Abstract
OSCA/TMEM63 channels are the largest known family of mechanosensitive channels1,2,3, playing critical roles in plant4,5,6,7 and mammalian8,9 mechanotransduction. Here we determined 44 cryogenic electron microscopy structures of OSCA/TMEM63 channels in different environments to investigate the molecular basis of OSCA/TMEM63 channel mechanosensitivity. In nanodiscs, we mimicked increased membrane tension and observed a dilated pore with membrane access in one of the OSCA1.2 subunits. In liposomes, we captured the fully open structure of OSCA1.2 in the inside-in orientation, in which the pore shows a large lateral opening to the membrane. Unusually for ion channels, structural, functional and computational evidence supports the existence of a ‘proteo-lipidic pore’ in which lipids act as a wall of the ion permeation pathway. In the less tension-sensitive homologue OSCA3.1, we identified an ‘interlocking’ lipid tightly bound in the central cleft, keeping the channel closed. Mutation of the lipid-coordinating residues induced OSCA3.1 activation, revealing a conserved open conformation of OSCA channels. Our structures provide a global picture of the OSCA channel gating cycle, uncover the importance of bound lipids and show that each subunit can open independently. This expands both our understanding of channel-mediated mechanotransduction and channel pore formation, with important mechanistic implications for the TMEM16 and TMC protein families.
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Data availability
The following cryo-EM maps and atomic coordinates have been deposited to EM Data Bank and Protein Data Bank: OSCA1.2-liposome-inside-in (EMD-38200, PDB 8XAJ), OSCA1.2-lipsosome-inside-out (EMD-38503, PDB 8XNG), OSCA3.1-1.1ver-open/open (EMD-38611, PDB 8XRY), OSCA3.1-1.1ver-open/‘desensitized’ (EMD-38612, PDB 8XS0), OSCA1.2-DOPC-1:20-contracted1 (EMD-38614, PDB 8XS4), OSCA1.2-DOPC-1:20-contracted2 (EMD-38615, PDB 8XS5), OSCA1.2-DOPC-1:20-expanded (EMD-38721, PDB 8XVX), OSCA1.2-DOPC-1:50-βCD (EMD-38722), OSCA3.1-2E-closed/open (EMD-38723, PDB 8XVY), OSCA3.1-2E-closed/‘desensitized’ (EMD-38724, PDB 8XVZ), OSCA3.1-GDN (EMD-38725, PDB 8XW0), OSCA3.1-liposome-inside-in (EMD-38726), OSCA1.2-V335W-DDM (EMD-38727, PDB 8XW1), OSCA1.2-DOPC-1-50-contracted (EMD-38728, PDB 8XW2), OSCA1.2-DOPC-1-50-expanded (EMD-38729, PDB 8XW3) and TMEM63B-Digitonin (EMD-38730, PDB 8XW4). Further data that support the findings of this study are available from the corresponding authors upon reasonable request.
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Acknowledgements
We thank T. Walz, J. J. Chou, N. Gao, N. Li and Y. Niu for critical reading and discussing this manuscript. We also thank P. Zhang for sharing A. thaliana cDNA. We thank the Cryo-Electron Microscopy Center at the Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry for help with data collection. Y.Z. is supported by STI2030-Major Projects (grant no. 2022ZD0207400), Shanghai Municipal of Science and Technology Project (grant no. 20JC1419500), Shanghai Key Laboratory of Aging Studies (grant no. 19DZ2260400), Shanghai Municipal Science and Technology Major Project (grant no. 2019SHZDZX02) and the Science and Technology Commission of Shanghai Municipality. C.D.C. is supported by an Australian Research Council Future Fellowship (grant no. FT220100159). R.J. and B.C. are supported by an Australian Research Council discovery project (grant no. DP200100860). This research project was undertaken with the assistance of resources and services from the National Computational Infrastructure and the Pawsey Supercomputing Research Centre, which are supported by the Australian Government and the Australian Government and the Government of Western Australia, respectively. X.Y. is supported by STI2030-Major Projects (grant no. 2022ZD0207400). Z.C. is supported by Young Scientists Fund of the National Natural Science Foundation of China (grant no. 32101213).
