Functional conservation and species-specific tuning of PIEZO1 mechanotransduction in birds and humans

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PIEZO1 is a mechanosensitive cation channel that mediates diverse physiological processes in mammals; however, its functional role in birds has not been directly established. Here, we present a comprehensive electrophysiological comparison of chicken PIEZO1 (gPIEZO1) and human PIEZO1 (hPIEZO1) using heterologous expression systems and native human cells. Using whole-cell and excised outside-out patch-clamp recordings, we show that both gPIEZO1 and hPIEZO1 are robustly activated by membrane stretch and by the synthetic PIEZO1 agonist Yoda1, generating characteristic mechanically activated currents that are potentiated by Yoda1 and inhibited by gadolinium. Single-channel analyses revealed comparable unitary currents for mechanically and chemically evoked currents in both orthologs, indicating conservation of core permeation properties. Notably, gPIEZO1 exhibited significantly greater sensitivity to both mechanical stimulation and Yoda1 than hPIEZO1, revealing species-dependent tuning of PIEZO1 mechanosensitivity. Importantly, in human skin keratinocytes, mechanical stimulation and Yoda1 elicited PIEZO1-like currents that were abolished by siRNA-mediated knockdown of PIEZO1, confirming physiological relevance in a native cellular context. Together, these findings establish PIEZO1 as a functional mechanosensor in birds and illustrate conserved yet quantitatively tuned mechanotransduction across vertebrates. General Cell Biology & Physiology PIEZO1 birds mechanosensor human skin keratinocytes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Mechanical forces are fundamental regulators of cellular and tissue physiology across diverse biological systems, contributing to processes such as touch sensation, audition, tissue morphogenesis, and vascular homeostasis. In multicellular organisms, cells continuously experience mechanical stimuli arising from membrane tension, fluid flow, and changes in extracellular matrix stiffness, and convert these forces into biochemical and electrical signals through mechanotransduction mechanisms. Identifying how these mechanisms are conserved and diversified across species is essential for understanding the evolutionary principles underlying mechanosensory biology. Birds experience unique and extreme mechanical demands associated with powered flight, rapid locomotion, and specialized sensory behaviors, making them an informative vertebrate model for exploring how mechanotransduction pathways are evolutionarily tuned to meet distinct biomechanical challenges. PIEZO proteins constitute a family of mechanically activated cation channels that play central roles in mechanotransduction. Since their initial identification, PIEZO1 and PIEZO2 have been shown to mediate mechanically evoked ionic currents in a wide range of cell types and organisms 1 , 2 . PIEZO1, originally characterized in neuronal cells, is now known to be broadly expressed in both neuronal and non-neuronal tissues, including vascular endothelial cells, where it regulates vascular development, blood flow–dependent signaling, and myogenic tone 3 , 4 . Consistent with these diverse functions, PIEZO channels are evolutionarily conserved across vertebrates and invertebrates, and homologs have also been identified in plants and protozoa, indicating that PIEZO-mediated mechanotransduction represents a fundamental biological process 5 . PIEZO channels are directly activated by multiple forms of mechanical force, including membrane stretch, cell-poking by a mechanical probe, shear stress generated by fluid flow, and alterations in membrane tension or stiffness 1 , 3 , 4 , 6 . The functional roles of PIEZO channels have been extensively investigated in several animal models, including mouse 3 , 4 , zebrafish 7 , drosophila 8 , 9 , and duck 10 , revealing their involvement in sensory transduction, development, and tissue homeostasis. The discovery of the synthetic PIEZO1 agonist Yoda1 11 has further accelerated functional studies by enabling chemical activation of PIEZO1 independent of mechanical stimulation, thereby uncovering its contributions to diverse physiological processes such as bladder distention sensing 12 , mechanically induced osteoclastogenesis 13 , and red blood cell volume regulation 14 – 17 . Despite major advances in the structural, biophysical, and physiological characterization of PIEZO channels, comparative analyses of their electrophysiological properties across species remain