WO2021016941A1

WO2021016941A1 - Utilizing structure and mechanogating mechanism of piezo channels for preparation of medicaments and technologies

Info

Publication number: WO2021016941A1
Application number: PCT/CN2019/098698
Authority: WO
Inventors: Bailong XIAO; Xueming LI; Li Wang; Heng ZHOU; Mingmin Zhang; Wenhao Liu
Original assignee: Tsinghua University
Priority date: 2019-07-31
Filing date: 2019-07-31
Publication date: 2021-02-04

Abstract

Utilizing the newly determined three-dimensional structure of Piezo2 and the revealed key structural domains, residues and mechnogating mechanisms critical for modulating the activity of the Piezo channels for developing Piezo channel-targeted medicine and technology.

Description

[Title established by the ISA under Rule 37.2] UTILIZING STRUCTURE AND MECHANOGATING MECHANISM OF PIEZO CHANNELS FOR PREPARATION OF MEDICAMENTS AND TECHNOLOGIES

FIELD

The present disclosure relates to the development of Piezo channel-targeted medicine and technology on the basis of the newly determined three-dimensional structure of Piezo2 and the revealed key structural domains and mechnogating mechanisms critical for modulating the activity of the Piezo channels.

BACKGROUND

The evolutionarily conserved Piezo family of proteins, including Piezo1 and Piezo2, has been established as the long-sought mammalian Mechanosensitive (MS) cation channels. Piezo1 mediates mechanically activated currents in various cell types, including endothelial cells, red blood cells, smooth muscle cells and epithelial cells. Studies of Piezo1 knockout mice have revealed its critical roles in a broad range of mammalian physiologies involving mechanotransduction. For instance, Piezo1 serves as a key mechanotransduction channel for converting flow-associated shear stress into cation permeation in the vascular and lymphatic systems, which controls blood and lymphatic vessel development, arterial remodeling, blood pressure regulation and red blood cell volume homeostasis. Furthermore, together with its homologue Piezo2, Piezo1 has been identified as the long-sought mechanosensor in baroreceptor neurons for baroreflex control of blood pressure and heart rate. In line with its physiological importance, either gain-or loss-of-function mutations in the human Piezo1 gene have been linked to hereditary xerocytosis or congenital lymphatic dysplasia, respectively.

Piezo2 mediates mechanically activated and rapidly inactivating cationic currents in primary sensory neurons. Studies of Piezo2 knockout mice or human patients carrying loss-of-function mutations in the Piezo2 gene have unequivocally demonstrated its essential role in sensing gentle touch, tactile pain, proprioception, airway stretch and lung inflation, as well as in baroreceptors for sensing blood pressure and regulating heart rate. Furthermore, gain-of-function mutations in human Piezo2 gene have been associated with distal arthrogryposis 5 (DA5) , a genetic disorder characterized by severe joint contractures and restrictive lung diseases.

Despite its physiological and pathophysiological significance and validated potential as a drug target for developing novel therapeutics such as analgesics for treating tactile pain, the structural basis and molecular mechanisms underlying the ability of Piezo2 to serve as an effective mechanically activated cation channel remain unknown. Mammalian Piezo2 proteins are large membrane proteins containing over 2800 residues and only share ～42%of sequence homology with Piezo1, the other member of the Piezo channel family, which has about 2500 residues and forms a remarkable three bladed, propeller-like homotrimeric structure comprising a central ion-conducting pore module and three peripheral blades with 24 resolved transmembrane helices (TM) folded into 6 repeated transmembrane helical units (THUs) . While both Piezo1 and Piezo2 sufficiently form mechanically activated cation channels, they differ in tissue expression, physiological function and biophysical properties, including inactivation kinetics, responsiveness to stretch-evoked or chemical-induced activation.

SUMMARY

Embodiments of the present disclosure seek to solve at least one of the problems existing in the prior art to at least some extent, or to provide a consumer with a useful commercial choice.

Here combining recombinant protein expression, purification, single-particle cryo-electron microscopy (cryo-EM) and electrophysiology, the inventors have for the first time determined the homo-trimeric structure of the full-length 2822-residue mouse Piezo2 to a resolution of

resulting in revelation of a total of 114 resolved TMs (38 in each protomer) and identification of the closed transmembrane and cytosolic gates along the central pore. TM1-36 are folded into nine tandem four-TM helical units (THU) to form the unusual non-planar Blade, three of which are curved into a nano-bowl shape of 28 nm-diameter and 10 nm-depth, with the extracellular Cap-like structure embedded in the center. Surrounded with the Anchor domain and TM37, TM38 and the C-terminal domain enclose the central pore with closed transmembrane and cytoplasmic gates. Importantly, structural comparison between Piezo2 and Piezo1, which has a dilated transmembrane pore, has allowed us for the first time to identify the transmembrane gate and analyze the specific mechanogating mode that might lead to opening of the transmembrane gate. Specifically, the transmembrane and cytosolic gates might be respectively controlled by the top Cap and the non-planar Blade, which has a 9 nm-long Beam connecting to the cytoplasmic gate. Together, the studies have not only for the first time determined the structure of Piezo2 with a completely resolved 38-TM topology, but also provided important insights into the ion-permeating and mechanogating mechanism of the mechanically activated Piezo channel family. The Piezo2 structure will also serve as a solid structural foundation for charactering the human disease-causing mutations and developing Piezo channel-based novel therapeutics and technologyies.

According to embodiments of a first broad aspect of the present disclosure, there is provided use of regulatoror technologyin preparation of a medicament 、kit or technology for activating, potentiating or inhibiting themechanicallyactivated Piezo channels, wherein the regulator、kit or technologyis used for regulating the Cap domain, the 9 THU-constituted Blade domain, the Blade /Latch/Anchor/outer helix (OH) domains, the inner helix (IH) and the cytosolic C-terminal domain (CTD) that contain the identified transmembrane and cytosolic gates which allow cation permeation (IH-CTD-constituted pore domain) . According to the embodiments, the inventor found that surrounded with the Anchor domain and the OH (TM37) , the IH (TM38) and the CTD enclose the central pore with closed transmembrane and cytoplasmic gates. The inventors also found that specific motion modes of the Cap might lead to close or open of the transmembrane gate residing in the IH, while specific motion modes of the Blade, the Beam and its associated CTD/Latch/Anchor/OH components might collectively control the close or open of the cytosolic gate residing in the CTD. The regulator、kit or technology acting on the Cap-IH-transmembrane gate or the Blade/Beam/CTD/Latch/Anchor/OH-cytosolic gate to open or close the channel can be used in preparation of a medicament or technology for activating or inhibiting the mechanically activatedPiezo channels.

According to embodiments of present disclosure, the above mentioned use may possess at least one of the following additional features:

In some embodiments, wherein the Piezo is Piezo1 orPiezo2.

In some embodiments, wherein the Piezo is from mouse or human beings and their amino acid sequences are shown in Fig. 7.

In some embodiments, wherein regulating the close or open of the IH-CTD-constituted pore domain is achieved by acting onthe OH-Cap-IH or THU1-9-Beam-Latch-CTD-Anchor-OH-IH structural domains.

In some embodiments, wherein regulating the close or open of the IH-CTD-constituted pore domain is achieved by acting onat least one of the following sites or functional domains of Piezo: (1) the Cap structural domain; (2) IH-CTD domains; (3) THU1-THU2-THU3 of the Bladedomain; (4) residues L2743, V2750, K2753, F2754, E2757, G2761, M2767, P2801 or E2811 ofthe IH-CTD structural domains of mouse Piezo2 (Uniprot ID: Q8CD54) or the corresponding residues L2673, V2680, K2683, F2684, E2687, G2691, M2697, P2731 or E2741 of human Piezo2 (Uniprot ID: Q9H5I5) ; (5) residues L2469, V2476, K2479, F2480, G2483, E2487, M2493, P2536, E2537of the IH-CTD structural domains of mouse Piezo1 (Uniprot ID: E2JF22) or the corresponding residues L2443, V2450, K2453, F2454, G2457, E2461, M2467, P2510, E2511 of human Piezo1 (Uniprot ID: Q92508) .

In some embodiments, whereinthe medicament, kit or technology is used for inhibiting the mechanically-activated Piezo2 channel, wherein the regulator is used to induce conformational changes of the Cap, theIHdomain and the transmembrane gate comprising residues L2743, V2750, K2753, F2754, E2757, G2761 of mouse Piezo2 or the corresponding domains and residues of human Piezo2 to close the Piezo2 channel; or wherein the regulator、kit or technology is used to induce conformational changes of the Blade/Beam/Latch/Anchor/OH domains and the cytosolic gate comprising residues M2767, P2801 or E2811 of the CTD structural domains of mouse Piezo2 or the corresponding domains and residues of human Piezo2 to close the Piezo2 channel.

In some embodiments, wherein the medicament 、kit or technology is used for inhibiting the mechanically-activated Piezo1 channel, wherein the regulator is used to induce conformational changes of the Cap, theIHdomain and the transmembrane gate comprising residues L2469, V2476, K2479, F2480, G2483, E2487 of mouse Piezo1 or the corresponding domains and residues of human Piezo1 to close the Piezo1 channel; or wherein the regulator is used to induce conformational changes of the Blade/Beam/Latch/Anchor/OH domains and the cytosolic gate in the CTD domain comprising residues M2493, P2536, E2537 of mouse Piezo1 or the corresponding domains and residues of human Piezo1 to close the Piezo1 channel.

In some embodiments, wherein the medicament 、kit or technology is used for inhibiting the mechanically-activated Piezo2 channel, wherein theregulator is a small molecule, antibody, nano-material, mechanical force, light, temperature, ultrasound, or magnet; or oligonucleotide to induce deletion, insertion or mutations of the coding sequence of the Cap, the IH-CTD, the Blade/Beam/Latch/Anchor/OHdomains and residues L2743, V2750, K2753, F2754, E2757, G2761, M2767, P2801 or E2811 of the mouse Piezo2 or the corresponding domains and residues of human Piezo2.

In some embodiments, wherein the medicament , kit or technology is used for inhibiting the mechanically-activated Piezo1 channel, wherein theregulator is a small molecule, antibody, nano- material, mechanical force, light, temperature, ultrasound, or magnet; or oligonucleotide to induce deletion, insertion or mutations of the coding sequence of the Cap, the IH-CTD, the Blade/Beam/Latch/Anchor/OHdomains and residues L2469, V2476, K2479, F2480, G2483, E2487, M2493, P2536, E2537of the mouse Piezo1 or the corresponding domains and residues of human Piezo1.

In some embodiments, wherein the medicament or technology is used for inhibiting the mechanically-activated Piezo2 channel, wherein the medicament or technology is used for treating and preventing tactile pain (mechanical allodynia) , touch and proprioceptive defects, bone deformation, hypertension, respiratory diseases.

In some embodiments, wherein the medicament or technology is used for inhibiting the mechanically-activated Piezo1 channel, wherein the medicament or technology is used for treating and preventing hypertension, defects of blood vessel development, defects of lymphatic vessel development, red blood cell diseases including ammonia and malaria infection, bone loss, urinary dysfunction, cancer development and metastasis.

In some embodiments, wherein the medicament 、kit or technology is used for activating or potentiatingthe mechanically-activated Piezo2 channel, wherein the regulator is used to induce conformational changes of the Cap, theIHdomain and the transmembrane gate comprising residues L2743, V2750, K2753, F2754, E2757, G2761 of mouse Piezo2 or the corresponding domains and residues of human Piezo2 to activate or potentiate the Piezo2 channel; or wherein the regulator is used to induce conformational changes of the Blade/Beam/Latch/Anchor/OH domains and the cytosolic gate comprising residues M2767, P2801 or E2811 of the CTD structural domains of mouse Piezo2 or the corresponding domains and residues of human Piezo2 to activate or potentiate the Piezo2 channel.

In some embodiments, wherein the medicament , kit or technology is used for activating or potentiatingthe mechanically-activated Piezo1 channel, wherein the regulator is used to induce conformational changes of the Cap, theIHdomain and the transmembrane gate comprising residues L2469, V2476, K2479, F2480, G2483, E2487 of mouse Piezo1 or the corresponding domains and residues of human Piezo1 to activate or potentiate the Piezo1 channel; or wherein the regulator is used to induce conformational changes of the Blade/Beam/Latch/Anchor/OH domains and the cytosolic gate in the CTD domain comprising residues M2493, P2536, E2537 of mouse Piezo1 or the corresponding domains and residues of human Piezo1 to activate or potentiate the Piezo1 channel.

In some embodiments, wherein the medicament, kit or technology is used for activating or potentiatingthe mechanically-activated Piezo2 channel, wherein theregulator is a small molecule, antibody, nano-material, mechanical force, light, temperature, ultrasound, or magnet; or oligonucleotide to induce deletion or mutations of the coding sequence of the Cap, the IH-CTD, the Blade/Beam/Latch/Anchor/OHdomains and residues L2743, V2750, K2753, F2754, E2757, G2761, M2767, P2801 or E2811 of the mouse Piezo2 or the corresponding domains and residues of human Piezo2.