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Y.H. performed protein purification, nanodiscs reconstitution, cryo-EM sample preparation, data collection and image processing. Y.H., Y.S. and Y.Z. performed structural analysis. F.D. assisted the protein purification of OSCA 3.1. X.J., X.M. and Y.H. optimized the proteoliposome reconstitution conditions for cryo-EM study. Y.G., Z.C. and L.Y. assisted in the lifetime measurement of tension probe. R.J., S.H. and B.C. performed molecular dynamic simulations. Z.Z. and C.D.C. conducted and analysed electrophysiology experiments. Y.W., W.Y. and X.Y. assisted in functional studies. Y.Z., C.D.C. and B.C. conceived and supervised the project. All authors wrote and approved the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Global architecture of OSCA channels and data processing of OSCA1.2-DOPC-βCD.
a, Atomic model of OSCA1.2 (PDB:6MGV) shown in side (left) and top (right) views. The numbering of transmembrane helices TM0-TM10 and the cytoplasmic helices H3 and H4 are labeled. The cleft angle is measured between the two TM3 helices (green dashed line) in each subunit. b, A representative cryo-EM image of OSCA1.2 in DOPC nanodiscs after βCD treatment. Some particles are circled. Scale bar: 50 nm. c, Image-processing workflow of OSCA1.2-DOPC-βCD with Relion and CryoSPARC. See Methods and Extended Data Table 1, 2 for details. d, Gold-standard FSC curves calculated between the independently refined half maps of OSCA1.2-DOPC-βCD indicate a resolution of 6.28 Å (FSC = 0.143).
Extended Data Fig. 2 Data processing of OSCA1.2 in nanodiscs.
a, A representative cryo-EM image of OSCA1.2 in DOPC nanodiscs with a msp:lipid ratio of 1:50. Some particles are circled. Scale bar: 50 nm. b, Image-processing workflow of OSCA1.2-DOPC-1:50 with Relion and CryoSPARC. See Methods and Extended Data Table 1, 2 for details. c, Gold-standard FSC curves calculated between the independently refined half maps of OSCA1.2-DOPC-1:50 in the contracted state indicate a resolution of 3.59 Å (FSC = 0.143) and in the expanded state indicate a resolution of 3.63 Å (FSC = 0.143). d, Image-processing workflow of OSCA1.2 in DOPC nanodiscs reconstituted with a msp:lipid ratio of 1:20. Green box represents the contracted2 state. Salmon box represents contracted1 state. Blue box represents the expanded state. e, Gold-standard FSC curves calculated between the independently refined half maps for OSCA1.2-DOPC-1:20 in contracted2 state (left) and expanded state (right). f, The tentative density of TM6. A rod-shaped density (magenta) is located above the dilated pore in the contracted2 state of OSCA1.2. The map was low-pass filtered to facilitate the visualization of this density. This density is only visible at a high threshold, indicating its weak density level. Based on the location and orientation, this density is tentatively assigned as TM6. g, Dilation of the pore. The pore forming helices TM3-7 in closed (salmon) and activated (green) subunits are shown in ribbon. The residues on the ion permeation pathway are shown in magenta stick. h-i, Image-processing workflow of OSCA1.2 in DOPC (h) and azolectin (i) nanodiscs reconstituted with different msp:lipid ratios. Asterisk represents the class in which one protomer exhibits weaker density, but the overall conformation remains the same as that of the contracted1 state. j, The particle distribution of OSCA1.2 in different conformations was analyzed using the particles in green, salmon, and blue boxes shown in the image-processing workflow. k, The gel filtration profiles of nanodiscs-embedded OSCA1.2 reconstituted with different MSP:lipid ratios.