limited. Studies of other ion channel families highlight the importance of such cross-species comparisons, as channel gating, pharmacological sensitivity, and ion permeability can vary substantially among orthologs. For example, species-dependent differences in agonist sensitivity and ion permeation have been reported for transient receptor potential channels, illustrating how evolutionary divergence can fine-tune channel function while preserving overall mechanistic frameworks 18 , 19 . Such differences not only provide insight into channel structure–function relationships but also inform the development of species-selective pharmacological tools. Among avian species, mechanosensory function of PIEZO channels has been only partially explored. PIEZO2 is functionally expressed in trigeminal ganglia of tactile-foraging ducks, where it responds to mechanical stimulation in a manner comparable to human PIEZO2 10 . In contrast, the expression patterns and functional properties of avian PIEZO1 channel remain largely unexplored, and direct electrophysiological characterization of avian PIEZO1 has not been reported. Here, we compare the electrophysiological characteristics of chicken PIEZO1 (gPIEZO1) and human PIEZO1 (hPIEZO1) using heterologous expression in HEK cells, and we further examine endogenous hPIEZO1 in human keratinocytes as a native cellular model. We show that gPIEZO1 mediates mechanically activated currents with biophysical properties similar to those in hPIEZO1, yet exhibits significantly greater sensitivity to both mechanical stimulation and Yoda1. These findings establish PIEZO1 as a functional mechanosensor in birds and reveal quantitative, species-dependent tuning of PIEZO1 mechanosensitivity, providing new insight into the evolutionary diversification of mechanically gated ion channels. Results Native HEK cells show negligible PIEZO1 activity and the undetectable protein expression Prior to comparing the electrophysiological properties of gPIEZO1 and hPIEZO1 heterologously expressed in HEK cells, we first tested the effects of Yoda1 and positive mechanical pressure in native HEK cells without exogenous PIEZO1 expression (nHEK). As shown in Figures 1A and 1B, application of 3 μM Yoda1 failed to evoke PIEZO1-like currents in nHEK cells. Moreover, when positive mechanical pressures ranging from 2.5 to 100 mmHg were applied to excised outside-out membrane patches from nHEK cell, pressure-induced current activation was minimal and inconsistent (Figure 1C). Even in the presence of 3 μM Yoda1, currents elicited by 20 mmHg were not significantly different from those recorded at 0 mmHg (Figure 1D), suggesting that negligible endogenous PIEZO1 activity in nHEK cells. Consistent with these functional observations, PIEZO1 protein was undetectable in nHEK cells by western blot (WB) analysis (Figure 1E). In contrast, robust hPIEZO1 expression was detected in HEK cells transiently expressing hPIEZO1 (HEK-hPIEZO1) as well as in HaCaT cells, a human keratinocyte cell line (Figure 1E). Together, these data indicate that endogenous hPIEZO1 expression in nHEK cells is minimal, justifying the use of HEK cells as a heterologous expression system for direct comparison of gPIEZO1 and hPIEZO1. Chicken PIEZO1 shows sequence divergence The full-length amino acid (AA) sequence of gPIEZO1 was aligned with that of hPIEZO1 (Supplementary Figure S1). Two structurally important regions of PIEZO1—the Beam-central plug linker and the central plug —were further compared among mouse, human, and chicken orthologs (Figure 1F). While hPIEZO1 exhibited relatively high sequence identity with mouse PIEZO1 (mPIEZO1), gPIEZO1 displayed greater divergence within these regions. Consistent with this sequence divergence, gPIEZO1 expressed in HEK cells (HEK-gPIEZO1) was not detected by an antibody recognizing an hPIEZO1 epitope spanning residues 1275-1540, whereas both heterologously expressed hPIEZO1 in HEK-hPIEZO1 and endogenous hPIEZO1 in HaCaT cells were readily detected (Figure 1F). To assess whether this sequence divergence affects overall channel architecture, we generated a three-dimensional (3D) full-length protomer model of gPIEZO1 (XP_015148150.2, 2492 AAs; orange, Figure 1G) and superimposed it onto the AlphaFold2-predicted structure of hPIEZO1 (AF-Q92508-F1-v4, 2521 AAs; blue). In addition, a trimeric homology model of full-length gPIEZO1 was also constructed (Supplementary FigureS2). These structural analyses propose strong overall conservation of PIEZO channel architecture despite localized sequence divergence. [Figure 1, here] Human PIEZO1 is robustly activated by Yoda1 and positive pressure in heterologous expression To establish a reference for cross-species comparison, we first characterized the electrophysiological properties of hPIEZO1 (Figures. 