Mechanosensory transduction for senses such as proprioception, touch, balance, acceleration, hearing and pain relies on mechanotransduction channels, which convert mechanical stimuli into electrical signals in specialized sensory cells1. How force gates mechanotransduction channels is a central question in the field, for which there are two major models. One is the membrane-tension model: force applied to the membrane generates a change in membrane tension that is sufficient to gate the channel, as in the bacterial MscL channel and certain eukaryotic potassium channels2,3,4,5. The other is the tether model: force is transmitted via a tether to gate the channel. The transient receptor potential (TRP) channel NOMPC is important for mechanosensation-related behaviours such as locomotion, touch and sound sensation across different species including Caenorhabditis elegans6, Drosophila7,8,9 and zebrafish10. NOMPC is the founding member of the TRPN subfamily11, and is thought to be gated by tethering of its ankyrin repeat domain to microtubules of the cytoskeleton12,13,14,15. Thus, a goal of studying NOMPC is to reveal the underlying mechanism of force-induced gating, which could serve as a paradigm of the tether model. NOMPC fulfils all the criteria that apply to mechanotransduction channels1,7 and has 29 ankyrin repeats, the largest number among TRP channels. A key question is how the long ankyrin repeat domain is organized as a tether that can trigger channel gating. Here we present a de novo atomic structure of Drosophila NOMPC determined by single-particle electron cryo-microscopy. Structural analysis suggests that the ankyrin repeat domain of NOMPC resembles a helical spring, suggesting its role of linking mechanical displacement of the cytoskeleton to the opening of the channel. The NOMPC architecture underscores the basis of translating mechanical force into an electrical signal within a cell.
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We thank R. Mackinnon, T. Xiao and W. Wang for advice on protein purification, S. Gründer for sharing construct, and members from laboratories of D. Minor, D. Julius and R. Vale for sharing equipment and reagents. We also thank colleagues in our laboratories for discussions, M. Braunfeld and C. Kennedy for technical support, and Z. Yu and his colleagues at the HHMI Janelia Cryo-EM Facility for help with data acquisition. This work was supported by grants from the NIH (R01NS069229 to L.Y.J., 5R37NS040929, 1R35NS097227 to Y.-N.J., R01GM098672 and S10OD020054 to Y.C.), and by a UCSF Program for Breakthrough Biomedical Research (Y.C.). L.Y.J., Y.-N.J. and Y.C. are Investigators with the Howard Hughes Medical Institute.
The authors declare no competing financial interests.
Reviewer Information Nature thanks E. McCleskey, K. Vinothkumar and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, Pressure-induced mechanogated current measured at −60 mV from outside-out patches excised from HEK293 cells untransfected (left) or transfected (right) with the NOMPC construct used for structure determination. b, TIRF microscopy images of surface anchored and fluorescently labelled microtubules (pseudo-coloured in magenta) are not decorated by purified strep-GFP alone (top) but by strep-GFP-tagged NOMPC (bottom), demonstrating that purified NOMPC is capable of binding microtubules, and that this binding is not through interaction between the Strep tag and residual streptavidin on the surface.
a, Size-exclusion chromatography of NOMPC after exchange from DDM into amphipol A8-35. The peak fraction corresponding to NOMPC tetramer (indicated by arrow) was examined by SDS–polyacrylamide gel electrophoresis. The NOMPC monomer (approximately 190 kDa) band is indicated by an arrowhead. The top bands may correspond to incompletely disassociated NOMPC oligomers. b, Size-exclusion chromatography of NOMPC reconstituted into lipid nanodisc with MSP2N2. The peak fraction corresponding to NOMPC is indicated by arrow. c, d, Raw micrographs of NOMPC–amphipol (c) and NOMPC–nanodisc (d) samples examined by negative-stain EM. Both showed mono-dispersed and homogeneous particles. e, f, 2D class averages of NOMPC particles in amphipol (e) or nanodisc (f) by negative-stain EM, with the nanodisc sample showing better ordered features than the amphipol sample.
a, A raw cryo-EM micrograph of NOMPC recorded as described in Methods. b, 2D class averages of the cryo-EM micrographs with a particle box size of 400 pixels (486 Å). c, Euler angle distribution of all particles used for the final 3D reconstruction. The size of the sphere is proportional to the amount of particles visualized from that specific orientation. d, Final unsharpened 3D density map of NOMPC-amphipol colored with local resolution at a low isosurface level (top left) to enable visualization of the amphipol layer, and at a high isosurface level in side (top right), top (bottom right) and bottom (bottom left) views. e, FSC curves between two independently refined half maps before (red) and after (blue) post-processing in RELION, indicated with resolutions corresponding to FSC = 0.143. f, Density maps of NOMPC in amphipol (top) and in nanodisc (bottom) with a C1 symmetry derived from all particles picked. Comparison of the maps shows NOMPC-nanodisc is closer to a four-fold symmetric structure.
