PIEZO channels respond to piconewton-scale forces to mediate critical physiological and pathophysiological processes1,2,3,4,5. Detergent-solubilized PIEZO channels form bowl-shaped trimers comprising a central ion-conducting pore with an extracellular cap and three curved and non-planar blades with intracellular beams6,7,8,9,10, which may undergo force-induced deformation within lipid membranes11. However, the structures and mechanisms underlying the gating dynamics of PIEZO channels in lipid membranes remain unresolved. Here we determine the curved and flattened structures of PIEZO1 reconstituted in liposome vesicles, directly visualizing the substantial deformability of the PIEZO1–lipid bilayer system and an in-plane areal expansion of approximately 300 nm2 in the flattened structure. The curved structure of PIEZO1 resembles the structure determined from detergent micelles, but has numerous bound phospholipids. By contrast, the flattened structure exhibits membrane tension-induced flattening of the blade, bending of the beam and detaching and rotating of the cap, which could collectively lead to gating of the ion-conducting pathway. On the basis of the measured in-plane membrane area expansion and stiffness constant of PIEZO1 (ref. 11), we calculate a half maximal activation tension of about 1.9 pN nm−1, matching experimentally measured values. Thus, our studies provide a fundamental understanding of how the notable deformability and structural rearrangement of PIEZO1 achieve exquisite mechanosensitivity and unique curvature-based gating in lipid membranes.
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The coordinates of the curved and flattened PIEZO1 structures derived from proteoliposomes were deposited in the Protein Data Bank under accessions 7WLT and 7WLU, respectively. The corresponding cryo-EM maps were deposited in the Electron Microscopy Data Bank (EMDB) under accessions EMD-32592 and EMD-32593, respectively.
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We thank H. Wang, N. Liu and J. Xu for providing the graphene grids; C. Yan for sharing the scripts for deep-2D; L. Wang, H. Zhou, X. Yao, X. Fan, J. Lei and X. Li for technical help; T. Zhao and X. Zhang for developing the Epicker software; the Beijing Frontier Research Center for Biological Structure and Beijing Advanced Innovation Center for Structural Biology for facility and financial support; and the Protein Preparation and Identification Facility at Technology Center for Protein Science in Tsinghua University for facility support. This work was supported by grant numbers 2021ZD0203301, 31825014, 32130049, 32021002, 31630090, 2016YFA0500402 and 2015CB910102 to B.X.; 31570730, 2016YFA0501102 and 2016YFA0501902 to X.L.; from either the Ministry of Science and Technology of the People’s Republic of China or the National Natural Science Foundation of China.
The authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Optimization of PIEZO1 proteoliposome reconstitution and cryo-EM sample preparation.
a, The 38-TM topological model of PIEZO1 showing the indicated structural domains. THU: Transmembrane Helical Unit. b, A trace of gel filtration of purified PIEZO1 proteins using the detergent C12E10. UV, ultraviolet. Dash lines indicate the peak fractions collected for subsequent studies. c, A negative staining EM image of PIEZO1 purified in C12E10. (Scale bar = 50nm). d, Cryo-EM images of PIEZO1 proteoliposomes using Bio-beads SM-2 for removing the indicated detergents. The white star indicates the aggregation. e and f, Cryo-EM images of PIEZO1 proteoliposomes using either dialysis to remove 2% glyco-diosgenin (GDN) (d) or SEC for decyl maltoside (DM) removal (e). g, Negative staining EM images of the indicated fractions after gradient centrifugation. DM solubilized soy lipid were either mixed with PIEZO1 proteins or the same volume of buffer. After detergent removal, samples were processed into an iodixanol-based gradient centrifugation. h, A Cryo-EM image of liposomes sticking on the carbon film. 4 µl liposome solution was applied to a holey Quantifoil Au grid with a standard 25 s glow discharge pre-treatment. After 30 s incubation, the grid was blotted and plunged into liquid ethane. The image was focused on the border between the hole and carbon film to exhibit the biased liposome distribution. i, 3D segmentation of a constructed tomogram showing PIEZO1 proteoliposomes clustered in the air-water interface instead of diffusion into the water. j, Distributions of PIEZO1 proteoliposomes on Quantifoil Au grids treated with different glow discharge time. The cryo-samples were prepared by multi-application method descried in Method, and prolonged glow-discharge time facilitated the abundant distribution of liposomes in the hole area suitable for cryo-EM imaging.
a, Cryo-EM micrograph of PIEZO1 proteoliposomes prepared on the graphene grid (Scale bar = 10nm). b, Cryo-EM micrograph of empty liposomes prepared on the Quantifoil Au grid (Scale bar = 10nm). c, Analyses of the radii of the PIEZO1-residing side and the opposite pole of the 2D averaged proteoliposome vesicles of varied sizes. The inner and outer bilayers are approximated with the red and yellow dashed circles that are based on the PIEZO1-residing pole and the opposite pole of the same vesicle, respectively.
