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3D-MINFLUX nanoscopy reveals distinct allosteric mechanisms for activation and modulation of PIEZO1 by Yoda1

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Verkest, Clement, Roettger, Lucas, Zeitzschel, Nadja, Lechner, Stefan G.·2025·Nature Communications
DOI 10.1038/s41467-025-67610-xPMC PMC12712055

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Abstract

The small molecule Yoda1 has become an indispensable tool for dissecting the role of the mechanically activated ion channel PIEZO1 in physiological and pathological contexts. Previous studies proposed that Yoda1 first binds to a hydrophobic pocket at the interface of the transmembrane helical units 8 and 9 close to the inner leaflet of the membrane to induce conformational flattening and thus channel activation and then transitions to a deeper located binding site with higher affinity in the open conformation. Here, using site-directed mutagenesis, electrophysiology, computational modelling and 3D-MINFLUX nanoscopy, we refine this model by demonstrating that mutation of the previously proposed Yoda1 binding site-I, solely abolishes Yoda1-induced activation and channel flattening but preserves modulation of mechanically-evoked PIEZO1 currents, whereas mutation of F1715, which lines a transient binding cavity accessible only in the flattened PIEZO1 conformation, eliminated modulation without affecting Yoda-induced calcium entry. Thus, our data support a revised model for Yoda’s mode of action that distinguishes discrete allosteric pathways for PIEZO1 activation versus modulation and provides a framework for the design of next-generation use-dependent PIEZO1 modulators.

