Piezo1 and Piezo2 are mechanically activated ion channels that mediate touch perception, proprioception and vascular development. Piezo proteins are distinct from other ion channels and their structure remains poorly defined, which impedes detailed study of their gating and ion permeation properties. Here we report a high-resolution cryo-electron microscopy structure of the mouse Piezo1 trimer. The detergent-solubilized complex adopts a three-bladed propeller shape with a curved transmembrane region containing at least 26 transmembrane helices per protomer. The flexible propeller blades can adopt distinct conformations, and consist of a series of four-transmembrane helical bundles that we term Piezo repeats. Carboxy-terminal domains line the central ion pore, and the channel is closed by constrictions in the cytosol. A kinked helical beam and anchor domain link the Piezo repeats to the pore, and are poised to control gating allosterically. The structure provides a foundation to dissect further how Piezo channels are regulated by mechanical force.
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We thank H. Turner and G. Ozorowski for assistance with electron microscopy data collection. We acknowledge early efforts to characterize Piezo1 using electron microscopy by E. Wilson-Kubalek and R. Milligan, and S. Kakuda for screening purification conditions. We thank A. Sobolevsky for critical reading of the manuscript, and members of the Ward and Patapoutian laboratories for discussion. This work was supported by a Ray Thomas Edwards Foundation grant to A.B.W. and National Institutes of Health (NIH) grants NS083174 and DE022358 to A.P. Computational analyses were performed using shared instrumentation funded by NIH 1-S10OD021634. A.P. is an investigator of the Howard Hughes Medical Institute. This is manuscript 29582 from The Scripps Research Institute.
The authors declare no competing financial interests.
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Extended data figures and tables
a, Preparative gel filtration chromatogram of mouse Piezo1 after affinity purification and proteolytic removal of the GST tag. b, SDS–PAGE analysis of Piezo1 following affinity purification (pre-SEC) and after subsequent size-exclusion chromatography (SEC) steps (SEC fractions). Purifications of Piezo1 have been repeated more than three times with similar results. c, Aligned micrograph of purified Piezo1 embedded in a thin layer of vitrified ice. Scale bar, 100?nm. d, Representative 2D classes of Piezo1 showing different particle orientations.
a, Data processing flow chart. b, Local resolution maps calculated by the locres program in RELION 2.0. Colour key for local resolution (in Å) is shown. c, Angular distributions of the particles after the final step of refinement in RELION. The radius of the sphere is proportional to the number of particles with a given orientation. Only one-third of the sphere is shown due to applied C3 symmetry. d, FSC plots calculated using relion_postprocess for unmasked maps and maps with a soft mask applied to remove the contribution of scattered detergent density.
a, Duplicate unsharpened maps of Piezo1 refined without symmetry imposed, with the blue map rotated approximately 120° around the central axis of pseudosymmetry relative to the pink map, then superimposed with the ‘fit in map’ function of UCSF Chimera. The left panel shows top view, while the right panel shows a horizontal slice through the transmembrane region. b, Flow chart of propeller blade-focused classification and refinement procedure. c, Local resolution maps calculated by the locres program in RELION 2.0. Colour key for local resolution (in Å) is shown and is the same as Extended Data Fig. 2b. d, Angular distributions of the particles after the final step of refinement in RELION. The radius of the sphere is proportional to the number of particles with a given orientation. e, FSC plots calculated using relion_postprocess for unmasked maps and maps with a soft mask applied to remove the contribution of scattered detergent density.
Select regions of the molecular model are shown as yellow cartoon, with superimposed electron density as blue mesh. The density is derived from the C3-symmetry core masked map for the top two rows, and derived from blade class 1 for the bottom row.
Extended Data Figure 5 Structural comparisons of current and previous (EMDB 6343) mouse Piezo1 structures.
For each panel, the ‘fit in map’ function of UCSF Chimera was used to align uncropped maps. In a–e, unsharpened map of C3 refinement is shown. a–c, Top (a), bottom (b) and side (c) views of current Piezo1 (green) and EMDB accession EMD-6343 (grey) structurally aligned. d, Expanded top view of the cap domain to illustrate an approximately 15° rotation of the cap between the two maps. e, Expanded sliced top view of pore-proximal transmembrane region. Subtle conformational rearrangements are present between the current and previous structures in the inner and outer helices. f, g, Top and side views of EMDB accession EMD-6343 (grey), blade class 1 (blue), and blade class 2 (orange). The trajectories of the propeller blades are broadly similar across the maps in both the membrane-parallel and membrane-normal directions.