Extended Data Fig. 3 Structural determination and analyses of the curved PIEZO1 structure derived from proteoliposomes.
a, 2D class averages of PIEZO1 proteoliposomes with a circular mask of 25 nm diameter, showing the cap domain located inside the vesicles. On the basis of the abruptly changed curvature, the red arrows might indicate the boundary between the PIEZO1-residing side and the opposite side lacking proteins. b, Local resolution maps of the curved PIEZO1 structure viewed from the top, side and bottom. c, Gold-standard Fourier shell correlation (FSC) curve of the indicated density map. The reported resolution was based on the FSC = 0.143 criteria. d, Side view of the 3D density map of the water-drop-shaped proteoliposome showing the resolved lipid bilayer and the embedded PIEZO1 at the indicated contour level. e, Cryo-EM map of the curved PIEZO1 structure derived from proteoliposomes at the contour level of 4 and 6. Compared to the density map resolved in detergent micelles (EMD: 6865) (f), the PIEZO1 map derived from proteoliposomes contains extra patches of densities in-between the blades and in parallel to the inner membrane layer. These densities are noticeable even at a high contour level of 4 and consist of the modeled α1-helix of the clasp domain (Claspα1), the α-helix preceding TM33 (TM33pre-α1), and potentially unmodeled intracellular loop regions that are stabilized by interacting with lipid layers. This layer of membrane-parallel structure might help to stabilize the bowl-shaped structure of PIEZO1 in lipid membranes. f, Cryo-EM map of the PIEZO1 structure derived from detergent micelles (EMD 6865) at the contour level of 6. g, The cartoon model of the curved PIEZO1 structure derived from proteoliposomes. The featured structural domains are labeled. The resolved blade lipids and pore lipids are shown in blue and green, respectively. h, Pore radius along the central axis of the ion conduction pathway of the indicated PIEZO1 structures derived from either detergent micelles (5Z10 and 6B3R) or the curved structure derived from proteoliposomes. The residues forming the TM gate and intracellular constriction neck are labeled. i, Surface presentation of the curved PIEZO1 structure colored based on lipophilicity, from gold (lipophilic) to blue (hydrophilic) and surface presentation of the blade lipids and the pore lipid enclosed in the dashed box, which is enlarged in the right box either in surface presentation or in cartoon model. The lateral portal is indicated by the red dashed box.
Extended Data Fig. 4 Flowchart of EM data processing of the proteoliposomes with PIEZO1 being reconstituted in the outside-in configuration.
Details of data processing were described in the Imaging processing part of Methods.
Extended Data Fig. 5 Local EM density of the indicated domains of the curved PIEZO1 structure derived from proteoliposomes.
a, The helices are shown in cartoon representation with side chains as sticks. The cryo-EM density is shown as gray mesh. b, A side view of the curved PIEZO1 structure showing the blade lipid densities at the boundary of neighboring THU6 to THU9. c and d, A side view of the OH-IH enclosed central pore module showing the pore lipid densities either in the curved (c) or the flattened state (d).
Extended Data Fig. 6 Structural determination and analyses of the flattened PIEZO1 structure derived from proteoliposomes.
a, 2D class averages of the D-shaped PIEZO1 proteoliposome vesicles with a circular mask of 28 nm diameter. b, Gold-standard Fourier shell correlation (FSC) curve of the flattened PIEZO1 structure. The reported resolution was based on the FSC = 0.143 criteria. c, Local resolution maps of the flattened PIEZO1 structure viewed from the top, side and bottom. d, The alphaFold2 predicted PIEZO1 structure with the wedge domain shown in orange. e, The indicated views of the cartoon model of the flattened PIEZO1 structure. The featured domains are labeled. f, A bottom view of the overlaid curved and flattened cryo-EM maps showing the difference of the central plug density. g, A transparent view of the cryo-EM density of the lateral plug and latch domain showing the density variation of the lateral plug between 3D classified structures. h, Hydrophobic surface presentation of the flattened PIEZO1 structure and surface presentation of the pore lipid enclosed in the dashed box, which is enlarged in the right box either in the hydrophobic surface presentation or in cartoon model.
Extended Data Fig. 7 Flowchart of EM data processing of the proteoliposomes with PIEZO1 being reconstituted in the outside-out configuration.
Details of data processing were described in the Imaging processing part of Methods.
a, Top view of the superimposed PIEZO1 structures as indicated. For clarity, the extracellular cap and loops are omitted. The displacement of the THU4 is labeled. b, Top view of the superimposed central pore region of the curved and flattened PIEZO1 structures (salmon red and cyan, respectively). The wedge domain of the flattened structure is shown in orange. c, Top view of the superimposed cap of the curved and flattened PIEZO1 structures (salmon red and cyan, respectively) and the PIEZO2 structure (PDB: 6KG7) (gray). The displacement of the Capα1-helix domain is labeled. d, A bottom view of the superimposed central pore of the curved and flattened structures of PIEZO1 showing the unchanged residues that form the intracellular constriction neck.
Expansion of the PIEZO1 channel. Top view of the structural rearrangement from the curved to the flattened state of PIEZO1.
Flattening of the PIEZO1 channel. Side view of the structural rearrangement from the curved to the flattened state of PIEZO1.
Gating of the PIEZO1 channel. Top view of the central pore dilation from the curved to the flattened state of PIEZO1.
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Yang, X., Lin, C., Chen, X. et al. Structure deformation and curvature sensing of PIEZO1 in lipid membranes. Nature 604, 377–383 (2022). https://doi.org/10.1038/s41586-022-04574-8