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Figures

Fig. 1 Mutation of the proposed Yoda1 binding site abolishes activation but not modulation of PIEZO1. A Proposed mechanism of action of Yoda1 on PIEZO1 and close-up view of the previously proposed Yoda1 binding sites in sphere representation. B time courses of mean ± SEM Ca2+-influx (F/F0, GCamp8) evoked by 10 and 100 µM Yoda1 in cells expressing PIEZO1 (left) and P1-A2094W (center) and comparison (two-sided Mann–Whitney test) of the mean ± SEM maximum responses (right). “N” denotes the numbers of coverslips and “n” denotes the total number of cells. Bars (right panel) represent means ± SEM and circles show the mean population responses of individual coverslips (N-numbers provide in bars). C Modulation of PIEZO1 (top) and P1.A2094W (bottom) stretch-evoked currents by 30 µM Yoda1. Example traces evoked by incrementing pressure stimuli (left), comparison of peak/sustained ratio (middle) using two-sided Mann–Whitney test (PIEZO1: CTL = 4.02, N = 14 vs Yoda1 = 1.38, N = 15, P = 0.0000131) and Student’s t-test (P1.A2094W: CTL = 8.35, N = 10 vs Yoda1 = 1.19, N = 10, P = 0.0033), normalized pressure-response curves (bottom right) and comparison of P50 values with and without of Yoda1 using two-sided Student’s t-test (PIEZO1: CTL = 26.1 ± 8.9 mmHg, N = 14 vs Yoda1 = −16.9 ± 6.65 mmHg, N = 13, P = 0.006) and Mann–Whitney test (P1.A2094W: CTL = −43.7 ± 10.04 mmHg, N = 10 vs Yoda1 = −27.4 ± 6.7 mmHg, N = 10, P = 0.0005). D Modulation of PIEZO1 (top) and P1.A2094W (bottom) poking-evoked currents by 30 µM Yoda1. Example traces evoked by incrementing poking stimui (Δ 400 nm, left), comparison of inactivation time constants obtained with exponential decay fit (middle) using two-sided Mann–Whitney test (PIEZO1: CTL = 15.9 ± 4.3 ms, N = 14 vs Yoda1 = 41.1 ± 21.6, N = 12, P = 0.000258) and Student’s t-test (P1.A2094W: CTL = 6.39 ± 2.8 ms, N = 18 vs Yoda1 = 8.3 ± 2.7, N = 20, P = 0.0405), displacement-response curves (bottom, right) and comparison of mechanical activation thresholds using two-sided Mann–Whitney test (PIEZO1: CTL = 4.1 ± 1.2 µm, N = 14 vs Yoda1 = 2.8 ± 1.4 µm, N = 12, P = 0.029; P1.A2094W: CTL = 4.8 ± 1.04 µm, N = 18 vs Yoda1 = 3.8 ± 1.1 µm, N = 20, P = 0.0069). N-numbers in (C, D) refer to biological replicates (number of recorded cells). Response amplitudes at different displacements were compared using two-sided Mann–Whitney test without adjustments for multiple comparison and P-values are provided next to symbols in graphs.
Fig. 1 Mutation of the proposed Yoda1 binding site abolishes activation but not modulation of PIEZO1. A Proposed mechanism of action of Yoda1 on PIEZO1 and close-up view of the previously proposed Yoda1 binding sites in sphere representation. B time courses of mean ± SEM Ca2+-influx (F/F0, GCamp8) evoked by 10 and 100 µM Yoda1 in cells expressing PIEZO1 (left) and P1-A2094W (center) and comparison (two-sided Mann–Whitney test) of the mean ± SEM maximum responses (right). “N” denotes the numbers of coverslips and “n” denotes the total number of cells. Bars (right panel) represent means ± SEM and circles show the mean population responses of individual coverslips (N-numbers provide in bars). C Modulation of PIEZO1 (top) and P1.A2094W (bottom) stretch-evoked currents by 30 µM Yoda1. Example traces evoked by incrementing pressure stimuli (left), comparison of peak/sustained ratio (middle) using two-sided Mann–Whitney test (PIEZO1: CTL = 4.02, N = 14 vs Yoda1 = 1.38, N = 15, P = 0.0000131) and Student’s t-test (P1.A2094W: CTL = 8.35, N = 10 vs Yoda1 = 1.19, N = 10, P = 0.0033), normalized pressure-response curves (bottom right) and comparison of P50 values with and without of Yoda1 using two-sided Student’s t-test (PIEZO1: CTL = 26.1 ± 8.9 mmHg, N = 14 vs Yoda1 = −16.9 ± 6.65 mmHg, N = 13, P = 0.006) and Mann–Whitney test (P1.A2094W: CTL = −43.7 ± 10.04 mmHg, N = 10 vs Yoda1 = −27.4 ± 6.7 mmHg, N = 10, P = 0.0005). D Modulation of PIEZO1 (top) and P1.A2094W (bottom) poking-evoked currents by 30 µM Yoda1. Example traces evoked by incrementing poking stimui (Δ 400 nm, left), comparison of inactivation time constants obtained with exponential decay fit (middle) using two-sided Mann–Whitney test (PIEZO1: CTL = 15.9 ± 4.3 ms, N = 14 vs Yoda1 = 41.1 ± 21.6, N = 12, P = 0.000258) and Student’s t-test (P1.A2094W: CTL = 6.39 ± 2.8 ms, N = 18 vs Yoda1 = 8.3 ± 2.7, N = 20, P = 0.0405), displacement-response curves (bottom, right) and comparison of mechanical activation thresholds using two-sided Mann–Whitney test (PIEZO1: CTL = 4.1 ± 1.2 µm, N = 14 vs Yoda1 = 2.8 ± 1.4 µm, N = 12, P = 0.029; P1.A2094W: CTL = 4.8 ± 1.04 µm, N = 18 vs Yoda1 = 3.8 ± 1.1 µm, N = 20, P = 0.0069). N-numbers in (C, D) refer to biological replicates (number of recorded cells). Response amplitudes at different displacements were compared using two-sided Mann–Whitney test without adjustments for multiple comparison and P-values are provided next to symbols in graphs.CC BY · PubMed Central
Fig. 2 PIEZO1 flattening exposes putative alternative Yoda1 binding sites lined by F1715. A Side view of curved (PDB:7WTL, top left) and flattened (PDB:7WTU, bottom left) PIEZO1 cryo-EM structure with close-up side (middle) and top (right) view of transmembrane helical units (THU) 8–9 interfaces with the putative binding pockets detected by DoGSite3 algorithm shown in isomesh representation. Amino acid side chains that line the pockets are shown in stick representation. B Close-up views of Yoda1 binding poses detected by AutoDock Vina in the curved (top) and flattened (bottom) PIEZO1 conformation. Note, only the binding poses with the highest scores that align with the binding pockets detected in (A) are shown. Residues that are within a distance of 3.5 Å of Yoda1 are shown in stick representation. Note, F1715 appears to be involved in Yoda1 binding in pocket 2 and pocket 3 in the flat conformation. C AutoDock Vina detects multiple possible docking poses for Yoda1. The bar graph shows the percentage of docking poses in which the indicated amino acids are located within 3.5 Å of Yoda1. Note, F1715 is in close proximity of Yoda1 in 78% of all possible binding poses indicating an important role of F1715 in Yoda1 binding in the flat state.