In some embodiments, wherein the medicament 、kit or technology is used for activating or potentiatingthe mechanically-activated Piezo1 channel, wherein theregulator is a small molecule, antibody, nano-material, mechanical force, light, temperature, ultrasound, or magnet; or oligonucleotide to induce deletion、insertionor mutations of the coding sequence of the Cap, the IH-CTD, the Blade/Beam/Latch/Anchor/OHdomains and residues L2469, V2476, K2479, F2480, G2483, E2487, M2493, P2536, E2537of the mouse Piezo1 or the corresponding domains and residues of human Piezo1.

In some embodiments, wherein the medicament or technologyis used for activating or potentiatingthe mechanically-activated Piezo2 channel, wherein the medicament or technology is used for treating and preventing tactile pain (mechanical allodynia) , touch and proprioceptive defects, hypertension, respiratory diseases.

In some embodiments, wherein the medicament or technology is used for activating or potentiatingthe mechanically-activated Piezo1 channel, wherein the medicament or technology is used for treating and preventing hypertension, defects of blood vessel development, defects of lymphatic vessel development, red blood cell diseases including ammonia and malaria infection, bone loss urinary dysfunction, cancer development and metastasis.

According to embodiments of a second broad aspect of the present disclosure, there is provided a method of screening for an agent that modulates the activity of the mechanically activated Piezo channel, the method comprising: (1) contacting candidate agents with cells expressing Piezo channels, wherein the cells are from mouse or human beings ; (2) detectingmodulation or conformational changes of key structural domains such as the Cap and the gates located in the IH and CTDof Piezo1 or Piezo2 before and after the contact of an agent; wherein thechange in conformation, activity or expressionof key structural domain changes indicates that the candidate agents act as the desired agents. The agent selected by the above method according to the embodiment can be used to effectively modulate the activity of the mechanically activated Piezo channel.

According to embodiments of present disclosure, the above mentioned method may possess at least one of the following additional features:

In some embodiments, wherein the Piezo is Piezo1 or Piezo2.

In some embodiments, wherein thecells are prepared as follows:

(1) preparation of the recombinant Piezo1 or Piezo2 proteins using the Piezo2-pp-GST-IRES-GFP or Piezo1-pp-GST-IRES-GFP expression plasmids, which include the mammalian expression promoter, cDNA encoding Piezo2 or Piezo1, a C-terminal Glutathione S-transferase (GST) tag or other affinity tag with a precision protease cleavage site in between, followed with the green fluorescent protein (GFP) -coding sequence driven by an internal ribosome entry site (IRES) , which is designated for monitoring the efficiency of transient transfection and protein expression; (2) the selection of the detergent glycol-diosgenin, a synthetic substitute for digitonin, for obtaining optimized purified Piezo2 or Piezo1protein samples.

In some embodiments, wherein the modulation or conformational changes ofPiezo1 or Piezo2 is observed using the cryo-electron microscopy.

In some embodiments, wherein the expression vector described above is heterologous to the cell.

In some embodiments, wherein the activity of the mechanically activated Piezo channel is determined by measuring the electrophysiological change.

In some embodiments, wherein the electrophysiological change is a change inmembrane potential, a change in current, or an influx of a cationincluding calcium.

In some embodiments, wherein the electrophysiological change is measured with a patch-clamp assay.

In some embodiments, wherein the activity of the mechanically activated Piezo channel is measured by the change of the membrane potential using amembrane potential dye assay.

In some embodiments, wherein the activity of the mechanically activated Piezo channel is determined by measuring the change of intracellular calcium using calcium-sensitive dye or genetically encoded sensors.

In some embodiments, wherein the cellsare eukaryotic cells.

In some embodiments, wherein the cellsare neuron.

In some embodiments, wherein the modulation of the mechanically activated Piezo channel is measured by determining the interaction of an agent with the Piezo proteins.

In some embodiments, wherein the modulation of the mechanically activated Piezo channel is indicated by conformational changes of the key structural domains of Piezo1 or Piezo2, including (1) the Cap domain; (2) the IH-CTD domains; (3) the THU-constituted Blade domain; (4) the Beam/CTD/Latch/Anchor/OH domains; (5) residues L2743, V2750, K2753, F2754, E2757, G2761, M2767, P2801 or E2811 of the IH-CTD structural domains of mouse Piezo2 or the corresponding domains and residues of human Piezo2, (6) residues L2469, V2476, K2479, F2480, G2483, E2487, M2493, P2536, E2537of the IH-CTD structural domains of mouse Piezo1 or the corresponding domains and residues of human Piezo1.

According to embodiments of a third broad aspect of the present disclosure, there is providedan antibody that binds and modulates the activity of the mechanically activated Piezo channel, including mouse and human Piezo1 and mouse and human Piezo2. wherein the antibody can specifically bind to polypeptide having at least 70%amino acid sequence identity the Cap, THU1 to THU9, The Beam, the Latch, the CTD, the Anchor and the IH to activate or potentiate or inhibit the opening of thePiezochannel.

According to embodiments of present disclosure, the above mentioned antibody may possess at least one of the following additional features:

In some embodiments, wherein the polypeptide comprises SEQ ID NO: 1～4.

In some embodiments, wherein the antibody recognize amino acids of the structural domains such as the CED domain with the following amino acid composition and those shown in Fig. 7.

The Cap of mouse Piezo2:

The Cap of human Piezo2:

The Cap of mouse Piezo1:

The Cap of human Piezo1:

In some embodiments, wherein the antibody is a monoclonal antibody.

In some embodiments, wherein the antibody is a humanized antibody.

In some embodiments, wherein the antibody is a chimeric antibody.

According to embodiments of a forth broad aspect of the present disclosure, there is providedan isolated antisense oligonucleotide, small interfering RNA (siRNA) , or guide RNA for CRISPR-Cas9-mediated genetic modifiationwherein the antisense oligonucleotide, siRNA or guide RNA inhibits or potentiates production or changes the activity of themouse and human Piezo1 and Piezo2, wherein the antisense oligonucleotide, siRNA or guide RNA described above can specificallytarget the Cap, IH, THU1-9, the Blade/Latch/CTD/Anchor/OH, residues L2743, V2750, K2753, F2754, E2757, G2761, M2767, P2801 or E2811 of the IH-CTD structural domains of mouse Piezo2 or the corresponding domains and residues of human Piezo2, and residues L2469, V2476, K2479, F2480, G2483, E2487, M2493, P2536, E2537of the IH-CTD structural domains of mouse Piezo1 or the corresponding domains and residues of human Piezo1.

In some embodiments, wherein the antisense oligonucleotide or siRNA targeting the homologous genetic regions of mouse or human Piezo1 or Piezo2 encoding the specified structural domains, such as the region encodingCap of mouse Piezo2 or the homologous region of human Piezo2 or mouse Piezo1 or human Piezo1, IH of mouse Piezo2 or the homologous region of human Piezo2 or mouse Piezo1 or human Piezo1, THU1 of mouse Piezo2 or the homologous region of human Piezo2 or mouse Piezo1 or human Piezo1, THU2 of mouse Piezo2 or the homologous region of human Piezo2 or mouse Piezo1 or human Piezo1, THU3 of mouse Piezo2 or the homologous region of human Piezo2 or mouse Piezo1 or human Piezo1.

The region encoding Cap of mouse Piezo2:

The region encoding IH of mouse Piezo2:

The region encoding THU1 of mouse Piezo2:

The region encoding THU2 of mouse Piezo2:

The region encoding THU3 of mouse Piezo2:

According to embodiments of a fifth broad aspect of the present disclosure, there is provided a vector comprising the expression cassette of the antisense oligonucleotide or siRNA described above.

According to embodiments of a sixth broad aspect of the present disclosure, there isprovided a method of treating Piezo related disease, comprise: administrating regulator to a subject in need thereof, wherein the regulator is used for regulating thekey structural domains of Piezo, wherein the key structural domains of Piezo including at least one of the following: Cap, IH, THU1-9, the Blade/Latch/CTD/Anchor/OH, residues L2743, V2750, K2753, F2754, E2757, G2761, M2767, P2801 or E2811 of the IH-CTD structural domains of mouse Piezo2 or the corresponding domains and residues of human Piezo2, and residues L2469, V2476, K2479, F2480, G2483, E2487, M2493, P2536, E2537of the IH-CTD structural domains of mouse Piezo1 or the corresponding domains and residues of human Piezo1. According to the embodiments, the regulator used for regulating thekey structural domains of Piezo can regulate the activity of the Piezochannel. Administrating the regulator to a subject in need thereof can effectively treat or prevent Piezo related diseases.

In some embodiments, wherein the Piezo related disease includes at least one of the following: dehydrated hereditary stomatocytosis, generalized lymphatic dysplasia, distal arthrogryposis type 5, Gordon syndrome and Marden-Walker syndrome, tactile pain, hypertension, respiratory diseases.

In some embodiments, wherein the regulator used for regulating key structural domains by acting on at least one of the following sites or functional domains of Piezo: (1) the Cap structural domain; (2) IH-CTD domains; (3) THU1-THU2-THU3 of the Bladedomain; (4) residues L2743, V2750, K2753, F2754, E2757, G2761, M2767, P2801 or E2811 of the IH-CTD structural domains of mouse Piezo2 or the corresponding domains and residues of human Piezo2; (5) residues L2469, V2476, K2479, F2480, G2483, E2487, M2493, P2536, E2537of the IH-CTD structural domains of mouse Piezo1 or the corresponding domains and residues of human Piezo1.

In some embodiments, wherein the regulator is antibody described above or isolated antisense oligonucleotide or siRNA or guide RNA described above.

The above summary of the present disclosure is not intended to describe each disclosed embodiment or every implementation of the present disclosure. The Figures and the detailed description more particularly exemplify the illustrative embodiments.

Additional aspects and advantages of embodiments of present disclosure will be given in part in the following descriptions, become apparent in part from the following descriptions, or be learned from the practice of the embodiments of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other aspects and advantages of embodiments of the present disclosure will become apparent and more readily appreciated from the following descriptions made with reference to the accompanying drawings, in which:

Fig. 1 shows the structure determination of Piezo2, wherein

a, Western blotting of cell lysates derived from HEK293T cells transfected with the indicated constructs. The anti-GST antibody detected the Piezo1-GST and Piezo2-GST proteins, while the anti-β-actin antibody detected β-actin for a loading control.

b, A representative trace of gel filtration of the purified full-length Piezo2 with the GST affinity tag cleaved in the GDN detergent. UV, ultraviolet.

c, Coomassie blue staining showing the purified Piezo2 proteins separated on a 8%SDS-PAGE.

d, A representative cryo-electron micrograph of Piezo2.

e, Power spectrum of the micrograph in d, with the

frequency indicated.

f, Representative 2D class averages of Piezo2 particles, showing the three-bladed propeller-like top view and a bowl-like side view.

g, Gold-standard Fourier shell correlation (FSC) curves of the indicated density maps. Reported resolutions were based on the FSC = 0.143 criteria.

h -k, The local resolution maps of the indicated density maps.

Fig. 2 shows overall architecture and a nano-bowl configuration of the Piezo2 trimer, wherein

a, The indicated view of the sharpened map (2.78 σ contour level) with a resolution of

with each subunit color-coded and the major domains labeled. The diameter

and height

of the trimeric structure, the height of the cap

and the outwardly curved TM blade

and the length of the beam

are labeled.

b, Cartoon models with the major structural domains labeled. The 36 TMs shown in the blade are numbered.

c, A side view of the surface electrostatic potential showing the unusual non-planar and curved hydrophobic transmembrane region (marked by dashed lines) with positively charged residues localized at the intracellular side, three of which might deform the residing membrane into a nano-bowl shape of 28 nm-diameter and 10 nm-depth as outlined by the approximated sphere, which produce a bowl-surface area of 880 nm ² and a projected in-plane area of 600 nm ².

d and e, top views zoomed in at either the top of the Cap (d) or the extracellular end of the outer helix (OH) -inner helix (IH) pair (e) to illustrate the non-planar configuration of the distal TM blade. The TMs are labeled .

Fig. 3 showsthe 38-TM topology and structure of the Piezo2 protomer, wherein

a, A 38-TM topology color-coded to match the cartoon models in b. The 9 repetitive THUs and major structural domains are labeled. Dashed lines indicate unresolved regions. EL: extracellular loop; OH: outer helix; IH: inner helix; CED: C-terminal extracellular domain; CTD: C-terminal domain.

b, Structure of a Piezo2 protomer presented as either ribbon diagram or cylindrical helices showing individual THUs and major structural domains. Dashed lines indicate unresolved regions;

Fig. 3shows structural organization of the central region, wherein

a and b, Side (a) or bottom (b) views of the trimeric central region comprising the proximal end of the Beam, Latch, Anchor and the OH-CED-IH-CTD pore module. Two subunits are presented in surface electrostatic potential, while the other subunit in ribbon diagram

c, A bottom view ofsurface representation showing organization of the indicated domains. The central purple dots indicate the constricted intracellular exit of the central pore.

d, An enlargedribbon representation showing positively and negatively charged residues that contribute to the negative surface potential of the Latch and positive surface potential of the Beam and CTD shown in panel b. Residue Y1568 from the Latch might form hydrogen bond with residue E2811 from the CTD that forms the constricted intracellular exit of the central pore.

e, An enlargedribbon representation showing the intertwined polar interactions of the Anchor-OH-linker, Anchor ^α2-3-turn, CTD ^α1-2-turn and the Latch domain. Polar residues contributing to the positive and negative surface potential shown in the boxed region of panel a are labeled. Several pairs of polar interactions are indicated in red dashed lines.

f, Ribbon representation showing a top view of the structural organization of the Anchor, OH, IH and CTD.