2, 3, and 6). Under whole-cell patch-clamp conditions, Yoda1 elicited PIEZO1-like currents in HEK-hPIEZO1 cells in a concentration-dependent manner (Figures 2A and 2B). In excised outside-out patch recordings, positive pressure applied to the membrane patch induced PIEZO1-like currents that increased with pressure magnitude (Figures 2C and 2D). Moreover, current activity evoked by 20 mmHg was significantly potentiated by 3 μM Yoda1 and effectively inhibited by 10 μM Gd 3+ , a non-selective cation channel blocker (Figures 2E and 2F). In some outside-out patches, discrete single-channel events were observed at -50 mV following mechanical stimulation or in the presence of Yoda1 (Figure 3A). The unitary current amplitudes induced by mechanical stimulation were comparable to those evoked by Yoda1 (Figure 3B), suggesting that both stimuli activate the same hPIEZO1 channel population. [Figure 2, here], [Figure 3, here] Chicken PIEZO1 is robustly activated by Yoda1 and positive pressure in heterologous expression We next examined the electrophysiological properties of gPIEZO1 using an experimental paradigm analogous to that used for hPIEZO1 (Figures 4, 5, and 6). In whole-cell recordings, Yoda1 induced PIEZO1-like currents in HEK-gPIEZO1 cells in a concentration-dependent manner (Figures 4A and 4B). Similarly, in excised outside-out patches, positive pressure induced PIEZO1-like currents in a pressure-dependent manner (Figures 4C and 4D). Currents evoked by 20 mmHg were significantly potentiated by Yoda1 at concentrations ranging from 0.1 to 3 μM and were inhibited by 3 μM Gd 3+ (Figures 4E and 4F). Single-channel activity was detected at -50 mV in some excised patches following mechanical stimulation or in the presence of Yoda1 (Figure 5A). As observed for hPIEZO1, the unitary current amplitudes induced by positive pressure were comparable to those induced by Yoda1 (Figure 5B), indicating that both stimuli activate gPIEZO1 channels. [Figure 4, here], [Figure 5, here] Chicken PIEZO1 exhibits enhanced mechanical and Yoda1 sensitivity To directly compare gPIEZO1 and hPIEZO1, we analyzed current-voltage (I-V) relationships and stimulus sensitivities (Figure 6). During the application of graded positive pressures, voltage ramp protocols were applied to excised outside-out patches from HEK cells expressing gPIEZO1 or hPIEZO1 (Figure 6A). The resulting I-V relationships were highly similar between the two channels (Figure 6B), indicating conserved permeation properties. In contrast, both mechanical sensitivity (Figure 6C) and Yoda1 sensitivity (Figure 6D) were significantly greater for gPIEZO1 than for hPIEZO1. These results demonstrate that, while gPIEZO1 shares fundamental electrophysiological properties with hPIEZO1, it exhibits enhanced responsiveness to both mechanical stimulation and Yoda1. To explore the evolutionary conservation of gain-of-function (GOF)-associated substitutions, we performed AA sequence alignments across 100 avian PIEZO1 orthologs (Supplementary Table S2), This analysis revealed that six residues corresponding to known GOF-associated positions in hPIEZO1—706E (G718S), 1086V (S1117L), 1554I (T1589I), 2212N (R2245Q), 2351D (G2384S), and 2377R (E2407K) —are recurrently substituted in multiple avian species (Supplementary Fig.S4), suggesting that enhanced PIEZO1 mechanosensitivity may represent an evolutionarily selected feature in birds. [Figure 6, here] Endogenous PIEZO1 mediates mechanically and chemically evoked currents in human keratinocytes We next investigated endogenous hPIEZO1 activity in HaCaT cells (Figures 7 and 8). In whole-cell recordings, application of 3 μM Yoda1 induced PIEZO1-like currents in HaCaT cells (Figures7A and 7B). In excised outside-out patches, positive pressure elicited PIEZO1-like currents in a pressure-dependent manner (Figures 7C and 7D). Currents evoked by 20 mmHg were significantly potentiated by 3 μM Yoda1 (Figures 7E and 7F). Because HaCaT cells also express TRPV4, a mechanoregulated cation channel 20 , 21 , we examined the effect of the TRPV4 agonist GSK1016790A (GSK) and positive pressure in this system. Although 10 nM GSK robustly activated TRPV4-like currents (Figure 7E), applying 20 mmHg did not alter these currents (Figures 7E and 7F). [Figure 7, here] siRNA knockdown confirms that keratinocyte mechanosensitive currents require PIEZO1 In some excised patches, single-channel activity was