Cryo-EM structure of the mechanically activated ion channel OSCA1.2
- The Scripps Research Institute, United States
- University of Oxford, United Kingdom
Abstract
Mechanically activated ion channels underlie touch, hearing, shear-stress sensing, and response to turgor pressure. OSCA/TMEM63s are a newly-identified family of eukaryotic mechanically activated ion channels opened by membrane tension. The structural underpinnings of OSCA/TMEM63 function are not explored. Here, we elucidate high resolution cryo-electron microscopy structures of OSCA1.2, revealing a dimeric architecture containing eleven transmembrane helices per subunit and surprising topological similarities to TMEM16 proteins. We locate the ion permeation pathway within each subunit by demonstrating that a conserved acidic residue is a determinant of channel conductance. Molecular dynamics simulations reveal membrane interactions, suggesting the role of lipids in OSCA1.2 gating. These results lay a foundation to decipher how the structural organization of OSCA/TMEM63 is suited for their roles as MA ion channels.
Data availability
Cryo-EM maps of OSCA1.2 in nanodiscs and LMNG have been deposited to the Electron Microscopy Data Bank under accession codes 9112 and 9113. Atomic coordinates of OSCA1.2 in nanodiscs and LMNG have been deposited to the PDB under IDs 6MGV and 6MGW. Due to their large size (300Gb+), the raw data files are available upon request to the corresponding author(s).
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Cryo-EM map of mechanically activated ion channel OSCA1.2 in nanodiscElectron Microscopy Data Bank, 9112.
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Cryo-EM map of mechanically activated ion channel OSCA1.2 in LMNGElectron Microscopy Data Bank, 9113.
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Structure of mechanically activated ion channel OSCA1.2 in nanodiscProtein Data Bank, 6MGV.
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Structure of mechanically activated ion channel OSCA1.2 in LMNGProtein Data Bank, 6MGW.
Article and author information
Author details
Sebastian Jojoa Cruz
Che Chun (Alex) Tsui
Funding
Howard Hughes Medical Institute
- Ardem Patapoutian
Croucher Foundation
- Che Chun (Alex) Tsui
National Institute of Neurological Disorders and Stroke (1R35NS105067)
- Ardem Patapoutian
Ray Thomas Edwards Foundation
- Andrew B Ward
Wellcome (208361/Z/17/Z)
- Mark SP Sansom
Biotechnology and Biological Sciences Research Council (BB/N000145/1)
- Mark SP Sansom
Biotechnology and Biological Sciences Research Council (BB/R00126X/1)
- Mark SP Sansom
Engineering and Physical Sciences Research Council (EP/R004722/1)
- Mark SP Sansom
Jane Coffin Childs Memorial Fund for Medical Research
- Kei Saotome
Skaggs-Oxford Scholarship
- Che Chun (Alex) Tsui
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2018, Jojoa Cruz 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.
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Cryo-EM structure of the mechanically activated ion channel OSCA1.2
eLife 7:e41845.
https://doi.org/10.7554/eLife.41845
Further reading
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- Evolutionary Biology
- Structural Biology and Molecular Biophysics
Mechanically activated (MA) ion channels convert physical forces into electrical signals, and are essential for eukaryotic physiology. Despite their importance, few bona-fide MA channels have been described in plants and animals. Here, we show that various members of the OSCA and TMEM63 family of proteins from plants, flies, and mammals confer mechanosensitivity to naïve cells. We conclusively demonstrate that OSCA1.2, one of the Arabidopsis thaliana OSCA proteins, is an inherently mechanosensitive, pore-forming ion channel. Our results suggest that OSCA/TMEM63 proteins are the largest family of MA ion channels identified, and are conserved across eukaryotes. Our findings will enable studies to gain deep insight into molecular mechanisms of MA channel gating, and will facilitate a better understanding of mechanosensory processes in vivo across plants and animals.
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- Structural Biology and Molecular Biophysics
Osmotic stress and chloride regulate the autophosphorylation and activity of the WNK1 and WNK3 kinase domains. The kinase domain of unphosphorylated WNK1 (uWNK1) is an asymmetric dimer possessing water molecules conserved in multiple uWNK1 crystal structures. Conserved waters are present in two networks, referred to here as conserved water networks 1 and 2 (CWN1 and CWN2). Here, we show that PEG400 applied to crystals of dimeric uWNK1 induces de-dimerization. Both the WNK1 the water networks and the chloride-binding site are disrupted by PEG400. CWN1 is surrounded by a cluster of pan-WNK-conserved charged residues. Here, we mutagenized these charges in WNK3, a highly active WNK isoform kinase domain, and WNK1, the isoform best studied crystallographically. Mutation of E314 in the Activation Loop of WNK3 (WNK3/E314Q and WNK3/E314A, and the homologous WNK1/E388A) enhanced the rate of autophosphorylation, and reduced chloride sensitivity. Other WNK3 mutants reduced the rate of autophosphorylation activity coupled with greater chloride sensitivity than wild-type. The water and chloride regulation thus appear linked. The lower activity of some mutants may reflect effects on catalysis. Crystallography showed that activating mutants introduced conformational changes in similar parts of the structure to those induced by PEG400. WNK activating mutations and crystallography support a role for CWN1 in WNK inhibition consistent with water functioning as an allosteric ligand.