Figure 4 | Structural organization of the central region

e, An enlargedribbon representation showing the intertwined polar interactions of the Anchor-OH-linker, Anchor ^α2-3-turn, CTD ^α1-2-turn and the Latch domain. Polar residues contribute to the positive and negative surface potential shown in the boxed region of panel a are labeled. Several pairs of polar interactions are indicated in dashed lines.

f, Ribbon representation showing the structural organization of the Anchor, OH, IH and CTD. Residues important for Piezo2 functions are labeled.

Fig. 5 shows the ion-conducting pore of Piezo2 and Piezo1, wherein

a, Sequence alignment of the pore-lining IH-CTD domains between Piezo1 and Piezo2. Residues shown in salmon red and cyan represent the transmembrane and cytosolic gate residues, while those in red are lateral portal-lining residues.

b, Ribbon diagram showing the IH-CTD-encoded central pore of Piezo2 and that of the Piezo1 structure (PDB: 6b3r) . Residues shown in a are labeled. The central solvent-accessible pathway is marked with a dotted mesh generated by the program HOLE (pore radius:

) . The membrane vestibule (MV) and intracellular vestibule (IV) are labeled.

c, Pore radius along the central axis of the ion conduction pathway of the indicated Piezo2 and Piezo1 pore structures.

d, Side view of superimposed IH-CTD derived from the Piezo2 and Piezo1. The red arrow indicates the outward shift of the IH main chain of Piezo1 relative to that of Piezo2.

e -i, Top view of superimposed IH-CTD derived from the Piezo2 and Piezo1 zoomed in near the positions of the indicated gate residues. Red and purple arrows respectively indicate the position shift of either the IH main chain or the side chain of the gate residues of Piezo1 relative to that of Piezo2.

Fig. 6 shows structural comparison between Piezo1 and Piezo2, wherein

a, c, d, g, Top view of the superimposed Piezo2 and Piezo1 (PDB: 6b3r) structures zoomed in at either the CED ^{α1-α2 helix} (a) , the Piezo2-2743 gate (c) , the Piezo2-F2754 gate (d) , or the cytosolic gate Piezo2-M2767/P2810/E2811 (g) positions. The boxed regions are presented in enlarged views as indicated. Arrows indicate the displacement direction of Piezo1 relative to Piezo2. Red arrows indicate the displacement of the Cap or the Blade, purple arrows indicate the displacement of the side chain of the gate residues.

b and e, Side view of the superimposed Piezo2 and Piezo1 (PDB: 6b3r) (in cyan) trimmers (b) or monomers (e) . Arrows indicate the displacement direction of Piezo1 relative to Piezo2. The dashed lines in panel e indicate the flatness of the curved transmembrane region of the Blade.

f shows the upward displacement of the cytosolic and transmembrane gate residues.

h, Scatter plot of the maximal poking-induced currents in the Piezo1-KO-HEK cells transfected with the indicated constructs. The structural domain corresponding to CED ^{α1-α2 helix} is shown in the right panel of a. Each bar represents mean ± s.e.m., and the number of recorded cells are labeled. One-way ANOVA comparison to Piezo1 or Piezo2. ^****P<0.0001.

Fig. 7 shows amino acid sequences of Piezo homologous.

Secondary structural elements of Piezo2 are labeled above the sequence alignment.

mPiezo2: mouse Piezo2;

hPiezo2: human Piezo2;

mPiezo1: mouse Piezo1;

hPiezo1: human Piezo1;

dPiezo: drosophila Piezo.

DETAILED DESCRIPTION

Reference will be made in detail to embodiments of the present disclosure. The embodiments described herein with reference to drawings are explanatory, illustrative, and used to generally understand the present disclosure. The embodiments shall not be construed to limit the present disclosure. The same or similar elements and the elements having same or similar functions are denoted by like reference numerals throughout the descriptions.

As used herein, the following terms have the meanings ascribed to them unlessspecified otherwise.

In addition, terms such as “first” and “second” are used herein for purposes of description and are not intended to indicate or imply relative importance or significance.

In addition, terms “acting on” refer to but not limited to direct or indirect action, i.e., the transmission to the target site after action is also involved.

The term "mechanically-activated cation channel" refers to an ion channel that opens toallow passage of positively charged ions (i.e. cations) into and out of a cell in response tomechanical force or pressure being applied, e.g., to a cell expressing the channel. In some embodiments, the mechanically-activatedcation channels of the present invention are involved in sensory transduction, such as paintransduction, including but not limited to, cells such as neurons.

"Inhibitors, " "activators, " and "regulators" of mechanically-activated cation channel activity are used interchangeably herein to refer to inhibitory, activating, ormodulating molecules identified using in vitro and in vivo assays for sensory transduction, e.g., ligands, agonists, antagonists, and their homologs and mimetics. The term "regulators " encompasses inhibitors and activators. Inhibitors arecompounds that, e.g., bind to, partially or totally block stimulation, decrease, prevent, delayactivation, inactivate, desensitize, or down regulate signal transduction, e.g., antagonists. Activators are compounds that, e.g., bind to, stimulate, increase, open, activate, facilitate, enhance activation, sensitize, or up regulate signal transduction, e.g., agonists. Regulatorsinclude naturally occurring and synthetic ligands, antagonists, agonists, small chemicalmolecules and the like. Such assays for inhibitors and activators include, e.g., expressing amechanically-activated cation channel polypeptide in cells or cell membranes, applying putativemodulator compounds, and then determining the functional effects on ion flux, membranepotential, electrophysiology, or mechanical activation. Samples or assays comprising amechanically-activated cation channel polypeptide that is treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, ormodulator to examine the extent of modulation. Control samples (untreated with inhibitors) areassigned a relative mechanically-activated cation channel polypeptide activity value of 100%. Inhibition of a mechanically-activated cation channel polypeptide is achieved when the mechanically-activatedcation channel polypeptide activity value relative to the control is about80%, optionally 75%, 50%, or 25-0%. Activation of the mechanically-activated cation channelpolypeptide is achieved when the mechanically-activated cation channel polypeptide activityvalue relative to the control is 110%, optionally 125%, optionally 150%, optionally 200-500%, or 1000-3000%higher.

"Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs) .

Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues. The term "nucleic acid" encompasses the terms gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

The terms "polypeptide, " "peptide, " and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, . gamma. -carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methioninesulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that havea structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations, " which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.

The following eight groups each contain amino acids that are conservative substitutions for one another:

1) Alanine (A) , Glycine (G) ;

2) Aspartic acid (D) , Glutamic acid (E) ;

3) Asparagine (N) , Glutamine (Q) ;

4) Arginine (R) , Lysine (K) ;

5) Isoleucine (I) , Leucine (L) , Methionine (M) , Valine (V) ;

6) Phenylalanine (F) , Tyrosine (Y) , Tryptophan (W) ;

7) Serine (S) , Threonine (T) ; and

8) Cysteine (C) , Methionine (M) (see, e.g., Creighton, Proteins (1984) ) .

The terms "isolated, " "purified, " or "biologically pure" refer to material that is substantially or essentially free from components that normally accompany it as found in itsnative state. Purity and homogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or high performance liquidchromatography. A protein that is the predominant species present in a preparation issubstantially purified. The term "purified" denotes that a nucleic acid or protein gives rise toessentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid orprotein is at least 85%pure, optionally at least 95%pure, and optionally at least 99%pure.

The term "recombinant" when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by theintroduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid orprotein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cellsexpress genes that are not found within the native (non-recombinant) form of the cell or expressnative genes that are otherwise abnormally expressed, under expressed, or not expressed at all.

The term "heterologous" when used with reference to a protein 's or nucleic acid 'srelationship to a cell indicates that the protein or nucleic acid is not found in the samerelationship to the cell (e.g., not expressed in the cell) in nature. The term "heterologous" whenused with reference to portions of a nucleic acid indicates that the nucleic acid comprises two ormore subsequences that are not found in the same relationship to each other in nature. Forinstance, the nucleic acid is typically recombinantly produced, having two or more sequencesfrom unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from onesource and a coding region from another source. Similarly, a heterologous protein indicates thatthe protein comprises two or more subsequences that are not found in the same relationship toeach other in nature (e.g., a fusion protein) .

An "expression cassette" is a nucleic acid construct, generated recombinantly orsynthetically, with a series of specified nucleic acid elements that permit transcription of aparticular nucleic acid in a host cell. The expression cassette can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression cassette includes a nucleic acid to be transcribed operably linked to a promoter.

The terms "identical" or percent "identity, " in the context of two or more nucleic acidsor polypeptide sequences, refer to two or more sequences or subsequences that are the same. Sequences are "substantially identical" of they have a specified percentage of amino acidresidues or nucleotides that are the same (i.e., 70%identity, optionally 75%, 80%>, 85%, 90%, or95%identity over a specified region) , when compared and aligned for maximum correspondenceover a comparison window, or designated region (aspecified length, or when not specified, theentire length) as measured using one of the following sequence comparison algorithms or bymanual alignment and visual inspection.

For sequence comparison, typically one sequence acts as a reference sequence, towhich test sequences are compared. When using a sequence comparison algorithm, test andreference sequences are entered into a computer, subsequence coordinates are designated, ifnecessary, and sequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. The sequence comparisonalgorithm then calculates the percent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

The phrase "selectively (or specifically) hybridizes to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringenthybridization conditions when that sequence is present in a complex mixture {e.g., total cellularor library DNA or R A) .

"Antibody" refers to a polypeptide comprising a framework region from animmunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. Therecognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, andmu constant region genes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa) . The N-terminus of each chain definesa variable region of about 100 to 110 or more amino acids primarily responsible for antigenrecognition. The terms variable light chain (VL) and variable heavy chain (V) refer to these lightand heavy chains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number of well-characterizedfragments produced by digestion with various peptidases. Thus, for example, pepsin digests anantibody below the disulfide linkages in the hinge region to produce F (ab) ', a dimer of Fabwhich itself is a light chain joined to VH-CHI by a disulfide bond. The F (ab) '2 may be reducedunder mild conditions to break the disulfide linkage in the hinge region, thereby converting theF (ab) 2 dimer into an Fab 'monomer. The Fab' monomer is essentially Fab with part of the hingeregion (see FUNDAMENTALIMMUNOLOGY (Paul ed., 3d ed. 1993) . While various antibodyfragments are defined in terms of the digestion of an intact antibody, one of skill will appreciatethat such fragments may be synthesized de novo either chemically or by using recombinant DNAmethodology. Thus, the term antibody, as used herein, also includes antibody fragments eitherproduced by the modification of whole antibodies, or those synthesized de novo usingrecombinant DNA methodologies (e.g., single chain Fv) or those identified using phage displaylibraries (see, e.g., McCaffertyet al, Nature 348: 552-554 (1990) ) .

For preparation of monoclonal or polyclonal antibodies, any technique known in the artcan be used (see, e.g., Kohler & Milstein, Nature 256: 495-497 (1975) ; Kozboret al., Immunology Today 4: 72 (1983) ; Cole et al., pp. 77-96 in Monoclonal Antibodies and CancerTherapy (1985) ) . Techniques for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanizedantibodies. Alternatively, phage display technology can be used to identify antibodies andheteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCaffertyet al., Nature348: 552-554 (1990) ; Marks et al, Biotechnology 10: 779-783 (1992) ) .

A "chimeric antibody" is an antibody molecule in which (a) the constant region, or aportion thereof, is altered, replaced or exchanged so that the antigen binding site (variableregion) is linked to a constant region of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portionthereof, is altered, replaced or exchanged with a variable region having a different or alteredantigen specificity.

A "humanized antibody" is an antibody that retains the reactivity of a non-humanantibody while being less immunogenic in humans. This can be achieved, for instance, byretaining the non-human CDR regions and replacing the remaining parts of the antibody withtheir human counterparts. See, e.g., Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 685 1-6855 (1984) ; Morrison and Oi, Adv. Immunol, 44: 65-92 (1988) ; Verhoeyenet al, Science, 239: 1534-1536 (1988) ; Padlan, Molec. Immun., 28: 489-498 (1991) ; Padlan, Molec. Immun., 31 (3) : 169-217 (1994) .

The phrase "specifically (or selectively) binds" to an antibody or "specifically (orselectively) immunoreactive with, " when referring to a protein or peptide, refers to a bindingreaction that is determinative of the presence of the protein in a heterogeneous population ofproteins and other biologies. Thus, under designated immunoassay conditions, the specifiedantibodies bind to a particular protein at least two times the background and do not substantiallybind in a significant amount to other proteins present in the sample. Specific binding to anantibody under such conditions may require an antibody that is selected for its specificity for aparticular protein. For example, polyclonal antibodies raised to Piezol or Piezo2 from specificspecies such as rat, mouse, or human can be selected to obtain only those polyclonal antibodiesthat are specifically immunoreactive with Piezol or Piezo2 and not with other proteins, exceptfor polymorphic variants and alleles of Piezol or Piezo2. This selection may be achieved bysubtracting out antibodies that cross-react with Piezol or Piezo2 molecules from other species. A variety of immunoassay formats may be used to select antibodies specifically immunoreactivewith a particular protein. For example, solid-phase ELISA immunoassays are routinely used toselect antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988) , for a description of immunoassay formats andconditions that can be used to determine specific immunoreactivity) . Typically a specific orselective reaction will be at least twice background signal or noise and more typically more than10 to 100 times background.

