic signaling in Parkinson mutant human induced dopaminergic neurons in vitro using nicotine, both rescue age-dependent dopaminergic neuron decline. Hence, our research uncovers that one of the earliest indicators of disease in five different models of familial Parkinsonism is synaptic dysfunction in higher-order cholinergic projection neurons and this contributes to the development of hyposmia. Furthermore, the shared pathways of synaptic failure in these cholinergic neurons ultimately contribute to dopaminergic dysfunction later in life.