a, A raw cryo-EM micrograph of NOMPC recorded as described in Methods. b, 2D class averages of the cryo-EM micrographs with a particle box size of 400 pixels (486 Å). c, Euler angle distribution of all particles used for the final 3D reconstruction. The size of the sphere is proportional to the amount of particles visualized from that specific orientation. d, Slices through the unsharpened density map at different levels along the channel symmetry axis. The slice numbers starting from the cytoplasmic side are marked. e, Final unsharpened 3D density map of NOMPC–nanodisc coloured with local resolution at a low isosurface level (top left) to enable visualization of the lipid bilayer, and at a high isosurface level in side (top right), top (bottom right) and bottom (bottom left) views. f, FSC curves between two independently refined half maps before (red) and after (blue) post-processing in RELION, indicated with resolutions corresponding to FSC = 0.143. g, Cross-validation using FSC curves of the density map calculated from the refined model versus half map 1 (work, green), versus half map 2 (free, pink) and versus summed map (blue).
a–j, Representative cryo-EM densities of various NOMPC domains as indicated are superimposed on the atomic model. k, Density map of pre-S1 elbow, S1 and S4. l, Density map of stacked S4–S5 linker, TRP domain and linker helices that couple ARs to the pore through domain interactions. All the density maps (a–l) are shown as cyan meshes, and the model is shown as sticks and coloured according to atom type (C: light grey; N: blue; O: red; S: yellow).
a–d, Ribbon diagrams of NOMPC atomic model for residues Asn 125–Trp 1602 and Gly1689–Arg1670. The entire AR domain of 29 ARs was resolved. e, Ribbon diagrams showing two views of one NOMPC subunit denoting specific domains.
a, Pore radius calculated by the CAVER program. b, I–V curves from the steady-state currents of whole-cell recording from S2 cells expressing NOMPC or NOMPC mutants suggesting that both W1572A and I1554A mutations resulted in large basal current in the absence of applied pressure (wild type: n = 7; W1572A: n = 7; I1554A: n = 3). Cells expressing I1554A mutant channels displayed very large basal currents and were not amenable to recording from excised patch recording of mechanogated current. c, Dose-dependent curves of pressure-induced mechanogated currents measured at −60 mV from outside-out patches excised from S2 cells expressing NOMPC or NOMPC mutants (n = 7). d, Representative images of unpermeabilized staining of S2 cells expressing control proteins or His1423Ala mutant. The surface staining signal from approximately 35 cells were visually surveyed under fluorescent microscope. All cells expressing wild-type (middle) and His1423Ala mutant (bottom) NOMPC showed similar levels of surface expression, indicating that the His1423Ala mutant is properly localized to the plasma membrane like the wild type. All error bars (b, c) denote s.d.
The flowchart of classification procedures with RELION is shown. After 1 round of 2D classification, 190,879 particles were subjected to 3D refinement, yielding a 3.7 Å map (C4 imposed). Following 3D classification of these particles, 175,314 particles were selected and refined, yielding the final map at 3.55 Å resolution (C4 imposed), which was used to build the ‘consensus model’. Further classification of these 175,314 particles using a mask to exclude all regions outside the AR domain gave 3 major classes, which were subsequently refined to around 3.8–4.0 Å resolution.
a, Superposition of NOMPC AR domains from three classes are shown in three views as indicated. Overall, the entire AR domains from all three classes overlap well with each other. There are small differences in some ARs, which are caused by a slight shift of individual ARs as a rigid body, suggesting small mobility and plasticity of the AR domain. b, Superposition of NOMPC AR domain (blue) with human ankyrin-B AR domain (pink) shown in three views as indicated, suggesting that elastic deformation of NOMPC AR domain under pressure could potentially be more notable than the shift presumably caused by thermal motion as shown in a. A peptide from the C-terminal region of ankyrin-R, which was added to stabilize ankyrin-B ARs for crystallization by forming an auto-inhibitory segment (AS) structure, is also shown (AnkR AS, in orange). c, Schematic of NOMPC (without precise depiction of domain swap between neighbouring subunits) showing the N terminus tethered to a microtubule. Mechanical force is transduced from the microtubule cytoskeleton to NOMPC, possibly causing lateral movement, extension, compression or torsion of the AR domain. d, e, Movement of the AR domain that immediately precedes the linker helices results in displacement of the TRP domain and S4–S5 linker that are connected to the ends of the pore domain, triggering channel opening.
Sequence homology of NOMPC orthologues were analysed by clustal omega. The conserved residues are highlighted. The two residues (His1423 and Trp1572), which were shown in this study to be crucial for mechanogating, are marked by red triangles. Secondary structure elements are indicated above the sequence.
This file contains a supplementary discussion and a table of the summary of cryo-EM data collection and model refinement.
This file contains the uncropped SDS PAGE gel for extended data figure 2a.
Animated video shows morph between atomic models generated from “class 1” and “class 2” maps (Ext. Data Fig. 7). Atomic models were generated by real space refinement of the consensus model into the maps for the corresponding classes in PHENIX.
Animated video shows morph between atomic models generated from “class 1” and “class 3” maps (Ext. Data Fig. 7). Atomic models were generated as in Extended Data Video 1.
Animated video shows morph between atomic models generated from “class 2” and “class 3” maps (Ext. Data Fig. 7). Atomic models were generated as in Extended Data Video 1.
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Jin, P., Bulkley, D., Guo, Y. et al. Electron cryo-microscopy structure of the mechanotransduction channel NOMPC. Nature 547, 118–122 (2017). https://doi.org/10.1038/nature22981
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