A "subject" or "individual" refers to an animal, including a human, non-human primate, mouse, rat, rabbit, dog, or other mammal.

The following examples are provided so that the invention might be more fully understood. However, it should be understood that these embodiments merely provide a method of practicing the present invention, and the present invention is not limited to these embodiments.

The related methods are described as follows:

Methods

Molecular cloning

Piezo2 was sub-cloned into the pcDNA3.1 expression vector encoding a C-terminal Glutathione S-transferase (GST) tag with a precision protease cleavage site in between, followed with the green fluorescent protein (GFP) -coding sequence driven by an internal ribosome entry site (IRES) , which is designated for monitoring the efficiency of transient transfection and protein expression. We refer this expression plasmid as Piezo2-pp-GST-IRES-GFP, which mediated similar poking-evoked whole-cell currents as the Piezo2-ires-GFP vector when transfected in the Piezo1-KO-HEK cells (the endogenous Piezo1 gene is disrupted) and assayed with a piezo-electrically driven glass probe to indent the cell membrane, suggesting that fusion of GST at the C-terminus of Piezo2 did not affect its functionality. All constructs including the Piezo2-E2757A-mRuby2, Piezo2-E2811A-mRuby2, Piezo1-E2537A-mRuby2, Piezo1-ΔCED ^{α1-α2 helix}-GST (replacement of the residues 2253-2274 with a GGGGG-linker) , Piezo2-ΔCED -mRuby2 (replacement of the 2502-2727 with a GGGG-linker) and Piezo2-ΔCED ^{α1-α2 helix}-mRuby2 (replacement of the residues 2536-2557 with a GGGG-linker) were subcloned by using the One Step Cloning Kit (Vazyme Biotech) according to the instruction manual and as described previously ³⁵, then sequenced to validate the desired mutations.

Protein expression and purification of Piezo2

The purification procedure of the Piezo2 protein was adapted from the previously described protocol for purification of Piezo1 with significant modifications. Due to much lower expression level of Piezo2 than Piezo1, we scaled up to 120 15-cm petri dishes of culture and transfection of HEK293T cells to obtain ～ 50 μg of purified Piezo2 proteins suitable for each trial of cryo-EM experiment following the purification procedure as described below. Furthermore, on the basis of FPLC gel filtration and negative EM staining, we found that the detergent glycol-diosgenin (GDN) , which is a synthetic substitute of digitonin and was used for determining the Piezo1 (6bpz) structure, was more suitable for Piezo2 proteins than the detergent C ₁₂E ₁₀ used for determining our previous Piezo1 (5z10) structure.

For each batch of protein purification, transiently transfected HEK293T cells cultured in 120 150 mm x 25 mm petri-dishes with a total of 2.4 liters of culture medium were harvested by centrifugation at 850 g and solubilized in buffer A containing 25 mMNaPIPES, pH 7.3, 150 mMNaCl, 3 mMdithiothreitol (DTT) , a mixture of detergents including 0.012% (w/v) glyco- diosgenin (GDN) , 0.1% (w/v) C ₁₂E ₉, 1% (w/v) CHAPS, 0.5% (w/v) L-a-phosphatidylcholine (Avanti) , a cocktail of protease inhibitors (Roche) and 3 mMphenylmethylsulfonyl fluoride (PMSF) at 4 ℃ for 1.5 h. After centrifugation at 13,000g for 35 min, the supernatant was collected and incubated with glutathione–sepharose beads (GE Healthcare) at 4 ℃ for 7 h. The resin was washed extensively with buffer B, containing 25 mM Na-PIPES, pH 7.3, 150 mMNaCl, 3 mM DTT, 0.05% (w/v) GDN. The GST-tagged Piezo2 was cleaved off by PreScission Protease (Amersham-GE) in buffer B at 4℃ for 16 h, and applied to size-exclusion chromatography (Superpose-6 10/300 GL, GE Healthcare) in buffer C, containing 25 mM Na-PIPES, pH 7.3, 150 mMNaCl, 3 mM DTT, and 0.02% (w/v) GDN. Peak fractions representing oligomeric Piezo2 were collected for electron microscopy analysis. Protein in GDN was used for final cryo-EM structure determination. All detergents used in this project were purchased from Anatrace.

Negative-staining electron microscopy

An aliquot of 4 μl Piezo2 (0.03 mg/ml) was applied to 15 s glow-discharged carbon-coated copper grids (300 mesh, Zhongjingkeyi, Beijing) . After the grid was incubated at room temperature for 1 min, excessive liquid was absorbed by filter paper. Grids containing the specimen were stained by applying droplets of 2%uranyl acetate for 30 s and air-dried. Micrographs were collected on a T12 microscope (FEI) operated at 120 kV, using a 4k x 4k CCD camera (UltraScan 4000, Gatan) . Images were recorded at a nominal magnification of 49,000x.

Sample preparation and cryo-EM data acquisition

Sample preparation procedure was optimized by following previously described protocols so that the Piezo2 proteins at a concentration of ～ 1 mg/ml could enter into the holes without a thin carbon film for absorbing and concentrating the proteins, which might reduce the background noise and improve the image quality. In brief, 3 μl of freshly purified mouse Piezo2 proteins (～1 mg/ml) each time were sequentially blotted for three times to 300-mesh holey carbon Au R2/1 grids treated with 25 s of glow discharge (Quantifoil, Micro Tools GmbH) . 0.65 mM fluorinated Fos-Choline-8 (Hampton) was added to freshly concentrated Piezo2 sample immediately prior to freezing. The grids were then blotted and plunged into liquid ethane using an FEI Mark IV Vitrobot operated at 8 ℃ and 100%humidity.

The grids were transferred to a 300 kV Titan Krios (FEI) electron microscope equipped with a Cs corrector and GIF Quantum energy filter (slit width 20 eV) . Images were recorded by a post-GIF K2 Summit direct electron detector (Gatan, Inc. ) working at the super-resolution mode. Data acquisition was performed using AutoEMation II with a nominal magnification of 81,000 X, which yields a super-resolution pixel size of

on image plane, and with defocus ranging from -1.5 μm to -2.5 μm. The dose rate on the detector was ～8.4 counts per physical pixel per second with a frame exposure time of 0.275 s and a total exposure time of 8.8 s. Each micrograph stack contains 32 frames. The total dose was approximately

for each micrograph.

Image processing

A simplified flowchart of the procedure for image processing is presented in. Three batches of cryo-EM data containing 8, 792, 8, 621 and 5, 661 micrograph stacks were processed respectively in the first several steps. The stacks were motion-corrected and dose-weighted using MotionCor2with 2×2 binning, resulting in a pixel size of

After whole image CTF estimation using CTFFIND3, 7, 243, 7, 685 and 4, 915 good micrographs were manually selected, respectively. A total of 1, 260, 085, 948, 941 and 512, 933 particles were respectively autopicked using RELION-3.0. After several rounds of two-dimensional (2D) classification, 184, 226, 271, 730 and 178, 808 particles were selected, then subjected to 3D refinement, respectively. The Piezo1 EM map (EMD-6865) low-pass filtered to

was used as an initial model. Each particle was re-centered and subjected to Gctf ⁵⁰ to refine the local defocus parameters. The well-centered particles with more accurate defocus parameters were re-extracted from the motion-corrected and dose-weighted micrographs then subjected to 3D refinement, which resulted in three electron density maps at

and

resolution.

A total of 634, 764 particles selected from the three datasets were pooled for further processing. Three parallel 3D classifications skipping alignment were performed with the numbers of classes of 3, 4 and 5, respectively. Particles from each class (or merged several classes with similar density maps) were subjected to 3D refinement, separately, resulting in multiple density maps. These density maps were thoroughly compared, and the maps with the highest resolution in each classification show the nearly same structures, indicating that they might represent a similar conformation of Piezo2. Particles from these highest-resolution classes were merged to a dataset with 281, 283 particles. All duplicated particles derived from the parallel routine of 3D classifications and 3D refinements were removed during data merging. Subjecting to 3D refinement, this dataset yielded a map at

resolution. To further improve the resolution, the random phase classification was used to remove heterogeneous particles, and resulted in a new map at

resolution after 3D refinement and post processing. All 3D refinement and 3D classification above except random phase classification were performed with C3 symmetry.

Since the CED domain and the distal blade were absent from the overall

map, the focused refinements were performed for these regions. The subroutine of relion_particle_symmetry_expand in RELION-3.0 was used to expand 281, 283 Piezo2 particles with C3 symmetry. Then 843, 849 blade particles were obtained using the subtraction of two adjacent blades in raw images. These blade particles were subjected to 3D refinement, resulting in a focused map of

resolution. After further 3D classification with local angular search of 0.9°step size and 5° search range, 386, 144 particles were selected and subjected to 3D refinement, resulting in the final focused map of the blade at

resolution. Since the density of THU1 were still fragmentary and unclear in this map, more surrounding regions of the distal blade were subtracted from the 386, 144 blade particles, and the following focused refinement led to a reconstruction at

resolution. After a 3D classification skipping alignment, 347, 108 particles were further selected from the 386, 144 particles and subjected to 3D refinement, yielding the final focused map of the distal blade at

resolution.

In another processing, the focused refinement of the central region of the 281, 283 particles were performed by subtracting the projections of most parts of three surrounding blades. C3 symmetry was imposed during the refinement and resulted in a focused map at

resolution. Since the density of the CED domain was still fragmentary and unclear in this map, 3D classification skipping alignment with a local mask of CED domain were performed. 108, 781 particles from the class with the best density of CED domain were selected and subjected to 3D refinement with a soft mask of the central region, resulting the final focused map of the central region at

resolution. The three final focused maps were fit together according to their overlapping area using Chimera then combined using PHENIX Combine Focused Maps. Three copies of the combined map were further combined into the final density map.

The reported resolutions are based on the gold-standard Fourier shell correlation 0.143 criterion. All density maps were sharpened by applying a negative B-factor that was estimated using automated procedures ⁵³. Local resolution variations were estimated using Resmap ⁵⁴.

Model building and structure refinement.

The cryo-EM structures of Piezo1 (PDB accession 5Z10 and 6B3R) and the crystal structure of Piezo1 CED domain (PDB accession 4RAX) were docked into the cryo-EM map in Chimera as the initial models. The structure was first refined in real space using PHENIX with secondary structure and NCS restraints. The model of THU1-3 was built de novo in COOT. The remaining part of the model was manually mutated from refined initial models. The model was refined in real space using PHENIX with secondary structure and NCS restraints. The final atomic model was evaluated using MolProbity ⁵⁷.

Western blotting

Western blotting was carried out as previously described. In brief, one 150 mm-sized petri dish of HEK293T cells transfected with either vector, mPiezo1-pp-GST-IRES-GFP or mPiezo2-pp-GST-IRES-GFP were harvested and lysed into 700 μl cell lysates. 10 μl of cell lysate from each sample was separated on 8%SDS-PAGE gels and transferred onto 0.45 μm PVDF membranes (Millipore) . The membranes were blocked by 5%non-fat milk (Bio-Rad) in TBST buffer (TBS buffer with 0.1%Tween-20) and incubated with the diluted primary antibodies overnight. After 3 washes with TBST buffer, the membranes were incubated with the peroxidase-conjugated anti-rabbit IgG secondary antibody (Cell Signaling Technichlogy, dilution 1: 10,000) at room temperature for 1 hour, followed with washing and detection using the enhanced chemiluminescence detection kit (Thermofisher Scientific) . The antibodies used for western blotting include rabbit anti-GST (Millipore, dilution 1: 3,000) and rabbit anti-β-actin (Cell Signaling Technology, dilution 1: 3,000) .

Cell surface biotinylation assay

To examine the plasma membrane expression of Piezo1-GST, Piezo1-ΔCED-GST, Piezo1-ΔCED ^{α1-α2 helix}-GST, Piezo2-mRuby2 and Piezo2-ΔCED ^{α1-α2 helix}-mRuby2, cell surface biotinylation assay was carried out according to the procedure previously described ²⁷. The anti-GST antibody (Millipore, dilution 1: 3,000) was used for detecting the Piezo1-GST, Piezo1-ΔCED-GST, Piezo1-ΔCED ^{α1-α2 helix}-GST fusion proteins, while a custom-generated Piezo2 antibody (dilution 1: 1000) using the synthesized peptide NH2-KAPSDSNSKPIKQC-CONH2 corresponding to residues 2662-2674 in the CED of mouse Piezo2 for detecting the Piezo2-mRuby2 and Piezo2-ΔCED ^{α1-α2 helix}-mRuby2 fusion proteins. The Piezo2 antibody did not recognize protein bands from the vector-transfected cells and the Piezo2-ΔCED protein (data not shown) , demonstrated the specificity of the Piezo2 antibody in recognizing the Piezo2 proteins in western blotting. To detect the Piezo2-ΔCED-mRuby2 protein, the anti-Flag antibody (Sigma, dilution 1: 3,000) was used to detect the Flag epitope located in the C-terminal of the Piezo2- mRuby2 or Piezo2-ΔCED-mRuby2 fusion proteins. The anti-rabbit IgG secondary antibody (CST, 1: 10,000) or anti-mouse IgG secondary antibody (pierce, 1: 20,000) were used.