Fig. 2 PIEZO1 flattening exposes putative alternative Yoda1 binding sites lined by F1715. A Side view of curved (PDB:7WTL, top left) and flattened (PDB:7WTU, bottom left) PIEZO1 cryo-EM structure with close-up side (middle) and top (right) view of transmembrane helical units (THU) 8–9 interfaces with the putative binding pockets detected by DoGSite3 algorithm shown in isomesh representation. Amino acid side chains that line the pockets are shown in stick representation. B Close-up views of Yoda1 binding poses detected by AutoDock Vina in the curved (top) and flattened (bottom) PIEZO1 conformation. Note, only the binding poses with the highest scores that align with the binding pockets detected in (A) are shown. Residues that are within a distance of 3.5 Å of Yoda1 are shown in stick representation. Note, F1715 appears to be involved in Yoda1 binding in pocket 2 and pocket 3 in the flat conformation. C AutoDock Vina detects multiple possible docking poses for Yoda1. The bar graph shows the percentage of docking poses in which the indicated amino acids are located within 3.5 Å of Yoda1. Note, F1715 is in close proximity of Yoda1 in 78% of all possible binding poses indicating an important role of F1715 in Yoda1 binding in the flat state.CC BY · PubMed Central
Fig. 3 Yoda-dependent activation and modulation involve different binding sites. A Time courses of mean normalized fluorescence ± SEM (F/F0) of Yoda2-evoked calcium influxes in N2a-P1KO cells co-transfected with jRGECO1a and PIEZO1 (black), P1.A2094W (yellow) and P1.V1714A_F1715A (red), respectively. “N” denotes the numbers of coverslips and “n” denotes the total number of cells. B Yoda2 dose-response curves, i.e., max. F/F0 (mean ± SEM) vs. Yoda2 concentration, and sigmoidal fits with corresponding EC50 values. N-numbers for (B) are the same as in (A). N-numbers not provided in (A) are: N/n, PIEZO1 (10 nM, 5/321; 300 nM, 3/257; 3 µM, 3/241; 30 µM, 3/238), P1.A2094W (30 nM, 3/277; 300 nM, 3/190; 3 µM, 3/179; 30 µM, 3/250), P1.V1714A_F1715A (10 nM, 4/281; 300 nM, 3/224, 3 µM, 3/267; 30 µM, 3/237). C The left panel shows representative traces of stretch-evoked PIEZO1 (top), A2094W (middle) and V1714A_F1715A (bottom) currents in the absence (CTL) and presence of 30 nM Yoda2. Middle panel shows comparisons of normalized pressure-response curves in the absence (CTL) and presence (Y2) of 30 nM Yoda2, and additional concentrations for PIEZO1 (CTL, N = 17; 10 pM, N = 5, 100 pM, N = 9; 3 nM, N = 7; 30 nM, N = 9; 300 nM, N = 12). Symbols represent means ± SEM normalized peak current amplitudes (I/Imax) and N-numbers indicated in the graphs refer to the number of different cells from which currents were recorded. Pressure-response relationships were fitted separately for each cell with a Boltzmann equation and the mean ± SEM pressures at which half maximal activation was reached (P50) are compared in the bar graphs in the right panel using two-sided Student’s T-test for PIEZO1 and two-sided Mann–Whitney test for A2094W and V1714A_F1715A. D Representative traces of poking-evoked whole-cell currents from PIEZO1 (top left), A2094W (middle left) and V1714A_F1715A (bottom left) without (CTL) and with 30 nM Yoda2. Displacement-response curves of mean ± SEM peak current amplitudes (middle) and comparison of the mean ± SEM threshold with two-sided unpaired t-test (PIEZO1, A2094W) and Mann–Witney test (V1714A_F1715A) (right). Response amplitudes at different displacements were compared using two-sided Mann–Whitney test without adjustments for multiple comparison and P-values are provided next to symbols in graphs.
Fig. 3 Yoda-dependent activation and modulation involve different binding sites. A Time courses of mean normalized fluorescence ± SEM (F/F0) of Yoda2-evoked calcium influxes in N2a-P1KO cells co-transfected with jRGECO1a and PIEZO1 (black), P1.A2094W (yellow) and P1.V1714A_F1715A (red), respectively. “N” denotes the numbers of coverslips and “n” denotes the total number of cells. B Yoda2 dose-response curves, i.e., max. F/F0 (mean ± SEM) vs. Yoda2 concentration, and sigmoidal fits with corresponding EC50 values. N-numbers for (B) are the same as in (A). N-numbers not provided in (A) are: N/n, PIEZO1 (10 nM, 5/321; 300 nM, 3/257; 3 µM, 3/241; 30 µM, 3/238), P1.A2094W (30 nM, 3/277; 300 nM, 3/190; 3 µM, 3/179; 30 µM, 3/250), P1.V1714A_F1715A (10 nM, 4/281; 300 nM, 3/224, 3 µM, 3/267; 30 µM, 3/237). C The left panel shows representative traces of stretch-evoked PIEZO1 (top), A2094W (middle) and V1714A_F1715A (bottom) currents in the absence (CTL) and presence of 30 nM Yoda2. Middle panel shows comparisons of normalized pressure-response curves in the absence (CTL) and presence (Y2) of 30 nM Yoda2, and additional concentrations for PIEZO1 (CTL, N = 17; 10 pM, N = 5, 100 pM, N = 9; 3 nM, N = 7; 30 nM, N = 9; 300 nM, N = 12). Symbols represent means ± SEM normalized peak current amplitudes (I/Imax) and N-numbers indicated in the graphs refer to the number of different cells from which currents were recorded. Pressure-response relationships were fitted separately for each cell with a Boltzmann equation and the mean ± SEM pressures at which half maximal activation was reached (P50) are compared in the bar graphs in the right panel using two-sided Student’s T-test for PIEZO1 and two-sided Mann–Whitney test for A2094W and V1714A_F1715A. D Representative traces of poking-evoked whole-cell currents from PIEZO1 (top left), A2094W (middle left) and V1714A_F1715A (bottom left) without (CTL) and with 30 nM Yoda2. Displacement-response curves of mean ± SEM peak current amplitudes (middle) and comparison of the mean ± SEM threshold with two-sided unpaired t-test (PIEZO1, A2094W) and Mann–Witney test (V1714A_F1715A) (right). Response amplitudes at different displacements were compared using two-sided Mann–Whitney test without adjustments for multiple comparison and P-values are provided next to symbols in graphs.CC BY · PubMed Central
