Force-induced conformational changes in PIEZO1


PIEZO1 is a mechanosensitive channel that converts applied force into electrical signals. Partial molecular structures show that PIEZO1 is a bowl-shaped trimer with extended arms. Here we use cryo-electron microscopy to show that PIEZO1 adopts different degrees of curvature in lipid vesicles of different sizes. We also use high-speed atomic force microscopy to analyse the deformability of PIEZO1 under force in membranes on a mica surface, and show that PIEZO1 can be flattened reversibly into the membrane plane. By approximating the absolute force applied, we estimate a range of values for the mechanical spring constant of PIEZO1. Both methods of microscopy demonstrate that PIEZO1 can deform its shape towards a planar structure. This deformation could explain how lateral membrane tension can be converted into a conformation-dependent change in free energy to gate the PIEZO1 channel in response to mechanical perturbations.

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Fig. 1: Proposed activation mechanisms of PIEZO1.
Fig. 2: Reconstitutions of PIEZO1 in vesicles exhibit various orientations in cryo-EM micrographs.
Fig. 3: PIEZO1 channels become flatter in large vesicles.
Fig. 4: HS-AFM experiments of PIEZO1.
Fig. 5: Mechanical response of PIEZO1 to applied force.

Data availability

Any data relating to the findings presented in this Article are available from the corresponding authors upon reasonable request.


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We thank M. Ebrahim and J. Sotiris at the Evelyn Gruss Lipper Cryo-EM Resource Center of Rockefeller University for assistance with cryo-EM data collection. Y.R.G. is a Howard Hughes Medical Institute Fellow of the Damon Runyon Cancer Research Foundation (DRG 2317-18). R.M. is an investigator in the Howard Hughes Medical Institute.

Author information




Y.-C.L., Y.R.G., R.M. and S.S. designed the study; Y.R.G. and J.L. purified and reconstituted the protein. Y.R.G. performed cryo-EM measurements. Y.-C.L. and A.M. performed HS-AFM experiments. Y.-C.L., Y.R.G., R.M. and S.S. analysed the data. Y.-C.L., Y.R.G., R.M. and S.S. wrote the paper.

Corresponding authors

Correspondence to Roderick MacKinnon or Simon Scheuring.

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The authors declare no competing interests.

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Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Peer review information Nature thanks Ardem Patapoutian, Victor Shahin and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Fig. 1 Architecture and topology of the mechanosensitive channel PIEZO1.

a, Top, bottom and side views of PIEZO1 (PDB 6B3R) in cartoon representation (top) and embedded in the micelle density map (EMD-7042), contoured at 6σ. CTD, C-terminal domain. b, Top, topology of PIEZO1 shown rainbow-coloured (with N terminus in blue and C terminus in red), except for the structurally unsolved TM1–TM12 regions (shown in grey). Helices are represented as cylinders, loops as solid lines and unresolved regions as dotted lines. Bottom, top view of transmembrane helices, labelled as in the topology. Red squares outline four-transmembrane units that constitute the arm. TM21–TM24 are at the ‘elbow’ of the arm. The hypothetical position of the unresolved units TM1–TM4, TM5–TM8 and TM9–TM12 are indicated (dashed outline).

Extended Data Fig. 2 Image processing procedure to determine the Rc of side-view PIEZO1 channels, and the intrinsic Rc of the vesicles in which they are embedded.

a, The input 2D-averaged image of one side of the vesicle (here the PIEZO1 occupied side). b, Edge detection output using the Canny method. c, Edge detection output overlaid with the selection polygon. d, Edges in the selected polygon region annotated with numbers in different colours for easier identification. e, f, Measured (blue circle) and fitted (red dotted line) circles of the edges corresponding to the outer (e) and inner (f) boundaries of the vesicle membrane. Centre coordinates, the radius and its 95% confidence interval are shown. The unit of all values is in pixels. g, Fitted circles (red dashed lines) overlaid onto the edge detection output. Radii with the confidence interval of outer and inner boundaries are shown in units of nm. h, Fitted circles (red dashed lines) overlaid onto the input 2D-averaged image.

Extended Data Fig. 3 Estimation of Fts in HS-AFM at various Aratio, on the basis of experimental tip motion analysis and numerical simulation.