Whole-cell electrophysiology and mechanical stimulation

The protocols for culture of Piezo1-KO-HEK cells in which the endogenous Piezo1 gene is disrupted, transient transfection and patch-clamp experiments with an Axopatch 200B amplifier (Axon Instruments) or HEKA EPC10 were essentially similar to those previously described, except that polyethylenimine (PEI) (Polysciences) was used for the transfection reagent ^18, 30. For whole-cell patch-clamp recordings, the recording electrodes had a resistance of 2-5 MΩ when filled with an internal solution composed of (in mM) 133 CsCl, 1 CaCl ₂, 1 MgCl ₂, 5 EGTA, 10 HEPES (pH 7.3 with CsOH) , 4 MgATP and 0.4 Na ₂GTP. The extracellular solution was composed of (in mM) 133 NaCl, 3 KCl, 2.5 CaCl ₂, 1 MgCl ₂, 10 HEPES (pH 7.3 with NaOH) and 10 glucose. All experiments were carried out at room temperature. The currents were sampled at 20 kHz, filtered at 2 kHz using the Clampex 10.4 software (Axon Instruments) or Patchmaster software. Leak currents before mechanical stimulation were subtracted off-line from the current traces.

Mechanical stimulation was delivered to the cell during the patch-clamp recording at an angle of 80° using a fire-polished glass pipette (tip diameter 3 –4 μm) as previously described ^1, 2. The downward movement of the probe toward the cell was driven by a Clampex controlled piezo-electric crystal micro-stage (E625 LVPZT Controller/Amplifier; Physik Instrument) . The probe had a velocity of 1 μm/ms during the downward and upward motion, and the stimulus was maintained for 150 ms. Aseries of mechanical steps in 1 μm increments was applied every 8 s, and the currents were recorded at a holding potential of -80 mV.

Measurement of P _Ca/P _Cs

The measurements were carried out as described ^26, 35. Briefly, P _Ca/P _Cs was measured in the internal solution consisting of (in mM) 149 Cs-methanesulfonate, 1 CsCl and 10 HEPES (pH7.3 with CsOH) and the extracellular solution containing (in mM) 50 Ca-gluconate, 0.5 CaCl ₂, 10 HEPES and 170 sucrose (pH 7.3 with CsOH) . For I-V relationship recordings, voltage steps were applied 700 ms before mechanical stimulation (150 ms) from a holding potential of -60 mV. Voltage steps were given from -37.1 mV to +22.9 mV in 10 mV increments (liquid junction potential of 7.1 mV was corrected) . Reversal potential for each cell under the specific recording solution was determined by linear regression fit of the whole-cell I-V curve. Permeability ratios were calculated by using the following Goldman-Hodgkin-Katz (GHK) equations described previously:

PCa/PCs:

Measurement of unitary conductance

Single-channel currents were recorded in the cell-attached patch clamp configuration using HEKA EPC10 as described previously ^1, 35. Currents were sampled at 20 kHz and filtered at 1 kHz. The recording electrodes had a resistance of 2-3 MΩ when filled with standard solution containing (in mM) 130 mMNaCl, 5 KCl, 1 CaCl ₂, 1 MgCl ₂, 10 TEA-Cl and 10 HEPES (pH 7.3 with NaOH) . External solution used to zero the membrane potential consisted of (in mM) 140 KCl, 1 MgCl ₂, 10 glucose and 10 HEPES (pH 7.3 with KOH) . Membrane patches were stimulated with 500 ms negative pressure pulses through the recording electrode using Patchmaster controlled pressure clamp HSPC-1 device (ALA-scientific) . Recordings with one to four channel opening were generally used. Single-channel amplitude at a given potential was measured from trace histograms of 10 to 80 repeated recordings. Histograms were fitted with Gaussian equations using Clampfit 10 software or multi-peak fitting analysis of IGOR Pro software. Single-channel slope conductance for each individual cell was calculated from linear regression curve fit to single-channel I-V plots.

EXAMPLES

Structure determination of Piezo2

To determine the structure of the full-length 2822-residue mouse Piezo2, we have adopted a similar strategy employed for the mouse Piezo1 structure determination ¹⁸, but with significant modifications to optimize its recombinant expression, purification and cryo-EM sample preparation. Piezo2 was cloned into an expression vector encoding a C-terminal Glutathione S-transferase (GST) tag with a precision protease cleavage site in between, followed with the green fluorescent protein (GFP) -coding sequence driven by an internal ribosome entry site (IRES) , which is designated for monitoring the efficiency of transient transfection and protein expression. We refer this expression plasmid as Piezo2-pp-GST-IRES-GFP. Using a piezo-electrically driven glass probe to indent the cell membrane, we detected similar mechanically activated whole-cell currents in HEK293T cells transfected with either Piezo2-ires-GFP or Piezo2-pp-GST-ires-GFP, suggesting that fusion of GST at the C-terminus of Piezo2 did not affect its functionality. However, due to much lower expression level of Piezo2 than Piezo1 (Fig. 1a) , we scaled up to 120 15-cm petri dishes of culture and transfection of HEK293T cells to obtain ～ 50 μg of purified Piezo2 proteins suitable for each trial of cryo-EM experiment following the purification procedure described in the Method section (Fig. 1b, c) . Furthermore, on the basis of FPLC gel filtration and negative EM staining (Fig. 1b) , we found that the detergent glycol-diosgenin (GDN) was more suitable for Piezo2 proteins than C ₁₂E ₁₀, which was used for our previous Piezo1 structure determination ¹⁸. Furthermore, the cryo-EM sample preparation procedure was optimized so that the Piezo2 proteins at a concentration of ～ 1 mg/ml could enter into the holes without a thin carbon film for absorbing and concentrating the proteins, which might reduce the background noises and improve the image quality.

The optimized Piezo2 samples were observed using a 300 kV Titan Krios (FEI Company) electron microscope equipped with a Cs corrector, GIF Quantum energy filter and a post-GIF K2 Summit direct electron detector (Gatan, Inc. ) . A total of 23, 074 micrographs with ～2.7 million particles (Fig. 1d-f) were collected and analyzed through the procedure summarized in the Method section. Two-dimensional (2D) classification shows an apparent three-bladed propeller-like structure with a central Cap in the top-view particles and a bowl-like structure in the side-view particles (Fig. 1f) . Three-dimensional (3D) reconstruction of Piezo2 led to an overall

-resolution structure (Fig. 1g, h) , which resembles the three-bladed, propeller-shaped structure of Piezo1. Except the extracellular Cap structure, all the other featured structural domains observed in Piezo1, including the 26 TMs, the intracellular helical Beam, the Anchor and the intracellular C-terminal domain (CTD) in each protomer, were resolved to

-resolution in the Piezo2 structure (Fig. 1h) . To better resolve the Cap structure of Piezo2, we performed focused refinement of the central Cap region by subtracting the surrounding regions in raw images, and obtained a

-resolution map showing much improved density of the Cap (Fig. 1g, i) . In both the Piezo1 and Piezo2 structures, the distal blade, which is encoded by the N-terminal 500-600 residues and predicted to have 12 TMs, was not resolved ^18, 20, 21. To better resolve this region, we performed focused refinement of the blade and obtained a

-resolution map showing 12 additionally resolved TMs (Fig. 1g, j) . Since the density of the first 4 TMs were still fragmentary in this map, we further processed the distal blade within a smaller region and obtained a

-resolution map with the first 4 TMs starting from the N-terminal residue glycine 8 better resolved (Fig. 1g, k) . Thus, we have for the first time resolved a complete 38-TM topology of Piezo2.

The three final focused maps (Central, Blade and Distal blade) (Fig. 1i-k) were fit together according to their overlapping area using Chimera, then combined using PHENIX to generate the final map for model building (Fig. 2a, b) . Using the Piezo1 models ^18, 20 and X-ray crystal structure of the CED of Piezo1 ¹⁷ as templates and de novo model building of the newly or better resolved regions in Piezo2, we built and refined a structure model for Piezo2 by assigning 1818 residues into the 36 peripheral TMs, the Anchor domain, the central pore module comprising TM37 (termed outer helix, OH) , the C-terminal extracellular domain (CED) that trimerizes to form the Cap, TM38 (termed inner helix, IH) and the CTD, and the Beam with the Latch and Clasp domains at its proximal and distal ends, respectively (Fig. 2b) . The extracellular loops (EL) including EL7-8, EL11-12, EL15-16, EL19-20, EL23-24 and portion of EL31-32 and EL35-36, as well as the intracellular loops (IL) including IL20-21, IL24-25, TM36-Anchor-linker, Anchor-OH-linker were also assigned (Fig. 3) . The unassigned regions including the N-terminal 7 residues and other loop regions are depicted as dot lines in the topology model shown in Fig. 3a.

A nano-bowl configuration of the curved TM Blades

The complete revelation of the 38 TMs in each protomer of Piezo2 has allowed us to fully appreciate the characteristic three-bladed, propeller structure and the highly curved and non-planar conformation of the Piezo channel family. The Piezo2 trimeric structure has an axial height of

and a diameter of

(Fig. 2a) . When viewed from the extracellular side, the three blades are attached to the central Cap and extended to the periphery in a clockwise manner with each forming a half-circled superhelical structure, resembling a typical three-bladed propeller or triskelion (Fig. 2a, b) . The proximal TM25-38 and the central region were supported by the

-long Beam positioned in a 30° angle relative to the normal plasma membrane plane, while the peripheral TM1-24 hang at the distal end of the Beam (Fig. 2b) . The lack of apparent intracellular supporting structures for TM1-24 might explain why they are more flexible to be resolved than TM25-36, which is bundled together by the underlying Beam that has extensive interactions at both its distal and proximal ends as described below.

When viewed parallel to the membrane plane, TM1-38 in each blade form an apparent hydrophobic belt, which is continuously curved out of the planar membrane plane extending from the central inner helix (IH) -outer helix (OH) pair to the periphery and follows a positive-charged residue inside-rule for membrane proteins (Fig. 2c) . Strikingly, the hydrophobic belt formed by the distal TMs of one subunit is located far above the belt formed by the proximal TMs of the neighboring subunit (Fig. 2c) . Thus, the peripheral TM1-24 adopt a non-planar configuration with the most distal TM1-12 exceeding the

-height extracellular Cap (Fig. 2b-d) , which is clearly appreciated when viewed from the extracellular side and zoomed in at the top of the Cap (Fig. 2d) . In contrast, TM25-38 roughly reside in the membrane plane as viewed from the top and zoomed in at the position of the extracellular end of the IH-OH pair (Fig. 2e) . Thus, the transmembrane regions of the three blades are curved into a nano-bowl shape with a top opening diameter of ～28 nm and a depth of ～10 nm, resulting in a full embedment of the extracellular Cap in the center (Fig. 2a-d) . Given the demonstrated ability of the purified Piezo1 proteins to curve the membrane to conform to the non-planar shape of Piezo1 when reconstituted into liposomes ²⁰, we expect that Piezo2 might similarly deform the residing membrane into a nano-bowl shape. The unique nano-bowl configuration of the Piezo-membrane system might represent the specialized structural basis for conferring exquisite mechanosensitivity to the Piezo channel family.

The 38-TM topological organization of the Piezo2 protomer

The Piezo2 structure reveals a complete 38-TM topology and its featured domains. While TM37 and TM38 constitute the outer helix and inner helix of the central pore module respectively, TM1-36 in 18 pairs are organized into 9 repetitive and left-handed 4 TM-containing transmembrane helical units (THUs) to form the highly curved blade (Fig. 3) . The resolved extracellular loops of 30 -50 residues in between the third and fourth TMs of each THU lean against each other and form a flattened mono-layer on the extracellular surface of the TM blade, except that the extracellular loop of TM19-20 sits on top of that of TM23-24. Such an organization might help stabilize the blade and facilitate conformational propagation from the distal blade to the central region. In line with this, studies of Piezo1 have shown that the extracellular loops of TM3-4, TM7-8 and TM11-12 are important for its proper expression and plasma membrane targeting, while those of TM15-16 and TM19-20 are critical for its mechanical-and chemical-activation ^18, 23. Local application of force to the extracellular loops of TM3-4 and TM7-8 of Piezo1 indicated that these two distal loops might be mechanosensitive.

Characteristically, except THU1 with a short N-terminus of 7 residues, each of the other 8 THUs is preceded with a membrane-parallel helix, which connects perpendicularly to the first TM (Fig. 3a, b) . These membrane-parallel helices are amphipathic with their hydrophobic side facing the lipid membrane, thus collectively form an intracellular helical layer immediately underneath the membrane. These helices might not only help to stabilize the non-planar transmembrane region of the Blade and the residing membrane, but also contribute to mechanosensitivity, in line with the proposed idea that parallel amphipathic helices represent a structural motif characteristic of inherently mechanosensitive channels.