a, Top view of Piezo1 depicted as cartoon, with amphipathic helices from Piezo repeats A, B and C shown in red. Extracellular cap domain is omitted for clarity. b–d, Ribbon models of amphipathic helices from Piezo repeat A (b), Piezo repeat B (c), and Piezo repeat C (d). Cα positions are shown as spheres and hydrophobic amino acids are coloured grey, basic amino acids are coloured blue, acidic amino acids are coloured red, and polar uncharged amino acids are coloured yellow. e, Cartoon model of a portion of the Piezo1 propeller blade, with Piezo repeats coloured separately. A strong density peak putatively representing a lipid head group is shown as a pink mesh. f, Expanded view of the putative lipid binding site sandwiched between Piezo repeats B and C (helices B4 and C1). Electron density (blue mesh) is superimposed onto the molecular model, and the putative lipid head group is shown as pink mesh. Side chains contributing to the binding pocket are shown as yellow sticks and labelled.
a, HEK293T cells deficient in Piezo1 (P1KO) that express the Piezo1 double mutant M2493A/F2494A do not show discernible currents activated by indentation with blunt pipette (left) or stretch (right). b, Fluorescence-detection size-exclusion chromatography traces of untransfected HEK293F cells (grey) or cells expressing C-terminal tdTomato fusions of wild-type Piezo1 or double mutant M2493A/F2494A injected into Superose 6 increase column. Similar to the wild type, the double mutant displays a single dominant peak eluting shortly after void volume, indicating proper trimeric expression. Data are from one independent experiment. c, Representative images of Myc labelling in HEK293T-P1KO cells transfected with Piezo1–IRES-GFP (top), Piezo1–Myc–IRES-GFP (middle) or Piezo1(M2493A/F2494A)–Myc–IRES-GFP (bottom). GFP, green fluorescent protein. IRES, internal ribozyme entry site. Myc tags were inserted at position 897. Immunostaining was performed before (left) or after (right) cell permeabilization. Piezo1(M2493A/F2494A)–Myc is labelled at the surface in live cells similar to Piezo1–Myc, indicating that surface expression is preserved in the non-functional mutant. Scale bar, 10?μm. Experiments were repeated three times with reproducible results. d, Left, representative traces of stretch-activated single channel currents recorded (−80?mV) from HEK293T-P1KO cells expressing wild-type, M2493A or F2494A Piezo1 channels (traces displayed after applying 1?kHz digital filter). The corresponding pressure stimulus used to elicit the response is illustrated above the current trace. For each condition, the amplitude histogram for the corresponding trace is depicted on the right. The Gaussian fit for the closed (black curve) and open (red curve) components is overlaid on the histograms. e, Left, average I–V relationship of stretch-activated single-channel currents from wild-type (n?=?4), M2493A (n?=?5) or F2494A (n?=?4) Piezo1 channels. Amplitude was measured as a difference in Gaussian fits of full-trace histograms. Right, mean unitary conductance calculated from the slope of linear regression line fit to individual cells in each condition. **P?<?0.01, one-way ANOVA with Dunn’s comparison. f, Mean I–V relationship curves of mechanically activated currents recorded from wild-type (n?=?6), M2493A (n?=?8) or F2494A (n?=?9) Piezo1 channels with 150?mM CsCl-based intracellular solution and 100?mM CaCl2-based extracellular solution. Currents were elicited from −69.6 to 50.4?mV (Δ20?mV). Inset, expanded view of curves between 5 and 15?mV. Values are mean?±?s.e.m. g, Mean reversal potential from individual cells; Piezo1: 8.8?±?1.3?mV (n?=?6), M2493A: 10.2?±?1.2?mV (n?=?8) and F2494A: 13.5?±?0.5?mV (n?=?9). **P?=?0.0063, one-way ANOVA with Dunn’s multiple comparison test. N denotes X individual cells.
Sequence alignment of Piezo orthologues. Amino acid sequence alignment of selected regions of mouse Piezo1 (mPiezo1; UniProt E2JF22), human Piezo1 (hPiezo1; UniProt Q92508), mouse Piezo2 (mPiezo2, UniProt Q8CD54), human Piezo2 (hPiezo2, UniProt Q9H5I5), fruit fly Piezo (dmPiezo, UniProt M9MSG8). Structural features are annotated above the mPiezo1 sequence. Unmodeled regions are depicted as dotted lines. (PDF 4081 kb)
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Saotome, K., Murthy, S., Kefauver, J. et al. Structure of the mechanically activated ion channel Piezo1. Nature 554, 481–486 (2018). https://doi.org/10.1038/nature25453
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