Electron cryo-microscopy structure of the mechanotransduction channel NOMPC

Journal name:
Nature
Volume:
547,
Pages:
118–122
Date published:
DOI:
doi:10.1038/nature22981
Received
Accepted
Published online

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.

At a glance

Figures

  1. 3D reconstruction of NOMPC.
    Figure 1: 3D reconstruction of NOMPC.

    ad, NOMPC density map in nanodisc (EMDB accession 8702). Unsharpened (transparent) and sharpened (solid and colour-coded) maps are shown, from the side (a, b), top (c) and bottom (d). e, Diagram illustrating major structural domains of one subunit, colour-coded to match ribbon diagrams in f. Dashed lines denote regions in which the density map is insufficient for model building. AR1–AR7 are coloured pale red because their density is insufficient to position side chains. f, Ribbon diagram showing one NOMPC subunit denoting specific domains (PDB accession 5VKQ).

  2. Architecture of the ion permeation pathway of NOMPC.
    Figure 2: Architecture of the ion permeation pathway of NOMPC.

    a, Solvent-accessible pathway along the ion permeation pore illustrated as blue surface. Residues located at the selectivity filter and lower gate are rendered as sticks. b, Top view of the NOMPC pore region is shown in surface representation, coloured by the electrostatic potentials (negative: red; positive: blue).

  3. Lipid–protein and linker domain interactions.
    Figure 3: Lipid–protein and linker domain interactions.

    a, The transmembrane domains of two adjacent subunits are shown in blue and red from the cytosolic face (left) and with the cytosolic domain facing down (right). The lipid bound to the S4–S5 linker of the red subunit is coloured green. b, Enlarged view of boxed region of NOMPC shows the density map (green mesh) of the lipid superimposed with an atomic model of the bound phospholipid. The side chain of His1423 from the S4–S5 linker is close (~2.9 Å) to the lipid head group, and the adjacent His1424 is further away (~4.8 Å). c, Representative traces of mechanogated current of wild-type NOMPC and NOMPC mutants (His1423Ala, His1424Ala, Lys1573Ala and Trp1572Ala) under pressure ranging from 10 mm Hg (1,300 Pa) to 50 mm Hg (6,700 Pa), with 10 mm Hg increments, recorded by outside-out patch clamp. The His1424Ala and Lys1573Ala mutations are negative controls showing that no random mutation in the S4–S5 linker or TRP domain abolish the mechanogated current of NOMPC. dg, Enlarged views from the boxed regions showing key interactions between residues from interacting domains. Hydrogen bonds are indicated by dashed lines (d, e). EM density is shown in mesh (f).

  4. Motion of the AR domain.
    Figure 4: Motion of the AR domain.

    a, Interactions between the adjacent ARs. Residues with charged and polar side chains are indicated as coloured balls (negative: red; positive: blue). b, Three sub-classes with different conformation in ARs. cf, Comparison of class 1 and 2 at different positions along the AR and linker domain. The ARs shifted vertically along the symmetry axis at the lower junction in the vicinity of AR9 (c) and laterally relative to the symmetry axis above the upper junction, at AR29 (d). While there is no obvious vertical movement at the linker regions, there is a small domain rotation (e and f).

  5. Verification of recombinant NOMPC activity.
    Extended Data Fig. 1: Verification of recombinant NOMPC activity.

    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.

  6. Negative-stain analysis of purified NOMPC.
    Extended Data Fig. 2: Negative-stain analysis of purified NOMPC.

    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.

  7. Cryo-EM analysis of NOMPC in amphipol.
    Extended Data Fig. 3: Cryo-EM analysis of NOMPC in amphipol.

    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.

  8. Cryo-EM analysis of NOMPC reconstituted in nanodisc.
    Extended Data Fig. 4: Cryo-EM analysis of NOMPC reconstituted in nanodisc.

    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).

  9. Selected segments of cryo-EM density.
    Extended Data Fig. 5: Selected segments of cryo-EM density.

    aj, 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 (al) 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).

  10. Atomic model of NOMPC.
    Extended Data Fig. 6: Atomic model of NOMPC.

    ad, 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.

  11. Pore profile and channel properties of NOMPC and NOMPC mutants.
    Extended Data Fig. 7: Pore profile and channel properties of NOMPC and NOMPC mutants.

    a, Pore radius calculated by the CAVER program. b, IV 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.