The loop of TM28-29 (THU7-8) of ～540 residues represents the largest intracellular loop in Piezo2 (Fig. 3a, b) , containing the Beam extending underneath the THU7-9 and the C-terminal domain (CTD) , the Latch (residues 1557-1577) , the Clasp (residues 1668-1727) , TM29 ^preα1-2 (1948-1991) . The distal part of the Beam is apparently kinked at the position of A1465/S1466 and buried within a space enclosed by the intracellular side of THU7 and the second and third α-helix of the Clasp (Clasp ^α2, 3) (Fig. 3a, b) . The Clasp domain is composed of two long membrane-parallel helices (Clasp ^α1-2) , which is kinked at residues S1686/I1687/S1688 to form an L-shaped helical structure, and a short helix (Clasp ^α3) positioned underneath the kink position of the Beam. These domains are intertwined together with hydrogen-bond interactions of D1457-R1702, S1466-R1717, R1467-E1701 for stabilization. The 280 unresolved residues (1728-1947) linking the Clasp ^α3 and TM29 ^preα1-2 might provide additional interactions and regulations at the distal end of the Beam.

The proximal end of the Beam directly contacts the hairpin-like CTD positioned on top and connects to the perpendicularly crossed Latch through 42 unresolved residues (1513-1556) (Fig. 3b and Fig. 4a-d) . The C-terminal portion of the Latch domain is rich in negatively charged and polar residues (1572-ETDSEE) and sandwiched in between the Beam and the CTD with clusters of positively charged residues (R1500, R1504, K1507, K1512 in the Beam and K2815, R2818, K2820 in the C-terminal tail) (Fig. 4b-d) . Y1568 in the Latch domain points toward the putative intracellular exit of the central pore and form hydrogen bond with E2811 (Fig. 4c, d) , which forms the putative cytoplasmic gate (see below Fig. 5b, c) . Given its extensive interactions at both the distal and proximal ends, the Beam might be strategically vital not only for structurally stabilizing the core region but also for functionally coupling the distal blade to the central ion-conducting pore.

The Anchor domain consists of three α-helices (Anchor ^α1-3) and penetrates into the inner leaflet of the membrane to form a cuff surrounding the inner portion of the inner helix-enclosed central pore (Fig. 4a, e, f) . It might be critical for maintaining the integrity and gating of the ion-conducting pore. Indeed, mutating the conserved residue Piezo1-E2133 and Piezo2-E2416 (Fig. 4f) in this region affected ion permeation properties ²⁶. The long membrane-parallel Anchor ^α3 sits right on top of the CTD-hairpin-plane (Fig. 4a, e, f) andconnects to the outer helix through the lysine-rich Anchor-OH-linker (2456-KRYPQPRGQKKKK) (Fig. 3b and Fig. 4a, e, f) , which forms interactions with the polar residue-rich anchor ^α2-3-turn (2426-TDTTT) , the glutamate-rich region of the CTD ^α1-2-turn (2789-ETGELELEED) and the N-terminal portion of the Latch (Fig. 4a, e) . Several pairs of hydrogen bonds, including D2427-K2465, T2428-E2796 and T2429-E2797 (Fig. 4e) , might help to facilitate the intertwined interactions. The corresponding Anchor-OH-linker in Piezo1 is critical for mediating the regulation by the Sarco/Endoplasmic Reticulum Ca ²⁺-ATPase (SERCA) , which binds to both Piezo1 and Piezo2 ²⁷. Overall, the organization of the proximal portion of the Beam, Latch, CTD, and Anchor might allow them to function in concert to relay the lever-like motion of the Blade-Beam to the central pore (see below Fig. 6) .

The ion-conducting pore

The C-terminal OH-CED-IH-CTD domain trimerizes to form the central pore module (Fig. 4a) , which resembles other trimeric channels including acid-sensing channel (ASIC) and ATP-gated P2X ion channelcomprising a large extracellular domain and three pairs of TMs. The connections between the N-and C-termini of the CED and the outer helix and inner helix (2493-2500 and 2732-2738, respectively) were unresolved (Fig. 4a) , indicating flexibility of these two linking regions. Despite the existence of an extracellular vestibule inside the Cap and potential opening of the top, the apparent fenestrations located in between the Cap and the membrane region might allow cations to directly enter into the transmembrane pore. Thus, we have focused on analyzing the ion-conducting pathway spanning from the extracellular end of the inner helix to the cytosolic side (Fig. 5) . Residues 2740-2759 encode the inner helix, which is highly conserved between Piezo2 and Piezo1 (Fig. 5a) . Three inner helices converge at the center to enclose the hydrophobic transmembrane pore (Fig. 5b) . In contrast to Piezo1 showing wide and long side cavity between two IHs, the membrane-facing side of the transmembrane region of Piezo2 is fully sealed. Notably, a membrane vestibule is flanked with a top constriction site of less than

formed by the residue L2743 and a bottom

-long narrow pore region formed by the pore-facing residue V2750, F2754, E2757 (Fig. 5a, b, c) , demonstrating a closed transmembrane pore. Interestingly, the two constriction sites of Piezo2 at L2743 and F2754/E2757 are apparently dilated in the corresponding positions of Piezo1 at L2469 and F2480/G2483, respectively (Fig. 5a-c) . Thus we term the two constriction sites as upper and lower transmembrane gates. The acidic side chain of E2757, which is the only negatively charged residue in the inner helix of Piezo2, was assigned to face the central pore axis (Fig. 5b) . Although no density corresponding to cations was observed being coordinated by the three E2757 residues, E2757 is involved in controlling the ion-permeating properties of Piezo2. Piezo1 and Piezo2 had modestly higher Ca ²⁺ permeability relative to Cs ⁺ (P _Ca/P _Cs) . When mutated to alanine, the E2757A mutant had ～50%reduced P _Ca/P _Cs (Piezo2 vs E2757A: 1.34 ± 0.01 vs 0.63 ± 0.04) and ～30%decreased single-channel conductance (Piezo2 vs E2757A: 24.1 ± 1.0 pSvs 16.7 ± 0.5 pS) .

The transmembrane pore is continued into the cytosol as an intracellular vestibule followed with

-long constriction neck formed by residues M2767 in the linker connecting the inner helix to the intracellular CTD and P2810 and E2811 at the CTD ^α2-3-turn (Fig. 5a, b, c) . A similar constriction neck formed by the corresponding residues M2493, P2536 and E2537 has been previously observed in the Piezo1 structures ^18, 20, 21 (Fig. 5b, c) . Thus, this constriction neck might function as the cytosolic gate of Piezo1 and Piezo2. In contrast to the role of E2757 in the inner helix in determining Ca ²⁺ permeability, neither Piezo2-E2811A nor Piezo1-E2537A had altered permeability of Ca ²⁺ over Cs ⁺, indicating that this position might not play a major role in controlling cation selectivity.

In addition to the continuous central pore along the vertical axis, the intracellular vestibule has three apparent fenestration sites, which are open to three lateral portals. Similar intracellular fenestrations and lateral portals exist in the Piezo1 structure. Residues E2769 and E2770 following the inner helix of Piezo2 or the corresponding E2495 and E2496 of Piezo1 form a negative electrostatic potential surface in the lateral portal connecting to the intracellular vestibule (Fig. 5a, b) . Mutating these residues affects the ion permeation properties of Piezo1 and Piezo2 ³⁰, suggesting that the lateral portal might form an ion-conducting pathway in addition to the central vertical pore. However, given that these two residues are located near the M2767 cytosolic gate (Fig. 5a, b) , it remains possible that they might allosterically affect ion conduction through the central pore.

Structural comparison of the transmembrane gate and the Cap between Piezo1 and Piezo2

Given the structural and functional resemblance between Piezo1 and Piezo2, we have reasoned that conformational difference between the Piezo1 and Piezo2 structures might provide insight into the mechanogating process of the Piezo channel. Therefore, we aligned the Piezo1 structures previously determined in either the detergent digitonin (PDB: 6b3r) , glycol-diosgenin (the synthetic substitute for digitonin) (PDB: 6bpz) , or C ₁₂E ₁₀ (PDB: 5z10) with the Piezo2 structure in glycol-diosgenin. Compared to Piezo2, the three Piezo1 structures show apparent displacement of the Cap, distal Blade, and Beam domains. The clockwise twist and upward displacement of the Cap is more robust in Piezo1 (PDB: 6b3r) and Piezo1 (PDB: 6bpz) than in Piezo1 (PDB: 5z10) , while the lateral displacement of the distal Blade of TM13-24 is relatively stronger in Piezo1 (PDB: 5z10) than in Piezo1 (PDB: 6b3r) [TM13-24 of Piezo1 (PDB: 6pbz) was not modeled] . Importantly, the transmembrane gate, but not the cytosolic gate, is dilated in all three Piezo1 structures, suggesting that the distinct conformational states of the transmembrane pore between the Piezo1 and Piezo2 structures are unlikely due to the use of different detergents. In contrast to Piezo1, Piezo2 was reported to be insensitive to stretch simulation. Indeed, in our studies, compared to near 100%of stretch-responsiveness of Piezo1-expressing cells, only ～25%of Piezo2-expressing cells (16 out of 62) generated stretch–activated currents, despite that Piezo1 and Piezo2 similarly mediated poking-evoked whole-cell currents (Fig. 6h) . Therefore, we have speculated the possibility that surface tension generated during cryo-EM sample preparation might lead to opening of the transmembrane pore of Piezo1, but not Piezo2. Nevertheless, dilation of the transmembrane gate of Piezo1 is closely correlated with the displacement of the Cap among the three Piezo1 structures. For instance, compared to Piezo1 (PDB: 5z10) , Piezo1 (PDB: 6b3r) and Piezo1 (PDB: 6bpz) show relatively stronger displacement of the Cap (eg. the CED ^{α1-α2 helix}) and concurrent larger expansion of the upper transmembrane gate of L2469. Given that both Piezo1 (PDB: 6b3r) ²⁰ and Piezo2 were determined using similar detergents (digitonin and its synthetic substitute glycol-diosgenin, respectively) and cryo-EM sample preparation procedures and that Piezo1 (PDB: 6b3r) has a relatively more expanded transmembrane pore than Piezo1 (PDB: 5z10) , we focused on making a detailed structural comparison between Piezo1 (PDB: 6b3r) and Piezo2 in Fig. 5 and Fig. 6.

When viewed from the side, the Capof Piezo1 is shifted upward and laterally toward the central axis, accompanied with an upward displacement of the outer helix and inner helix (Fig. 6b) . When viewed from the top and zoomed in at the Cap ^{α1-α2 helix}-positioned plane (Fig. 6a) , the Cap of Piezo1 twists in a clockwise direction relative to Piezo2 (Fig. 6a) . When zoomed in at the top position of the inner helix, the outer portion of the inner helix and outer helix as well as the upper transmembrane gate of L2743 show an apparent outward expansion in a clockwise direction, while the neighboring TM25-36 show relatively subtle and anti-clockwise motion (Fig. 6c) . Similar changes in the inner helix and the upper transmembrane gate are observed when only the inner helix and the following C-terminal domain of Piezo2 was superimposed with that of the Piezo1 structure (Fig. 5d) . Compared to Piezo2, the outer portion of the inner helix of Piezo1 is outwardly tilted, resulting in its most outer part with an outward expansion of about a helical width of

(Fig. 5d) . These changes result in an expansion of the upper gate of L2743 from a radius of

in Piezo2 to

of L2469 in Piezo1 (Fig. 5c, e) .

V2750 of Piezo2 shows an outward displacement associated with the slight displacement of the main chain, leading to an increase of the radius of

of this position in Piezo2 to

of V2476 in Piezo1 (Fig. 5f) . Based on the permeability of organic cations such as tetramethyl ammonium (TMA) and tetraethyl ammonium (TEA) to Piezo1, a conducting pore of Piezo1 is expected to have a radius of larger than

Thus the position at Piezo2-V2750/Piezo1-V2476 appears not to be fully expanded (Fig. 5b, f) . Piezo2-V2750/Piezo1-V2476 has been shown to control channel inactivation kinetics and therefore proposed to form a hydrophobic inactivation gate. Thus, Piezo1-V2476 could represent an inactivated state.

At the lower transmembrane gate, the bulky benzyl group of Piezo2-F2754 cuts right into the transmembrane pore, blocking the ion-conducting pathway of Piezo2 (Fig. 5g, 6d) . Interestingly, despite little change of the main chain at this position, the benzyl group of Piezo1-F2480 is rotated away from the central axis, resulting in an expansion of the pore radius from

of Piezo2 to

of Piezo1 (Fig. 5c, g and Fig. 6d) . Notably, Piezo2-F2754/Piezo1-F2480 might form a cation-πinteraction with Piezo2-K2753/Piezo1-K2479 from the neighboring subunit (Fig. 5g showing the density of F2754 and K2479) . Piezo2-K2753/Piezo1-K2479 appears to move along with the main chain, which might cause the rotation of the side chain of the neighboring Piezo2-F2754/Piezo1-F2480 (Fig. 5g, 6d) . Interestingly, K2479 has been identified as a critical component for controlling voltage-dependence of the inactivation kinetics, raising the intriguing possibility that K2479 might affect F2480-mediated gating in a voltage-dependent manner. The three Piezo2-E2757 residues form a constriction with a radius less than

in the closed state (Fig. 5b, c, h) . The corresponding residue in Piezo1 is G2483 (Fig. 5a, h) , which lacks side chain and therefore accounts for a pore radius of

at this position. Interestingly, the Piezo1-E2487 residue located one helical below is in a dilated position relative to Piezo2-E2757 (Fig. 5h) . The cytosolic gate of Piezo1-M2493/P2536/E2537 and Piezo2-M2767/P2810/E2811 are largely superimposed in a closed state (Fig. 5i) .