detected at -50 mV following mechanical stimulation or in the presence of Yoda1 and GSK (Figure 8A). The unitary current amplitudes induced by pressure were comparable to those evoked by Yoda1 (Figure 8B), indicating that mechanical stimulation and Yoda1 activate endogenous hPIEZO1 in HaCaT cells. To further confirm the functional contribution of hPIEZO1, we knocked down PIEZO1 expression in HaCaT cells using Stealth siRNA. After transfection with PIEZO1-trageting siRNA (siRNA), we tested the mechanosensitivity of HaCaT cells compared with negative control RNA (ncRNA). In excised outside-out patches, mechanically evoked responses were significantly attenuated in PIEZO1-knockdown cells relative to ncRNA-treated cells (Figures 8C and 8D), demonstrating that endogenous hPIEZO1 functions as a mechanosensor in HaCaT cells. [Figure 8, here] TRPV4 does not account for pressure-evoked currents under the present stimulation Finally, to assess the mechanosensitivity of hTRPV4 under our stimulation paradigm, we examined HEK cells heterologously expressing hTRPV4 (HEK-hTRPV4, Figure 9). In whole-cell recordings, application of 3 μM Yoda1 failed to evoke a current response, whereas 10 nM GSK robustly activated TRPV4-like currents (Figures 9A and 9B). Consistently, in excised outside-out patches, positive pressures did not induce pressure-sensitive currents in HEK-hTRPV4 cells (Figures 9C and 9D). Even in the presence of 3 μM Yoda1, 20 mmHg failed to evoke mechanically sensitive current components (Figs 9C and 9E). In contrast, 10 nM GSK reliably induced TRPV4-like currents (Figures 9C and 9E), suggesting that hTRPV4 is insensitive to positive pressure under these conditions. [Figure 9, here] Discussion In this study, we provide the first direct electrophysiological characterization of avian PIEZO1 and demonstrate that chicken PIEZO1 (gPIEZO1) functions as a mechanically activated ion channel with properties broadly conserved with human PIEZO1 (hPIEZO1). Using whole-cell and excised patch recordings, we show that gPIEZO1 is robustly activated by both mechanical stimulation and the synthetic PIEZO1 agonist Yoda1. Importantly, while the core electrophysiological features of gPIEZO1 closely resemble those of hPIEZO1, gPIEZO1 exhibits significantly greater sensitivity to both mechanical force and chemical activation. These findings establish PIEZO1 as a functional mechanosensor in birds and reveal species-dependent tuning of PIEZO1 mechanosensitivity. A critical prerequisite for cross-species comparison is an expression system with minimal endogenous PIEZO1 activity. We therefore first assessed whether native HEK cells express functional PIEZO1. Although occasional mechanically evoked currents were observed in excised patches from native HEK cells, these responses were inconsistent and not potentiated by Yoda1. Consistent with these functional observations, hPIEZO1 protein was undetectable by western blotting. These results indicate that endogenous PIEZO1 expression in native HEK cells is negligible, validating HEK cells as a suitable platform for comparative analysis of heterologously expressed human and chicken PIEZO1 channels. Yoda1 has been widely used as a chemical tool to probe PIEZO1 function across species 11 , 22-25 . In the present study, Yoda1 reversibly activated gPIEZO1 in both whole-cell and excised patch configurations, suggesting that Yoda1-responsive structural elements are largely conserved. Two of the three residues previously implicated in Yoda1 sensitivity in mouse PIEZO1 (A1719, A2091, and A2094 26 ) are conserved in gPIEZO1 (A1684 and A2045), whereas the third position is substituted by serine (S2042). Despite this substitution, gPIEZO1 exhibited robust activation at low Yoda1 concentrations, suggesting that this residue contributes only modestly to Yoda1 binding or efficacy. Notably, both gPIEZO1 and hPIEZO1 were significantly potentiated by submicromolar Yoda1 under our recording conditions, whereas higher EC 50 values have been reported in other systems 11 . Differences in experimental conditions, including solution flow-induced shear stress and the high lipophilicity of Yoda1, may contribute to these apparent differences in potency. Despite differences in sensitivity, unitary currents activated by Yoda1 or mechanical stimulation were comparable between gPIEZO1 and hPIEZO1. This similarity is consistent with the conservation of negatively charged residues within the inner helix region that determine unitary conductance, Ca 2+ permeability, and blocker sensitivity 27 . In contrast, residues surrounding the extracellular fenestration