a, b, Average tip motions observed at different Aratio in buffer solution during HS-AFM imaging on mica (a) and a supported lipid bilayer (1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC):DOPS, 4:1) (b). c, d, Forces caused by the elastic response of the cantilever (top), the hydrodynamic damping with the medium (middle) and the total force that governs the tip motion (bottom). e, f, Sum of the drive force and tip-sample interaction force. g, h, Reconstructed Fts trajectories during a single oscillation cycle, based on the point-mass model (equation (7), equation (8), Methods). i, Comparison between peak forces obtained from reconstructed Fts trajectories on mica (blue dashed line) and membrane (red dashed line), and peak forces simulated using different surface stiffness using VEDA38 (dashed lines and grey shadowed area). The VEDA simulation is performed by using the amplitude-modulation-approach curve tool with the following settings: discrete approach steps within a defined z-range, acoustic excitation, Afree = 2 nm, Hertz contact model (Etip = 130 GPa, νtip = 0.3, νsample = 0.5, ν is the Poisson’s ratio) with a tip radius of 1 nm, and other HS-AFM experimental parameters (for example, k, Q and ω0). j, Comparison between average forces obtained from reconstructed Fts trajectories on mica (blue dashed line) and membrane (red dashed line) and values calculated through equation (1) (thick black dashed line). k, Comparison between peak forces obtained from experimental Fts trajectories on membrane at different Afree values. Using second-order polynomial fitting, the peak force reconstructed in the condition of Afree = 1.5 nm can be well-described by y = −688.7x2 + 633.4x + 55 (black dashed line) with R2 = 0.99. This fitting allows us to estimate the upper bound of force (peak force) applied to PIEZO1 channels at any given Aratio. Tip trajectories are representative of ≥5 independent experiments using ≥3 different HS-AFM cantilevers.

Extended Data Fig. 4 Classification of PIEZO1 channels using cross-correlation analysis on characteristic dimensions measured upon force application.

a, b, Cross-correlation density maps of smallest halo radius (Rmin) at low applied force versus largest halo radius (Rmax) at highest applied force (a) and of smallest halo radius (Rmin) versus maximum central height (Hmax) at lowest applied force (b). Along the diagonal direction of the cross-correlation density maps, the molecules separate into two peaks. This suggests the existence of two subtypes of PIEZO1 with different sizes in HS-AFM force-sweep movies. Therefore, we assigned the molecules in the peaks as type-1 (about 70%, white dashed circle) and type-2 (about 30%, yellow dashed circle) PIEZO1, respectively. The same molecules populate in the same subtype according to all analysed criteria. The total number of analysed PIEZO1 particles is 143, from 11 HS-AFM movies acquired on ≥5 different samples, days and HS-AFM tips.

Extended Data Fig. 5 Mechanical response of type-2 PIEZO1 to applied force.

a, HS-AFM image at \(\left\langle {F}_{{\rm{HS-AFM}}}\right\rangle \) of about 52 pN, of type-2 PIEZO1 viewed from the extracellular face. b, Height section profile (top) and radial height profile (bottom) of the topography displayed in a. c, Dimensional analysis of single type-2 PIEZO1 particle in a force-sweep HS-AFM experiment. Similar to the type-1 PIEZO1 (as reported in the main text), each single molecule (left) is 360-fold-symmetry averaged (centre) and a kymograph (right) across the centre profile (dashed line in 360-fold image) is calculated. The kymograph highlights the halo expansion (dashed line) as a function of force. Bottom, force as function of frame acquisition or time during the force-sweep HS-AFM movie acquisition. The yellow coloured area corresponds to the image acquisition of the particle shown (left). d, Normalized probability density maps of halo radius (R) as a function of force. Type-2 PIEZO1 also shows structural reversibility. The total number of analysed type-2 PIEZO1 particles is 43, from 11 HS-AFM movies acquired on ≥5 different samples, days and HS-AFM tips.

Extended Data Fig. 6 PIEZO1 has an exceptional low 2D density of transmembrane helices.

Transmembrane-helix 2D-density analysis for channels and transporters of known 3D structure. The transmembrane-helix density is estimated by the total number of transmembrane helices divided by the occupied membrane area of each known structure. A total of 201 channels and transporters structures have been analysed. A general trend seems to be that the channels are somewhat less densely packed than transporters, which is possibly a signature of the existence of a protein-free pore region. The PIEZO1 structure is an outlier: it is of exceptional size and has an exceptionally low density of transmembrane helices, (less than 0.2 of a helix per square nanometre. This unique feature might be a signature, and prerequisite for PIEZO1 mechanosensing.

Supplementary information

Supplementary Information

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Reporting Summary

Video 1

HS-AFM force-sweep video of the extracellular face of Piezo1 channels undergoing reversible conformational changes under force application. The top right molecule is the molecule shown in the middle panel in Fig. 4c. The bottom graph displays the calculated <FHS-AFM> during video acquisition. The HS-AFM video was recorded at 187 nm x 187 nm image size (280 x 280 pixels) and 1.0 s per frame.

Video 2

HS-AFM force-sweep video of Piezo1 channels exposed to a reverse force cycle. The bottom graph displays the calculated <FHS-AFM> (red), Aset/Afree ratio (blue) and the Z-piezo displacement (green) as a function of frame acquisition or time during the force-sweep HS-AFM video acquisition shown in Fig. 5a. The HS-AFM video was recorded at 350 nm x 350 nm image size (350 x 350 pixels) and 1.0 s per frame.

Video 3

Example of lateral expansion analysis of a single Piezo1 particle shown in Fig. 5b. The single molecule (left) is 360-fold symmetry averaged (middle) and a kymograph (right) across the center of the Piezo1 calculated (dashed line in 360-fold image). The kymograph highlights the outer radius (halo) expansion as a function of force application.

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Lin, Y., Guo, Y.R., Miyagi, A. et al. Force-induced conformational changes in PIEZO1. Nature 573, 230–234 (2019).

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