Notably, the domains surrounding the lower transmembrane gate, including the Anchor, outer helix, TM33-36 and the Clasp domain, appear to move laterally as a whole in an anti-clockwise manner (Fig. 6d) , which is not in line with the motion feature of the transmembrane gates. However, the upward displacement associated with the Beam, CTD, Anchor and outer helix might contribute to the upward displacement of the inner helix and the transmembrane gates (Fig. 6e, f) . Thus, the expansion of the outer portion of the inner helix and opening of the transmembrane gate of Piezo1 relative to Piezo2 is closely associated with conformational change of the extracellular Cap.

In line with the structural analysis, we have previously found that the CED-deletion mutant, Piezo1-ΔCED (deletion of residues 2219-2453) , had drastically reduced mechanically activated currents when tested in wild-type HEK293T cells, despite its normal trimeric architectureand plasma membrane expression. To test whether the corresponding CED in Piezo2 might play a similar role, we generated the Piezo2-ΔCED deletion mutant (deletion of residues 2502-2727) . Additionally, given that the CED ^{α1-α2 helix} domain shows large conformational change between Piezo1 and Piezo2 (Fig. 6a) and might contact the extracellular loops of TM31-32 and TM35-36 of the Blade, we also generated the Piezo1-ΔCED ^{α1-α2 helix} (deletion of residues 2253-2274) and Piezo2-ΔCED ^{α1-α2 helix} (deletion of residues 2536-2557) . Remarkably, when tested in the Piezo1 knockout HEK293 cells (Piezo1-KO-HEK) in which the endogenous Piezo1 gene is disrupted ³⁶, none of the deletion mutants, including Piezo1-ΔCED, Piezo1-ΔCED ^{α1-α2 helix}, Piezo2-ΔCED and Piezo2-ΔCED ^{α1-α2 helix}, generated poking-evoked whole-cell currents (Fig. 6h) . Cell surface biotinylation assay revealed relatively normal expression of the mutants in the plasma membrane. Together, these functional results support the structural analysis that motion of the Cap might control the opening of the transmembrane gate of Piezo1 and Piezo2.

Structural comparison of the cytosolic gate and the Blade-Beam pathway between Piezo1 and Piezo2

Compared to Piezo2, the Blades of Piezo1 are curved toward the 3-fold central axis with graded increase in lateral displacement from THU6 to THU4 (Fig. 6a) . For instance, TM16 of Piezo1 has a lateral displacement of about

relative to that of Piezo2 (Fig. 6a) . Furthermore, THU4-6 together with the extracellular loops of TM15-16, TM19-20 and TM23-24 of Piezo1 also show apparent downward displacement (Fig. 6b, e) . The extracellular loop of TM15-16 of Piezo1 has a downward displacement of about

which appears to push TM13-16 of THU4 downward (Fig. 6e) . Together with an upward displacement of the central inner helix and outer helix, the downward displacement of the distal THU4-6 of Piezo1 renders its TM blade flatter than that of Piezo2 (Fig. 6e) . Based on the projection trajectory of the full TM blade of Piezo2, the unresolved THU1-3 of Piezo1 are expected to have an even more dramatic displacement toward the central axis and downward. The intracellular membrane-parallel TM13 ^preα, TM17 ^preα and TM21 ^preα helices of Piezo1 mainly show a lateral displacement toward the central axis relative to that of Piezo2 (Fig. 6c, e) . Overall, such displacement of Piezo1 relative to Piezo2 would effectively reduce the non-planar curvature of the blade of Piezo1, which might concurrently flatten the residing membrane.

The intracellular Beam shows an uneven lever-like displacement with the distal end remaining relatively stable, while the proximal end shifting both laterally and vertically about a helical-width (Fig. 6b) . This results in a switch of the clockwise and downward displacement of the distal THU4-6 (Fig. 6a, b) into a collective anti-clockwise and upward displacement of the central region, including the proximal Beams, THU7-9, Anchors, CTDs, outer helices and inner helices (Fig. 6b, c, d, g) . Such displacement can lead to an upward displacement of both the cytosolic and transmembrane gates of Piezo1 relative to those of Piezo2 (Fig. 6f) . Furthermore, the three Beams positioned underneath the peripheral edges of the CTD planes appear to push the CTDs in an anticlockwise manner, which might twist the centrally located cytosolic gate to open (Fig. 6g) . Despite the apparently observed conformational difference between Piezo1 and Piezo2, the cytosolic gate remains in a closed state (Fig. 5i and 6g) .

The mechanically activated Piezo channel family, including Piezo1 and Piezo2 in mammals, plays essential roles in a broad range of physiological and pathophysiological processes involving mechanotransduction, and represents validated drug targets given their association with human genetic diseases. Thus, it is pivotal to understand how Piezo channels effectively convert mechanical force into ion permeation. Following our previous effort to determine the three-bladed, propeller structure of mouse Piezo1 trimer with 26 resolved TMs in each protomer, here we have for the first time determined the mouse Piezo2 structure with a completely resolved 38-TM topology (Fig. 1-3) . Given their sequence homology and structural resemblance, we conclude that Piezo1 shares a 38-TM topology as well. By possessing a striking 114 TMs in the trimeric channel complex, Piezo channels are membrane proteins with the largest number of TMs. The highly curved and non-planar TM blade composed of 9 repeated THUs represents the signature feature of the Piezo channel family. The Piezo2 structure and its comparison with the previously determined Piezo1 structures have provided important insights into the mechanogating mechanism of the Piezo channel family.

First, the Piezo2 structure has allowed us to clarify the intriguing “membrane dome” and “membrane footprint” hypotheses proposed by Mackinnon and colleagues for explaining the exquisite mechanosensitity of Piezo channels. The “membrane dome” mechanism predicts that the non-planar configuration of the Piezo-membrane system deforms upon channel opening. On the basis of the Piezo1 structure with an incompletely resolved TM blade (lacking the distal 12 TMs in the N-terminal region) , a Piezo1-shaped membrane dome with an under-sized opening diameter of 18 nm and a depth of 6 nm has been estimated to produce a total mid-plane surface area of 400 nm ² and a projected in-plane area of 280 nm ², giving rise to a maximal change of projection area of 120 nm ² when Piezo1 becomes completely co-planar with the membrane. Now on the basis of the complete Piezo2 structure of ～28 nm-diameter and ～10 nm-depth, a bowl-shaped mid-plane surface area of ～880 nm ² and a projected in-plane area of ～600 nm ² are expected to be generated, which could give rise to a maximal projection area of ～280 nm ² when Piezo2 becomes fully planar. Thus, the mechanosensitivity of Piezo channels associated with in-plane membrane expansion is much greater than the previous estimation. Also based on the incomplete structure of Piezo1, membrane mechanical calculations of the Piezo1-membrane system have predicted that Piezo1 deforms the membrane shape outside the perimeter of the channel into a large curved “membrane footprint” , which might amplify the sensitivity of Piezo channels to changes in membrane tension ³⁷. Since the predicted membrane footprint depends on the actual radius of the Piezo shape, an actual larger radius of the Piezo2 structure would make the membrane footprint less prominent. Nevertheless, the nano-bowl shaped Piezo-membrane system might potentially allow a much larger change of membrane area than that of the well-characterized bacteria mechanosensitive channel of large conductance (MscL) , which has an estimated in-plane membrane expansion of ～20 nm ² upon opening. In line with this, Piezo1 has a measured half maximal activation tension (T ₅₀) of ～1.4 mN/m, while MscL has a much larger T ₅₀of ～ 10 mN/m.

Second, the availability of both Piezo1 and Piezo2 structures has now laid a solid structural foundation not only for uncovering their general working principle, but also for dissecting out mechanistic differences that underlie their distinct channel properties and regulations. On the one hand, the similar architecture shared by Piezo1 and Piezo2 indicates similar ion permeation and mechanogating mechanisms. On the other hand, detailed structural difference might account for their distinct channel properties and regulations. Indeed, functional studies have verified the conserved roles of featured structural domains such as the CED (Fig. 6h) and the transmembrane pore in controlling the mechanosensitivity and ion permeation properties of Piezo1 and Piezo2. Even at the physiological level, Piezo1 is sufficient to replace the in vivo function of Piezo2 in mediating the gentle touch and proprioception behaviours of mice. Therefore, it is reasonable to consider that structural comparison between Piezo1 and Piezo2 might reflect the gating process of Piezo channels. Indeed, structural comparison among the Piezo1 structures in different conformations appears to be in line with the structural comparison between Piezo1 and Piezo2. Interestingly, dilation of the transmembrane gates of all the three previously determined Piezo1 structures relative to that of Piezo2 in closed state is closely correlated with the displacement of the Cap, but not with the Blade and those structural components surrounding the inner part of the central pore (Fig. 6) . Thus, we propose that an upward and clockwise motion mode of the Cap might lead to opening of the transmembrane gates of the Piezo channel, which appears to be reminiscent of the gating mechanism of other trimeric channels such as ASIC and ATP-gated P2X, in which ligand binding to their large extracellular domains leads to opening of the pore-lining helices. In line with the importance of the Cap in gating, deleting either the CED or the CED ^α1-α2 ^helix domain abolished mechanical activation of Piezo1 and Piezo2 despite that the mutant proteins had relatively normal plasma membrane expression (Fig. 6h) . Furthermore, chimeric studies have identified that the extracellular CED and the inner helix account for distinct inactivation kinetics among different Piezo channels.

Third, the gating process of the cytosolic gate is relatively less clear because it remains closed in both Piezo1 and Piezo2. Given that all the three Piezo1 structures have open transmembrane gates, but closed cytosolic gate, an intriguing hypothesis is that the cytosolic gate might be separately gated from the transmembrane gate, a previously unrealized dual-gating mechanism. In light of the proposed origin of the tension-gating from the curved Blade-membrane system, we have observed a partially flattened TM blade of Piezo1 relative to that of Piezo2 (Fig. 6e) , which would give rise to an expansion of the projected in-plane membrane area and consequently generate the gating energy to open the cytosolic gate. Therefore, it is intuitive for us to hypothesize that the peripheral Blades might be designated to allosterically control the cytoplasmic gate via levering the intracellular Beam and its associated CTD/Latch/Anchor/outer helix components. In line with this hypothesis, previous functional characterizations have identified regions and residues along the Blade-Beam pathway critical for mechanical-and chemical-activation of Piezo1. Furthermore, numerous human disease-causing mutations are clustered at the Beam/CTD/outer helix/inner helix relaying interfaces. Nevertheless, since the cytosolic gate has not been observed in an open state, whether it is indeed subjected to the proposed gating process needs to be further tested. It is possible that a more robust flattening of the Blade might be needed to open the cytosolic gate. Alternatively, the lack of the residing lipid membrane of the detergent-solubilized Piezo1 or Piezo2 proteins might not allow the highly curved and non-planar Blade to effectively gate the cytosolic gate. It will be of great interest to test whether the Piezo-induced nano-bowl shape exists in native cell membrane and whether it might indeed respond to mechanical stimuli to gate the Piezo channel in living cells.

Reference throughout this specification to “an embodiment, ” “some embodiments, ” “one embodiment” , “another example, ” “an example, ” “aspecific example, ” or “some examples, ” means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present disclosure. Thus, the appearances of the phrases such as “in some embodiments, ” “in one embodiment” , “in an embodiment” , “in another example, ” “in an example, ” “in a specific example, ” or “in some examples, ” in various places throughout this specification are not necessarily referring to the same embodiment or example of the present disclosure. Furthermore, the particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples.

Although explanatory embodiments have been shown and described, it would be appreciated by those skilled in the art that the above embodiments can not be construed to limit the present disclosure, and changes, alternatives, and modifications can be made in the embodiments without departing from spirit, principles and scope of the present disclosure.