Extended Data Fig. 3 Data processing of OSCA1.2 in liposome.
a, Representative inside-out recordings of OSCA1.1 (blue, n = 6), OSCA1.2 (red, n = 6) and OSCA3.1 (olive, n = 6) expressed in Piezo1−/− HEK293T cells at −40 mV holding potential with vector only transfected cells as a negative control (black, n = 6). Inside-out patches were perfused with 250 µM of lysophosphatidylcholine 12:0 (LPC12:0) which elicited gating of OSCA1.1 and OSCA1.2 in the absence of applied pressure but failed to generate any current prior to patch rupture in OSCA3.1 or vector only transfected cells. Quantification of peak currents elicited by LPC12:0 from replicate cells are shown on the right side. The error bars represent the S.E.M. The p-values were determined using one way ANOVA with Dunnett’s multiple comparison. b, Image-processing workflow of OSCA1.2-azolectin-1:40-LPC with Relion and CryoSPARC. See Methods and Extended Data Table 1, 2 for details. c, Gold-standard FSC curves calculated between the independently refined half maps of OSCA1.2-azolectin-1:40-LPC in the expanded state indicate a resolution of 4.01 Å (FSC = 0.143) and in the contracted2 state indicate a resolution of 3.83 Å (FSC = 0.143). d, A representative cryo-EM image of OSCA1.2-liposome. Scale bar: 50 nm. e, Image-processing workflow of OSCA1.2-liposome with Relion and CryoSPARC. See Methods and Extended Data Table 1, 2 for details. f, Gold-standard FSC curves calculated between the independently refined half maps of OSCA1.2 in inside-in orientation indicate a resolution of 3.29 Å (FSC = 0.143) and in inside-out orientation indicate a resolution of 3.56 Å (FSC = 0.143). g, Cryo-EM map (upper panel, top view) and atomic model (lower panel, side view) of OSCA1.2 in inside-out and inside-in orientation. The membrane density is shown in transparent orange. h, Membrane blocks H3-H4 inwardly movement. The atomic model of OSCA1.2 closed subunit (grey) is aligned to OSCA1.2-inside-in open subunit (forest green). The membrane density is shown in transparent orange. i, TM6b moved 5 Å outward from closed state to open state.
Extended Data Fig. 4 Data processing of TMEM63B-digitonin, TMEM63B-nanodiscs, and OSCA1.2-V335W-DDM.
a, A rightward shift is observed in the gel filtration profile of TMEM63B as compared to OSCA1.2. TMEM63B proteins were examined by SDS-PAGE gel. b, Image-processing workflow of TMEM63B-digitonin with Relion and CryoSPARC. Scale bar: 50 nm. c, Gold-standard FSC curves (FSC = 0.143) calculated between the independently refined half maps for TMEM63B-digitonin. d, Image-processing workflow of TMEM63B in azolectin nanodiscs. Scale bar: 50 nm. Though the 2D averages and 3D maps show smeared features, a monomer class was observed in the 3D classes. e, Dimerization interface of OSCA1.2 cytoplasmic domain. A construct of V335W was designed to abolish OSCA1.2 dimerization. f, A rightward shift is observed in the gel filtration profile of OSCA1.2-V335W as compared to OSCA1.2-WT. g, A representative cryo-EM image of OSCA1.2-V335W-DDM. Some particles are circled. Scale bar: 50 nm. h, Image-processing workflow of OSCA1.2-V335W-DDM with Relion and CryoSPARC. i, Gold-standard FSC curves (FSC = 0.143) calculated between the independently refined half maps for OSCA1.2-V335W-DDM.
Extended Data Fig. 5 Permeation pathway of OSCA1.2 in open conformation.