Fig. 4 Direct observation of Yoda1-induced conformational changes in wildtype as well as activation- and modulation-deficient PIEZO1 mutants with 3D-MINFLUX. A Cartoon depicting the overall strategy to resolve Yoda1-induced conformational changes (flattening) measured through changes in interblade distance. Insets illustrate the labelling of PIEZO1 with ALFA tag inserted after H86, and the DNA-PAINT method (left). Individual bound fluorophore is located with high precision in 3 dimensions via 3D-MINFLUX and its iterative process (middle), leading to multiple localisations of the same molecule. B Workflow example showing confocal image of a PIEZO1-ALFA-mGL expressing N2a-P1KO cell (left) and corresponding 3D-MINFLUX localizations (right), colored by Z range. Inset shows a triple-labelled PIEZO1, with the 3D scatter plots of the raw localizations and a superimposed cryo-EM structure. The 2D in-plane projections of the 3D data were fitted with a bivariate Gaussian distribution, with their probability densities, enabling determination of the average interblade distance (right). C Time courses of mean normalized fluorescence ± SEM (F/F0) of 50 µM Yoda1-evoked calcium influxes in N2a-P1KO cells co-transfected with jRGECO1a and PIEZO1 (black), P1.A2094W (yellow) and P1.V1714A_F1715A (red), respectively. “N” denotes the numbers of coverslips in which the effect of Yoda1 was tested and “n” denotes the total number of cells that were tested. D–F In-plane projections of representative trimers examples of PIEZO1 (D), A2094W (E) and V1714A_1715A (F) from cells treated with cytochalasin-D (CTL) or with cytochalasin-D and Yoda1 (50 µM) (left). Comparison of the mean ± SEM interblade distance of the identified trimers after addition of Yoda1 for PIEZO1 (D, N = 93 and 110), A2094W (E, N = 86 and 82) and V1714A_1715A (F, N = 91 and 96), with two-sided Student’s unpaired t-test (right). N-numbers in (D–F) represent the numbers of biological replicates (i.e., numbers of triple-labelled PIEZO channels that were detected).
Fig. 4 Direct observation of Yoda1-induced conformational changes in wildtype as well as activation- and modulation-deficient PIEZO1 mutants with 3D-MINFLUX. A Cartoon depicting the overall strategy to resolve Yoda1-induced conformational changes (flattening) measured through changes in interblade distance. Insets illustrate the labelling of PIEZO1 with ALFA tag inserted after H86, and the DNA-PAINT method (left). Individual bound fluorophore is located with high precision in 3 dimensions via 3D-MINFLUX and its iterative process (middle), leading to multiple localisations of the same molecule. B Workflow example showing confocal image of a PIEZO1-ALFA-mGL expressing N2a-P1KO cell (left) and corresponding 3D-MINFLUX localizations (right), colored by Z range. Inset shows a triple-labelled PIEZO1, with the 3D scatter plots of the raw localizations and a superimposed cryo-EM structure. The 2D in-plane projections of the 3D data were fitted with a bivariate Gaussian distribution, with their probability densities, enabling determination of the average interblade distance (right). C Time courses of mean normalized fluorescence ± SEM (F/F0) of 50 µM Yoda1-evoked calcium influxes in N2a-P1KO cells co-transfected with jRGECO1a and PIEZO1 (black), P1.A2094W (yellow) and P1.V1714A_F1715A (red), respectively. “N” denotes the numbers of coverslips in which the effect of Yoda1 was tested and “n” denotes the total number of cells that were tested. D–F In-plane projections of representative trimers examples of PIEZO1 (D), A2094W (E) and V1714A_1715A (F) from cells treated with cytochalasin-D (CTL) or with cytochalasin-D and Yoda1 (50 µM) (left). Comparison of the mean ± SEM interblade distance of the identified trimers after addition of Yoda1 for PIEZO1 (D, N = 93 and 110), A2094W (E, N = 86 and 82) and V1714A_1715A (F, N = 91 and 96), with two-sided Student’s unpaired t-test (right). N-numbers in (D–F) represent the numbers of biological replicates (i.e., numbers of triple-labelled PIEZO channels that were detected).CC BY · PubMed Central
Fig. 5 Mechanistic model of Yoda’s mode of action. A In the closed state PIEZO1 adopts a curved conformation (PDB:7WLT) in which binding site-I is accessible. Yoda binding to binding site-I induces a conformational change that is characterized by downward tilting of the blade domains, resulting in a flattened conformation (top left and Fig. 4D, E). Flattening also occurs in the presence of mechanical force (bottom left and refs. 16,17). In the flat conformation binding site-II becomes accessible, such that Yoda can either transition from site-I to site-II (top right) or directly binds to site-II (bottom right).
Fig. 5 Mechanistic model of Yoda’s mode of action. A In the closed state PIEZO1 adopts a curved conformation (PDB:7WLT) in which binding site-I is accessible. Yoda binding to binding site-I induces a conformational change that is characterized by downward tilting of the blade domains, resulting in a flattened conformation (top left and Fig. 4D, E). Flattening also occurs in the presence of mechanical force (bottom left and refs. 16,17). In the flat conformation binding site-II becomes accessible, such that Yoda can either transition from site-I to site-II (top right) or directly binds to site-II (bottom right).CC BY · PubMed Central