Claims

Use ofregulatoror technology in preparation of a medicament , kit or technology foractivating, potentiatingor inhibiting the mechanically-activated Piezo channels, wherein theregulator, kit or technology is used for regulating the Cap domain, the 9 THU-constituted Blade domain, the Blade–Latch-Anchor-OHdomains, or the IH-CTD-constituted pore domain.
Use of claim 1, wherein the Piezo is Piezo1 orPiezo2.
Use of claim 2, wherein the Piezo is from mouse or human beings.
Use of claim 1, wherein regulating the close or open of the IH-CTD-constituted pore domain is achieved by acting on the OH-Cap-IH or THU1-9-Beam-Latch-CTD-Anchor-OH-IH structural domains.
Use of claim 1, wherein regulating the close or open of the IH-CTD-constituted pore domain is achieved by acting on at least one of the following sites or functional domains of Piezo:

(1) the Cap structural domain;

(2) IH-CTD domains;

(3) THU1-THU2-THU3 of the Blade domain;

(4) residues L2743, V2750, K2753, F2754, E2757, G2761, M2767, P2801 or E2811 of the IH-CTD structural domains of mouse Piezo2 or the corresponding residues L2673, V2680, K2683, F2684, E2687, G2691, M2697, P2731 or E2741 of human Piezo2;

(5) residues L2469, V2476, K2479, F2480, G2483, E2487, M2493, P2536, E2537 of the IH-CTD structural domains of mouse Piezo1 or the corresponding residues L2443, V2450, K2453, F2454, G2457, E2461, M2467, P2510, E2511 of human Piezo1.
Use of claim 5, wherein the medicament, kit or technology is used for inhibiting the mechanically-activated Piezo2 channel, wherein the regulator is used to induce conformational changes of the Cap, the IH domain or the transmembrane gate comprising residues L2743, V2750, K2753, F2754, E2757, G2761 of mouse Piezo2 or the corresponding domains and residues of human Piezo2 to close the Piezo2 channel;

or wherein the regulator, kit or technology is used to induce conformational changes of the Blade/Beam/Latch/Anchor/OH domains and the cytosolic gate comprising residues M2767, P2801 or E2811 of the CTD structural domains of mouse Piezo2 or the corresponding domains and residues of human Piezo2 to close the Piezo2 channel.
Use of claim 5, wherein the medicament, kit or technology is used for inhibiting the mechanically-activated Piezo1 channel, wherein the regulator is used to induce conformational changes of the Cap, the IH domain and the transmembrane gate comprising residues L2469, V2476, K2479, F2480, G2483, E2487 of mouse Piezo1 or the corresponding domains and residues of human Piezo1 to close the Piezo1 channel; or wherein the regulator is used to induce conformational changes of the Blade/Beam/Latch/Anchor/OH domains or the cytosolic gate in the CTD domain comprising residues M2493, P2536, E2537 of mouse Piezo1 or the corresponding domains and residues of human Piezo1 to close the Piezo1 channel.
Use of claim 5, wherein the medicament, kit or technology is used for inhibiting the mechanically-activated Piezo2 channel, wherein the regulator is a small molecule, antibody, nano-material, mechanical force, light, temperature, ultrasound or magnet; or oligonucleotide to induce deletion, insertion or mutations of the coding sequence of the Cap, the IH-CTD, the Blade/Beam/Latch/Anchor/OH domains or residues L2743, V2750, K2753, F2754, E2757, G2761, M2767, P2801 or E2811 of the mouse Piezo2 or the corresponding domains and residues of human Piezo2;

or wherein the medicament , kit or technology is used for inhibiting the mechanically-activated Piezo1 channel, wherein theregulator is a small molecule, antibody, nano-material, mechanical force, light, temperature, ultrasound or magnet; or oligonucleotide to induce deletion, insertion or mutations of the coding sequence of the Cap, the IH-CTD, the Blade/Beam/Latch/Anchor/OHdomainsor residues L2469, V2476, K2479, F2480, G2483, E2487, M2493, P2536, E2537of the mouse Piezo1 or the corresponding domains and residues of human Piezo1.
Use of any one claim 6～8, wherein the medicament or technology is used for inhibiting the mechanically-activated Piezo2 channel, wherein the medicament or technology is used for treating and preventing tactile pain, touch and proprioceptive defects, bone deformation, hypertension, respiratory diseases;

or wherein the medicament or technology is used for inhibiting the mechanically-activated Piezo1 channel, wherein the medicament or technology is used for treating and preventing hypertension, defects of blood vessel development, defects of lymphatic vessel development, red blood cell diseases including ammonia and malaria infection, bone loss, urinary dysfunction, cancer development or metastasis.
Use of claim 5, wherein the medicament, kit or technology is used for activating or potentiating the mechanically-activated Piezo2 channel, wherein the regulator is used to induce conformational changes of the Cap, the IH domain or the transmembrane gate comprising residues L2743, V2750, K2753, F2754, E2757, G2761 of mouse Piezo2 or the corresponding domains or residues of human Piezo2 to activate or potentiate the Piezo2 channel;

or wherein the regulator is used to induce conformational changes of the Blade/Beam/Latch/Anchor/OH domains or the cytosolic gate comprising residues M2767, P2801 or E2811 of the CTD structural domains of mouse Piezo2 or the corresponding domains and residues of human Piezo2 to activate or potentiate the Piezo2 channel.
Use of claim 5, the medicament, kit or technology is used for activating or potentiating the mechanically-activated Piezo1 channel, wherein the regulator is used to induce conformational changes of the Cap, the IH domain or the transmembrane gate comprising residues L2469, V2476, K2479, F2480, G2483, E2487 of mouse Piezo1 or the corresponding domains or residues of human Piezo1 to activate or potentiate the Piezo1 channel;

or wherein the regulator is used to induce conformational changes of the Blade/Beam/Latch/Anchor/OH domains or the cytosolic gate in the CTD domain comprising residues M2493, P2536, E2537 of mouse Piezo1 or the corresponding domains and residues of human Piezo1 to activate or potentiate the Piezo1 channel.
Use of claim 5, wherein the medicament , kit or technology is used for activating or potentiating the mechanically-activated Piezo2 channel, wherein the regulator is a small molecule, antibody, nano-material, mechanical force, light, temperature, ultrasound, or magnet; or oligonucleotide to induce deletion or mutations of the coding sequence of the Cap, the IH-CTD, the Blade/Beam/Latch/Anchor/OH domains and residues L2743, V2750, K2753, F2754, E2757, G2761, M2767, P2801 or E2811 of the mouse Piezo2 or the corresponding domains and residues of human Piezo2.
Use of claim 5, wherein the medicament, kit or technology is used for activating or potentiatingthe mechanically-activated Piezo1 channel, wherein theregulator is a small molecule, antibody, nano-material, mechanical force, light, temperature, ultrasound, or magnet; or oligonucleotide to induce deletion、insertionor mutations of the coding sequence of the Cap, the IH-CTD, the Blade/Beam/Latch/Anchor/OHdomains and residues L2469, V2476, K2479, F2480, G2483, E2487, M2493, P2536, E2537of the mouse Piezo1 or the corresponding domains and residues of human Piezo1.
Use of any one claim 10～13, wherein the medicament or technology is used for activating or potentiating the mechanically-activated Piezo2 channel, wherein the medicament or technology is used for treating and preventing tactile pain, touch or proprioceptive defects, hypertension, respiratory diseases;

Or wherein the medicament or technology is used for activating or potentiatingthe mechanically-activated Piezo1 channel, wherein the medicament or technology is used for treating or preventing hypertension, defects of blood vessel development, defects of lymphatic vessel development, red blood cell diseases including ammonia or malaria infection, bone loss urinary dysfunction, cancer development or metastasis.
A method of screening for an agent that modulates the activity of the mechanically activated Piezo channel, the method comprising:

(1) contacting candidate agents with cells expressing Piezo channels, wherein the cells are from mouse or human beings ;

(2) detecting modulation or conformational changes of the Cap or the gates located in the IH and CTD of Piezo1 or Piezo2 before and after the contact of an agent;

wherein the change in conformation, activity or expression of the cap or the gates located in the IH and CTD changes indicates that the candidate agents act as the desired agents.
Method of claim 15, wherein the PiezoisPiezo1 or Piezo2.
Method of claim 15, wherein the cells are prepared as follows:

(1) preparation of the recombinant Piezo1 or Piezo2 proteins using the Piezo2-pp-GST-IRES-GFP or Piezo1-pp-GST-IRES-GFP expression plasmids, which include the mammalian expression promoter, cDNA encoding Piezo2 or Piezo1, a C-terminal Glutathione S-transferase tag or other affinity tag with a precision protease cleavage site in between, followed with the green fluorescent protein (GFP) -coding sequence driven by an internal ribosome entry site IRES, which is designated for monitoring the efficiency of transient transfection and protein expression;

(2) the selection of the detergent glycol-diosgenin, a synthetic substitute for digitonin, for obtaining optimized purified Piezo2 or Piezo1 protein samples.
Method of claim 15, whereinthe modulation or conformational changes of Piezo1 or Piezo2 is observed using the cryo-electron microscopy.
Method of claim 15, wherein the expression vector described above is heterologous to the cell.
Method of claim 15, wherein the activity of the mechanically activated Piezo channel is determined by measuring the electrophysiological change.
The method of claim 20, wherein the electrophysiological change is a change inmembrane potential, a change in current, or an influx of a cationincluding calcium.
The method of claim 20, wherein the electrophysiological change is measured with a patch-clamp assay.
The method of claim 15, wherein the activity of the mechanically activated Piezo channel is measured by the change of the membrane potential using a membrane potential dye assay.
The method of claim 15, wherein the activity of the mechanically activated Piezo channel is determined by measuring the change of intracellular calcium using calcium-sensitive dye or genetically encoded sensors.
The method of claim 15, wherein the cellsare eukaryotic cells.
The method of claim 15, wherein the cellsare neuron.
The method of claim 15, further comprising theconformation or activity or expression of at least one of the following sites or functional areas of Piezo changes indicating that the candidate agents act as the target agents,

(1) the IH-CTD domains;

(2) the THU-constituted Blade domain;

(3) the Beam/CTD/Latch/Anchor/OH domains;

(4) residues L2743, V2750, K2753, F2754, E2757, G2761, M2767, P2801 or E2811 of the IH-CTD structural domains of mouse Piezo2 or the corresponding domains and residues of human Piezo2,

(5) residues L2469, V2476, K2479, F2480, G2483, E2487, M2493, P2536, E2537 of the IH-CTD structural domains of mouse Piezo1 or the corresponding domains and residues of human Piezo1.
An antibodythat binds and modulates the activity of the mechanically activated Piezo channel, including mouse and human Piezo1 and mouse and human Piezo2, wherein the antibody can specifically bind to polypeptide having at least 70%amino acid sequence identity tothe Cap, THU1 to THU9, The Beam, the Latch, the CTD, the Anchor and the IH to activate or potentiate or inhibit the open of the Piezo channel.
The antibody of claim 28, wherein the polypeptide comprisesSEQ ID NO: 1～4.
The antibody of claim 28, wherein the antibody is a monoclonal antibody.
The antibody of claim 28, wherein the antibody is a humanized antibody.
The antibody of claim 28, wherein the antibody is a chimeric antibody.
An isolated antisense oligonucleotide, small interfering RNA, or guide RNA for CRISPR-Cas9-mediated genetic modifiation wherein the antisense oligonucleotide, siRNA or guide RNA inhibits or potentiates production or changes the activity of the mouse and human Piezo1 and Piezo2, wherein the antisense oligonucleotide, siRNA or guide RNA described above can specifically target the Cap, IH, THU1-9, the Blade/Latch/CTD/Anchor/OH, residues L2743, V2750, K2753, F2754, E2757, G2761, M2767, P2801 or E2811 of the IH-CTD structural domains of mouse Piezo2 or the corresponding domains and residues of human Piezo2, and residues L2469, V2476, K2479, F2480, G2483, E2487, M2493, P2536, E2537 of the IH-CTD structural domains of mouse Piezo1 or the corresponding domains and residues of human Piezo1.
A vector comprising the expression cassette of any one claim of 33.
A cell comprising the expression cassette or expression vector of claim 34, wherein the expression cassette or expression vector is heterologous to the cell.
A method of treating Piezo related disease, comprise:

administrating regulator to a subject in need thereof, wherein the regulator is used for regulating wherein the regulator is used for regulating the key structural domains of Piezo, wherein the key structural domains of Piezo including at least one of the following:

Cap, IH, THU1-9, the Blade/Latch/CTD/Anchor/OH, residues L2743, V2750, K2753, F2754, E2757, G2761, M2767, P2801 or E2811 of the IH-CTD structural domains of mouse Piezo2 or the corresponding domains and residues of human Piezo2, or residues L2469, V2476, K2479, F2480, G2483, E2487, M2493, P2536, E2537 of the IH-CTD structural domains of mouse Piezo1 or the corresponding domains and residues of human Piezo1.
Method of claim 36, wherein the Piezo related disease includes at least one of the following:

dehydrated hereditary stomatocytosis, generalized lymphatic dysplasia, distal arthrogryposis type 5, Gordon syndrome and Marden-Walker syndrome, tactile pain, hypertension, respiratory diseases.
Method of claim 36, wherein the regulator used for regulatingthe key structural domainsby acting on at least one of the following sites or functional domains of Piezo:

(1) the Cap structural domain;

(2) IH-CTD domains;

(3) THU1-THU2-THU3 of the Blade domain;

(4) residues L2743, V2750, K2753, F2754, E2757, G2761, M2767, P2801 or E2811 of the IH-CTD structural domains of mouse Piezo2 or the corresponding domains and residues of human Piezo2;

(5) residues L2469, V2476, K2479, F2480, G2483, E2487, M2493, P2536, E2537 of the IH-CTD structural domains of mouse Piezo1 or the corresponding domains and residues of human Piezo1.
Method of claim 36, wherein the regulator is antibody described in claims 28～32 or isolated antisense oligonucleotide, siRNA or guide RNA described in claims 33.