Molecular mechanisms of gating in the calcium-activated chloride channel bestrophin
- Memorial Sloan Kettering Cancer Center, United States
Abstract
Bestrophin (BEST1-4) ligand-gated chloride (Cl-) channels are activated by calcium (Ca2+). Mutation of BEST1 causes retinal disease. Partly because bestrophin channels have no sequence or structural similarity to other ion channels, the molecular mechanisms underlying gating are unknown. Here, we present a series of cryo-electron microscopy structures of chicken BEST1, determined at 3.1 Å resolution or better, that represent the channel's principal gating states. Unlike other channels, opening of the pore is due to the repositioning of tethered pore-lining helices within a surrounding protein shell that dramatically widens a neck of the pore through a concertina of amino acid rearrangements. The neck serves as both the activation and the inactivation gate. Ca2+ binding instigates opening of the neck through allosteric means whereas inactivation peptide binding induces closing. An aperture within the otherwise wide pore controls anion permeability. The studies define a new molecular paradigm for gating among ligand-gated ion channels.
Data availability
Atomic coordinates and cryo-EM density maps of have been deposited with the PDB and Electron Microscopy Data Bank with the accession numbers: 6N23 (BEST1405, inactivated; EMD-9321), 6N24 (BEST1345 W287F mutant, Ca2+-free; EMD-9322), 6N25 (BEST1345 W287F mutant, Ca2+-bound; EMD-9323), 6N26 ( BEST1345 Ca2+-free closed state; EMD-9324), 6N27 (BEST1345 Ca2+-bound closed state; EMD-9325), and 6N28 ( BEST1345 Ca2+-bound open state; EMD-9326).
-
BEST1 in a calcium-bound inactivated stateProtein Databank, 6N23.
-
BEST1 open state W287F mutant, calcium-freeProtein Databank, 6N24.
-
BEST1 open state W287F mutant, calcium-boundProtein Databank, 6N25.
-
BEST1 calcium-free closed state (deactivated)Protein Databank, 6N26.
-
BEST1 in a calcium-bound inactivated stateElectron Microscopy Data Bank, EMD-9321.
-
BEST1 open state W287F mutant, calcium-freeElectron Microscopy Data Bank, EMD-9322.
-
BEST1 open state W287F mutant, calcium-boundElectron Microscopy Data Bank, EMD-9323.
-
BEST1 calcium-free closed state (deactivated)Electron Microscopy Data Bank, EMD-9324.
-
BEST1 calcium-bound closed stateElectron Microscopy Data Bank, EMD-9325.
-
BEST1 calcium-bound open stateElectron Microscopy Data Bank, EMD-9326.
Article and author information
Author details
Stephen Barstow Long
Funding
National Institutes of Health (R01-GM110396)
- Stephen Barstow Long
National Cancer Institute (P30 CA008748)
- Stephen Barstow Long
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2019, Miller 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
-
- 4,988
- views
-
- 717
- downloads
-
- 35
- 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)
Molecular mechanisms of gating in the calcium-activated chloride channel bestrophin
eLife 8:e43231.
https://doi.org/10.7554/eLife.43231
Further reading
-
- Biochemistry and Chemical Biology
Prions replicate via the autocatalytic conversion of cellular prion protein (PrPC) into fibrillar assemblies of misfolded PrP. While this process has been extensively studied in vivo and in vitro, non-physiological reaction conditions of fibril formation in vitro have precluded the identification and mechanistic analysis of cellular proteins, which may alter PrP self-assembly and prion replication. Here, we have developed a fibril formation assay for recombinant murine and human PrP (23-231) under near-native conditions (NAA) to study the effect of cellular proteins, which may be risk factors or potential therapeutic targets in prion disease. Genetic screening suggests that variants that increase syntaxin-6 expression in the brain (gene: STX6) are risk factors for sporadic Creutzfeldt–Jakob disease. Analysis of the protein in NAA revealed, counterintuitively, that syntaxin-6 is a potent inhibitor of PrP fibril formation. It significantly delayed the lag phase of fibril formation at highly sub-stoichiometric molar ratios. However, when assessing toxicity of different aggregation time points to primary neurons, syntaxin-6 prolonged the presence of neurotoxic PrP species. Electron microscopy and super-resolution fluorescence microscopy revealed that, instead of highly ordered fibrils, in the presence of syntaxin-6 PrP formed less-ordered aggregates containing syntaxin-6. These data strongly suggest that the protein can directly alter the initial phase of PrP self-assembly and, uniquely, can act as an ‘anti-chaperone’, which promotes toxic aggregation intermediates by inhibiting fibril formation.
-
- Biochemistry and Chemical Biology
Proteasomes are essential molecular machines responsible for the degradation of proteins in eukaryotic cells. Altered proteasome activity has been linked to neurodegeneration, auto-immune disorders and cancer. Despite the relevance for human disease and drug development, no method currently exists to monitor proteasome composition and interactions in vivo in animal models. To fill this gap, we developed a strategy based on tagging of proteasomes with promiscuous biotin ligases and generated a new mouse model enabling the quantification of proteasome interactions by mass spectrometry. We show that biotin ligases can be incorporated in fully assembled proteasomes without negative impact on their activity. We demonstrate the utility of our method by identifying novel proteasome-interacting proteins, charting interactomes across mouse organs, and showing that proximity-labeling enables the identification of both endogenous and small-molecule-induced proteasome substrates.