a-c, The membrane wall at the lateral opening of the pore shown in top (a), side (b), and lateral (c) views. The model of OSCA1.2-inside-in structure is shown in surface. The cryo-EM density of the membrane was lowpass filtered and shown in orange. d-f, The membrane wall formed at the lateral opening in MD simulation shown in top (d), side (e), and lateral (f) views. Averaged volumetric density of PC head groups is depicted in solid orange with a resolution of 0.5 Å. This data was calculated using simulation trajectories of all DOPC head groups within the simulation box. The averaged volumetric density map of water molecules inside the pore is displayed in solid cyan, with a resolution of 1 Å. g, Lateral view show the head groups of membrane lipids lining the pore. The head groups of wall lipids are represented in thick orange licorice while the lipidic acyl tails of wall lipids are represented in thin yellow licorice. The bulk lipids are shown in grey. The averaged volumetric density map of water molecules inside the pore is displayed in a transparent cyan surface. OSCA1.2 is depicted in dark green cartoon with only one subunit shown for a clear view. h-i, Permeation pathway in OSCA1.2 closed structure (h) and OSCA1.2-inside-in open structure with the membrane wall (i). j, Pore radius and hydrophobicity were plotted using MOLEonline for OSCA1.2 in closed and open conformations. k, Averaged number of wall lipids along the conduction pathway in MD simulation (n = 6). The error bars represent the standard deviations. The p-values were calculated using unpaired t-test. l, Summary of the averaged number of water molecules between the height of F515 and Y519 at different transmembrane potentials calculated by multiplying the height of the simulation cell by the field strength (0.017 V/nm, 0.033 V/nm, and 0.05 V/nm, respectively). Each condition of transmembrane potential is represented by n = 6 samples, and ns indicates non-significant results. The error bars represent the standard deviations. The p-values were calculated using unpaired t-test. m, Sodium ion permeation events recorded in MD simulations under different transmembrane potentials (cyan: 0.23 V, blue: 0.46 V, and dark blue: 0.69 V). n, Comparison of unitary conductance of OSCA1.2 reconstituted in azolectin (Azo) or dioleoylphosphatidylcholine (DOPC)/dioleoylphosphatidylethanolamine (DOPE)/ dioleoylphosphatidylglycerol (DOPG) at a 4:4:2 ratio and recorded in symmetrical KCl 200 mM (n = 5) or a buffer containing KCl 200 mM and MgCl2 40 mM (n = 6 for Azo, n = 3 for PE/PC/PG). The error bars represent the S.E.M. The p-values were calculated using unpaired t-test; ***P < 0.001, ****P < 0.0001. o, Comparison of single channel amplitudes at two holding voltages in azoelctin liposomes (C – closed, O – open).
Extended Data Fig. 6 Data processing of OSCA3.1 in detergent, nanodiscs, and liposomes.
a-g, Image-processing workflow of OSCA3.1-GDN (a), OSCA3.1-azolectin-1:40 (b), OSCA3.1-azolectin-1:20 (c), OSCA3.1-azolectin-1:10 (d), OSCA3.1-PG10-1:20 (e), OSCA3.1-LPC9-1:20 (f), and OSCA3.1-liposome (g) with Relion and CryoSPARC. Gold-standard FSC curves (FSC = 0.143) are displayed beside the refined maps. The atomic models of OSCA3.1 in closed (red) and open (green) states are fitted into the obtained OSCA3.1-inside-in structure, thereby showing that the structure is in a closed state (g). h, The particle distribution of OSCA3.1 in different conformations. AZO, azolectin. i, A Zoom-in of a dimer of dimer class obtained during 3D classification in OSCA3.1-GDN dataset (a). The top view of the cryo-EM map (left) and the side view of the model (right) shows the tetramer consists of two dimers, colored in gold and cyan, respectively.
Extended Data Fig. 7 The lipids in cryo-EM maps of OSCA channels.
a, Lipid and acyl chain densities (magenta) are visible in cryo-EM maps of OSCA channels in both detergent and nanodiscs. b, The pore exit lipids (magenta) in OSCA1.2 closed subunit (OSAC1.2-DOPC-1:20-contracted1 and OSCA1.2-liposome-inside-out-closed) and ‘activated’ subunit (OSAC1.2-DOPC-1:20-contracted2 and OSCA1.2-liposome-inside-in-open). c, The lipid bound in the lower cleft of OSCA3.1 in detergent and nanodiscs. d, The charged residues in the lower cleft of OSCA channels. The cyan circles indicate the unique positive charged residues in OSCA3.1 as compared to OSCA1.1 and OSCA1.2. The red circles indicate the unique negative charged residues in OSCA1.1 and OSCA1.2 as compared to OSCA3.1. e, Sequence alignment of residues on cleft facing helices in OSCA3.1, OSCA1.1 and OSCA1.2. The unique positive charged residues (cyan) in OSCA3.1 and unique negative charged residues (red) in OSCA1.1 and OSCA1.2 are highlighted. There residues are circled in (d). The residues sandwiching the bound lipid in OSCA3.1 are shown in blue boxes.
