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
-
- 5,804
- views
-
- 856
- 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
Cerebellar dysfunction leads to postural instability. Recent work in freely moving rodents has transformed investigations of cerebellar contributions to posture. However, the combined complexity of terrestrial locomotion and the rodent cerebellum motivate new approaches to perturb cerebellar function in simpler vertebrates. Here, we adapted a validated chemogenetic tool (TRPV1/capsaicin) to describe the role of Purkinje cells — the output neurons of the cerebellar cortex — as larval zebrafish swam freely in depth. We achieved both bidirectional control (activation and ablation) of Purkinje cells while performing quantitative high-throughput assessment of posture and locomotion. Activation modified postural control in the pitch (nose-up/nose-down) axis. Similarly, ablations disrupted pitch-axis posture and fin-body coordination responsible for climbs. Postural disruption was more widespread in older larvae, offering a window into emergent roles for the developing cerebellum in the control of posture. Finally, we found that activity in Purkinje cells could individually and collectively encode tilt direction, a key feature of postural control neurons. Our findings delineate an expected role for the cerebellum in postural control and vestibular sensation in larval zebrafish, establishing the validity of TRPV1/capsaicin-mediated perturbations in a simple, genetically tractable vertebrate. Moreover, by comparing the contributions of Purkinje cell ablations to posture in time, we uncover signatures of emerging cerebellar control of posture across early development. This work takes a major step towards understanding an ancestral role of the cerebellum in regulating postural maturation.
-
- Neuroscience
Human-specific cognitive abilities depend on information processing in the cerebral cortex, where the neurons are significantly larger and their processes longer and sparser compared to rodents. We found that, in synaptically connected layer 2/3 pyramidal cells (L2/3 PCs), the delay in signal propagation from soma to soma is similar in humans and rodents. To compensate for the longer processes of neurons, membrane potential changes in human axons and/or dendrites must propagate faster. Axonal and dendritic recordings show that the propagation speed of action potentials (APs) is similar in human and rat axons, but the forward propagation of excitatory postsynaptic potentials (EPSPs) and the backward propagation of APs are 26 and 47% faster in human dendrites, respectively. Experimentally-based detailed biophysical models have shown that the key factor responsible for the accelerated EPSP propagation in human cortical dendrites is the large conductance load imposed at the soma by the large basal dendritic tree. Additionally, larger dendritic diameters and differences in cable and ion channel properties in humans contribute to enhanced signal propagation. Our integrative experimental and modeling study provides new insights into the scaling rules that help maintain information processing speed albeit the large and sparse neurons in the human cortex.