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Research works printed but not linked: 1

  1. Smith, K. A. et al. Regulation of PIEZO1 channel force sensitivity by interblade handshaking. Sci. Adv. 11, eadt7046 (2025). · DOI

Metadata

Authors
Verkest, C., Roettger, L., Zeitzschel, N., & Lechner, S. G.
Affiliations
University Medical Center Hamburg-Eppendorf (DE)
Corresponding author
Lechner, Stefan G.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2025-12-17
Volume
16
Issue
1
Pages
11192-11192
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-025-67610-x
PMC
PMC12712055
OpenAlex
W4417412685
Cited by
12
References
48
Citations by year
2026: 11 · 2025: 1
FWCI
7.62
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Deutsche Forschungsgemeinschaft (LE3210/3-3)
Topics
  • Life Sciences › Neuroscience › Sensory Systems › Ion Channels and Receptors
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ion channel regulation and function
  • Health Sciences › Medicine › Physiology (medical) › Erythrocyte Function and Pathophysiology
Keywords
Piezo1, Yoda1, allosteric modulation, mechanosensitive ion channels, channel activation, binding site I, calcium influx, channel conformation
MeSH terms
Allosteric Regulation, Allosteric Site, Animals, Binding Sites, Calcium, Cell Line, Humans, Ion Channels, Models, Molecular, Mutation, Protein Conformation, Pyrazines, Thiadiazoles, Ion Channel Gating, Mutagenesis, Site-Directed, Mice, HEK293 Cells

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