Extended Data Fig. 8 Structure determination of OSCA3.1 mutants.
a-b, Image-processing workflows of OSCA3.1-R611E/R619E-DDM (a) and OSCA3.1- Y367N/G454S/Y458I-DDM (b). The red arrows indicate the classes exhibit a conformational change of TM6. c, The pore radius along the permeation pathway in OSCA3.1 closed subunit, OSCA3.1-1.1ver open subunit, OSCA3.1-1.1ver desensitized subunit, and OSCA1.2-liposome open subunit. d-e, Permeation pathway in OSCA3.1 closed structure (d), OSCA3.1-1.1ver open structure with the membrane wall (e). The membrane wall (orange) was taken from OSCA1.2-liposome-inside-in structure. f, The pore exit lipids (magenta) in OSCA3.1-azolectin closed subunit and OSCA3.1-1.1ver open subunit and OSCA3.1-2E open subunit. g, In the AlphaFold2-predicted TMEM63B structure (yellow), TM3-4 and TM5-6 exhibit bending and expansion compared to the experimentally obtained closed TMEM63B structure (grey). h, The residues at the kink regions on TM3–6 helices are conserved among OSCA/TMEM63 channels. Each of the TM3–6 helices in the OSCA1.2-liposome-open structure (green) is aligned with those in OSCA1.2-closed structures (grey) on the left. Partial sequences of TM3-6 in OSCA/TMEM63 channels are aligned on the right. The residues at the kink regions are colored in pink. i, The groove in activated OSCA channels, AlphaFold2-predicted TMEM63B, and activated TMEM16 family members. The atomic model of OSCA1.2-liposome-open, OSCA3.1-1.1ver-open, AlphaFold2-predicted TMEM63B, afTMEM16 (PDB: 7RXH), and nhTMEM16 (PDB: 6QM9) are shown in surface. j, In simulations of an OSCA1.2 monomer starting from the closed nanodisc structure (grey) under a surface tension of 60 mM/m, within 200 ns in 2 out of 10 replicates, the protein transitions to an open conformation (blue) very similar to that seen in the cryo-EM structure determined in liposomes (red, left). The distance between L434 and Y519 was plotted (CB atom to CB atom) against the simulation time (right). These results provide additional evidence that the open conformation we observed in OSCA1.2-liposome and OSCA3.1-1.1ver represents a real or likely open conformation of the pore in response to mechanical stimuli. k, Permeation pathway in OSCA3.1-1.1ver desensitized subunit. l, The orientation of TM6 helix in the lowpass filtered map of OSCA1.2-DOPC-1:20-contracted2 “smeared” subunit (left:solid density, right: transparent density) and in the atomic model of OSCA3.1-1.1ver desensitized subunit (orange ribbon). The cytoplasmic domain was used for fitting. m, The ‘smeared’ subunit in OSCA1.2-DOPC-1:20-contracted2 (orange) exhibits a more profound conformational change than the OSCA1.2-inside-in open structure (green). The structure of a closed OSCA1.2 subunit (grey) was overlaid as a reference. The magenta encircles the helices with only subtle movement.
Extended Data Fig. 9 Generating tension in nanodiscs.