  12. 3D classification of NOMPC-nanodisc particles.
    Extended Data Fig. 8: 3D classification of NOMPC-nanodisc particles.

    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.

  13. Flexibility of AR domains and model of NOMPC mechanogating.
    Extended Data Fig. 9: Flexibility of AR domains and model of NOMPC mechanogating.

    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.

  14. Sequence alignment of NOMPC orthologues.
    Extended Data Fig. 10: Sequence alignment of NOMPC orthologues.

    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.

Videos

  1. Class 1 to class 2 transition
    Video 1: Class 1 to class 2 transition
    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.
  2. Class 1 to class 3 transition
    Video 2: Class 1 to class 3 transition
    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.
  3. Class 2 to class 3 transition
    Video 3: Class 2 to class 3 transition
    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.

Accession codes

Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

Referenced accessions

Protein Data Bank

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Author information

  1. These authors contributed equally to this work.

    • Peng Jin &
    • David Bulkley

Affiliations

  1. Department of Physiology, University of California, San Francisco, California 94158, USA

    • Peng Jin,
    • Yanmeng Guo,
    • Wei Zhang,
    • Zhenhao Guo,
    • Shan Meltzer,
    • Tong Cheng,
    • Lily Yeh Jan &
    • Yuh-Nung Jan
  2. Department of Biochemistry and Biophysics, University of California, San Francisco, California 94158, USA

    • David Bulkley,
    • Lily Yeh Jan,
    • Yuh-Nung Jan &
    • Yifan Cheng
  3. Howard Hughes Medical Institute, University of California, San Francisco, California 94158, USA

    • Wei Zhang,
    • Shenping Wu,
    • Tong Cheng,
    • Lily Yeh Jan,
    • Yuh-Nung Jan &
    • Yifan Cheng
  4. Department of Cellular and Molecular Pharmacology, University of California, San Francisco California 94158, USA

    • Walter Huynh

Contributions

P.J. and D.B. designed and performed most biochemical and cryo-EM experiments. Y.G. performed electrophysiology experiments, and W.Z. performed mutagenesis and initial electrophysiology studies of NOMPC. Z.G. assisted with biochemical experiments. W.H. performed microtubule-binding assay. S.W. helped with cryo-EM experiments. S.M. performed surface staining, T.C. performed mutagenesis. L.Y.J., Y.-N.J. and Y.C supervised experiments and data analysis. P.J., D.B., L.Y.J., Y.-N.J. and Y.C. wrote the manuscript. All authors contributed to manuscript preparations.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to:

Reviewer Information Nature thanks E. McCleskey, K. Vinothkumar and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Verification of recombinant NOMPC activity. (823 KB)

    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.

  2. Extended Data Figure 2: Negative-stain analysis of purified NOMPC. (676 KB)

    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.

  3. Extended Data Figure 3: Cryo-EM analysis of NOMPC in amphipol. (714 KB)

    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.

  4. Extended Data Figure 4: Cryo-EM analysis of NOMPC reconstituted in nanodisc. (836 KB)

    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).

  5. Extended Data Figure 5: Selected segments of cryo-EM density. (889 KB)

    aj, 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 (al) 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).

  6. Extended Data Figure 6: Atomic model of NOMPC. (583 KB)

    ad, 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.

  7. Extended Data Figure 7: Pore profile and channel properties of NOMPC and NOMPC mutants. (402 KB)

    a, Pore radius calculated by the CAVER program. b, IV 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.

  8. Extended Data Figure 8: 3D classification of NOMPC-nanodisc particles. (470 KB)

    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.

  9. Extended Data Figure 9: Flexibility of AR domains and model of NOMPC mechanogating. (652 KB)

    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.

  10. Extended Data Figure 10: Sequence alignment of NOMPC orthologues. (1,128 KB)

    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.

Supplementary information

Video

  1. Video 1: Class 1 to class 2 transition (771 KB, Download)
    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.
  2. Video 2: Class 1 to class 3 transition (759 KB, Download)
    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.
  3. Video 3: Class 2 to class 3 transition (889 KB, Download)
    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.

PDF files

  1. Supplementary Information (187 KB)

    This file contains a supplementary discussion and a table of the summary of cryo-EM data collection and model refinement.

Image files

  1. Supplementary Figure (156 KB)

    This file contains the uncropped SDS PAGE gel for extended data figure 2a.

Additional data