a-b, The amount of lipids per nanodisc decreases as the lipid/MSP ratio in nanodisc assembly decreases. Representative NP80 spectra recorded for nanodiscs reconstituted with azolectin and Liss-Rhod-DOPE mixture (a). The ratio of absorbance at 280 nm (MSP) and at 580 nm (Liss-Rhod-DOPE) was measured to indicate the amount of lipids per nanodisc. Each measurement was repeated three times. The columns show the averages, and the error bars represent the standard deviations (b). The p-values were calculated using unpaired t-test without adjustments for multiple comparisons. c, The lifetime of tension sensitive probe Flipper-TR in nanodiscs assembled with different MSP/lipid ratios (n = 5). A shorter lifetime indicates a higher tensile stress within the nanodiscs. The error bars represent the s.d. of the lifetime at each MSP/lipid ratio. The p-values were calculated using unpaired t-test without adjustments for multiple comparisons. d, The representative 2D averages of MscS-DOPC-1:90, MscS-DOPC-1:30, and MscS-DOPC-1:10. In the sample of MscS-DOPC-1:90, all the side view averages showed a thick transmembrane domain. In the sample of MscS-DOPC-1:30, both thick and thin transmembrane domains were observed in the 2D averages. In the sample of MscS-DOPC-1:10, which contained only a few lipids per nanodiscs, only the 2D averages with a thin transmembrane domain were observed. e, The cartoon model depicts the OSCA channel embedded in small liposomes in both inside-out and inside-in orientations. The blue arrows indicate tension on the outer leaflet of the small liposome, while the red arrows indicate compression in the inner leaflet. Additionally, the OSCA channel in the inside-in orientation experiences curvature mismatch with the surrounding lipid bilayer. f, Image-processing workflow of OSCA1.2-PG10-nanodiscs with Relion and CryoSPARC. None of the 3D classes show an open conformation, indicating membrane thinning or hydrophobic mismatch alone is not enough for OSCA channel opening. Gold-standard FSC curves (FSC = 0.143) are displayed beside the refined maps.
Extended Data Fig. 10 The cleft in OSCA channels.
Characterization of the cleft in OSCA channels was performed on both published structures and the structures obtained in this study. Distances were measured between the residues labeled in the bottom structures. Areas were measured within the colored parallelogram in each model. Cleft angles were determined by measuring the angle between the TM3 helices on each monomer, using Chimera.
Supplementary information
Supplementary Information
Supplementary Text regarding: the unitary conductance of OSCA1.2 in liposomes; the strategies to activate OSCA channels for structural studies; diverse roles of lipid in OSCA mechanotransduction; the expansion and contraction of the OSCA dimer and Supplementary References.
Supplementary Video 1
Top view of the conformational change of OSCA1.2-DOPC-1:20 from the expanded state to the contracted2 state. The video shows the trajectory that morphs between the atomic models of OSCA1.2-DOPC-1:20 in the expanded state, contracted1 state, and contracted2 state. The closed and activated subunits are colored in pink and green, respectively. TM0, TM6, and TM8, which are missing in the contracted2 state, are shown in transparent. The video was recorded from a top view.
Supplementary Video 2
Side view of the conformational change of OSCA1.2-DOPC-1:20 from the expanded state to the contracted2 state. The video shows the trajectory that morphs between the atomic models of OSCA1.2-DOPC-1:20 in the expanded state, contracted1 state, and contracted2 state. The closed and activated subunits are colored in pink and green, respectively. TM0, TM6, and TM8, which are missing in the contracted2 state, are shown in transparent. The video was recorded from a side view.
Supplementary Video 3
Top view of the conformational change of OSCA1.2 from the closed state to the open state. The video shows the trajectory that morphs between the atomic models of OSCA1.2 in the closed state and OSCA1.2-liposome-inside-in open state. The model is colored as in Fig. 2d-e. The video was recorded from a top view.
Supplementary Video 4
Side view of the conformational change of OSCA1.2 from the closed state to the open state. The video shows the trajectory that morphs between the atomic models of OSCA1.2 in the closed state and OSCA1.2-liposome-inside-in open state. The model is colored as in Fig. 2d-e. The video was recorded from a side view.
Supplementary Video 5
Force-induced conformational landscape of OSCA1.2. The video shows the model of OSCA1.2 activation under force.
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Han, Y., Zhou, Z., Jin, R. et al. Mechanical activation opens a lipid-lined pore in OSCA ion channels. Nature 628, 910–918 (2024). https://doi.org/10.1038/s41586-024-07256-9
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DOI: https://doi.org/10.1038/s41586-024-07256-9
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