site—previously implicated in ion conduction efficiency—are partially diverged between species 27 . Such sequence differences may subtly modulate permeation without altering overall conductance, thereby preserving fundamental channel function while enabling species-specific fine-tuning. In addition to chemical activation, gPIEZO1 displayed greater sensitivity to positive pressure than hPIEZO1 in excised patch recordings. Although variability in mechanically evoked responses was observed across patches, likely reflecting differences in membrane geometry or local mechanical conditions 28 , several key features were conserved: mechanically evoked gPIEZO1 currents were potentiated by Yoda1, inhibited by Gd 3+ , and exhibited unitary currents indistinguishable from those of hPIEZO1. These findings support the conclusion that the core mechanogating mechanism of PIEZO1 is conserved, whereas quantitative differences in force sensitivity reflect evolutionary tuning rather than fundamental mechanistic divergence. Interestingly, several AA substitutions in gPIEZO1 map to positions corresponding to GOF-associated SNPs in hPIEZO1, including residues 706E (G718S 14 ), 1086V (S1117L 14 ), 1554I (T1589I 29 ), 2212N (R2245Q 30 ), 2246S (R2279C 29 ), and 2377R (E2407K 30 ). These substitutions align with mutations previously shown to potentiate PIEZO1 activity in humans (Supplementary Fig.S4). In addition, the polyglutamine-rich region spanning residues 739-750, which contains the mild GOF-associated Q745 deletion in hPIEZO1 (Supplementary Fig.S2), is absent in avian 31 . Together, these sequence features suggest that avian PIEZO1 may have evolved intrinsic structural determinants that favor enhanced channel activity. In mammalian systems, GOF mutations in PIEZO1 have been linked to increased physical performance 32 and altered mechanosensory phenotypes 33 . Although direct causal relationships remain to be established, the enrichment of GOF–associated substitutions in gPIEZO1 raises the possibility that enhanced PIEZO1 mechanosensitivity may contribute to biomechanical adaptations characteristic of birds. This hypothesis provides a testable framework for future studies linking PIEZO1 function to avian physiology and biomechanics. To assess physiological relevance beyond heterologous expression systems, we examined endogenous mechanotransduction in human keratinocytes. In these cells, both Yoda1 and mechanical stimulation elicited inward currents that were abolished by siRNA-mediated knockdown of PIEZO1, demonstrating that endogenous hPIEZO1 mediates these responses. Although keratinocytes also express other mechanosensitive channels such as TRPV4 20 , 21 , TRPV4-mediated currents were insensitive to the positive-pressure paradigm used here, indicating that PIEZO1 is the primary mediator of mechanically evoked currents under these conditions. These findings are consistent with previous reports of functional PIEZO1 expression in keratinocytes and support the relevance of our comparative analysis to a native cellular context 34-36 . In summary, our study establishes avian PIEZO1 as a functional mechanosensor and reveals that PIEZO1 mechanotransduction is both evolutionarily conserved and quantitatively tuned across species. By directly comparing chicken and human PIEZO1 electrophysiology and validating endogenous PIEZO1 function in human cells, we provide insight into how conserved ion channel architectures support diverse mechanosensory demands. These findings lay the groundwork for future studies exploring the evolutionary, structural, and physiological determinants of mechanosensitivity across vertebrates. Methods Reagents The following reagents were used: 2-[5-[[(2,6-Dichlorophenyl)methyl]thio]-1,3,4-thiadiazol-2-yl]-pyrazine (Yoda1, Tocris Bioscience, Bristol, UK), GSK1016790A (GSK, Sigma/Aldrich, St. Louis, MO), and gadolinium chloride (Gd 3+ , Fujifilm, Osaka, Japan). Each reagent was dissolved in the vehicle recommended by the manufacturer. Cell culture Human keratinocytes (HaCaT, American Type Culture Collection, Manassas, VA) and Human embryonic kidney (HEK) cells (Health Science Research Resources Bank, HSRRB, Osaka, Japan) were maintained in Dulbecco’s modified Minimum Essential Medium (D-MEM; Sigma-Aldrich, Tokyo, Japan) supplemented with 10% heat-inactivated fetal calf serum (FCS; Sigma-Aldrich), penicillin G (100 U/mL, Meiji Seika Pharma Co., Ltd., Tokyo, Japan), and streptomycin (100 mg/mL, Meiji Seika Pharma Co., Ltd.). Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO₂. Recombinant expression of PIEZO1and hTRPV4 in HEK cells HEK cells at 40-60% confluency were transiently transfected with pIRES2-AcGFP1 expression plasmids (Takara Bio INC., Shiga, Japan) encoding human PIEZO1 (hPIEZO1, NP_001136336.2, 2521AAs, 37 ), chicken PIEZO1 (gPIEZO1, XP_015148150.2, 2492AAs), or human TRPV4 (hTRPV4, NP_067638, 871AAs 38 ). Transfections were performed using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. The full-length gPIEZO1 coding sequence was amplified from cDNA prepared from total RNA isolated from whole chicken brain tissue (G. gallus domesticus (GSP (Fayoumi breed)). Tissue samples were provided by Avian Bioscience Research Center at Nagoya University through the National Bio-Resource Project of the MEXT, Japan 19 ). For RT-PCR amplification of the gPIEZO1 coding region, six sets of gene-specific primers were designed (primer sequences are listed in Supplementary Table S1). The initial assembly of the gPIEZO1 coding sequence (284 bp), in which identical AA residues are conserved, was performed by a commercial service (Supplementary Table S1, Integrated DNA Technologies, Tokyo, Japan). All constructs were verified by DNA sequencing. The cloned gPIEZO1 construct contained nine amino acid changes: I103V, I541V, S705A, R1150S, R1305H, S1450V, deletion of A1468, H1532R, and deletion of E1791 deletion (2490 AAs). In an alignment across 100 avian PIEZO1 orthologs (Supplementary Table S2), 103V (5%), 541V (67%), 705A (50%), 1305H (100%), deletion of A1468 (57%), and deletion of E1791 deletion (16%) were conserved. Electrophysiological and biochemical experiments were performed within 48 h after transfection. Western blot analysis HEK cells both with or without PIEZO1 expression and HaCaT cells were lysed in 50 μl of lysis buffer containing (in mM) 50 Tris-HCl (pH 8.0), 150 NaCl, 5 EDTA supplemented with 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS, as well as a protease inhibitor cocktail. Lysates were incubated on ice for 30 min with intermittent vortexing at 5-min intervals and subsequently centrifugated at 20000 × g for 30 min at 4 °C. Protein concentrations were determined, and equal amounts of protein (50 µg per lane) were separated on 8% polyacrylamide gels and electro-transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked for 2 h in Tris-buffered saline (TBS) containing 5% skim milk and 0.1% Tween-20 to prevent nonspecific antibody binding. For immunodetection, membranes were incubated overnight at 4 °C with a monoclonal antibody against human PIEZO1 recognizing AAs 1275-1540 (MA5-32876, Invitrogen, 1:1000 dilution). After three washes with TBS containing 0.1% Tween-20, membranes were incubated with a horseradish peroxidase-conjugated IgG antibody (1:5000 dilution). Immunoreactive bands were visualized using enhanced chemiluminescence reagents (Millipore Japan, Tokyo, Japan). To normalize PIEZO1 protein expression, membranes were re-probed with a monoclonal antibody against β-actin (A5441, Sigma-Aldrich, 1:5000 dilution) as a loading control. Chemiluminescent signals were detected using a FUSION SOLO. 7S. EDGE (M&S Instruments Inc., Osaka, Japan). Patch-clamp recordings Whole-cell and excised outside-out patch-clamp recordings were performed as described previously 38 , 39 . Patch pipettes had a resistance of 3–5 MW when filled with the pipette solution. The Cs + -based pipette solution contained (in mM): 110 Cs-aspartate, 30 CsCl, 1 MgCl 2 , 10 HEPES, 1 EGTA, and 2 Na 2 ATP (pH 7.2, adjusted with CsOH). The extracellular solution contained (in mM): 137 NaCl, 5.9 KCl, 2.2 CaCl 2 , 1.2 MgCl 2 , 14 glucose, 10 HEPES (pH7.4, adjusted with NaOH). Membrane currents were amplified using an EPC-800 patch-clamp amplifier (HEKA, Lambrechit, Germany) and digitized at 10 KHz with a 5 kHz low-pass filter using a PCI6229 A/D converter (National Instruments Japan, Tokyo, Japan). Data acquisition and analysis were performed using WinWCPV4.5 for whole-cell recordings and WINEDR3.38 for excised patch single-channel recordings (software developed by Dr. John Dempster, University of Strathclyde, UK). The liquid junction potential (−10 mV) was corrected. For whole-cell recordings, voltage ramp commands from −110 to +90 mV (300 ms) were applied every 5 s from a holding potential of −10 mV. For excised outside-out patch recordings, voltage ramps (300 or 100 ms) were applied every 5 or 10 s from holding potentials of −50 mV (−100 to +100 mV) or -60 mV (−110 to +90 mV), as indicated. Single-channel recordings were digitally filtered at 1 kHz for amplitude histogram analysis. Leak current components were not subtracted. All recordings were performed at 25 ± 1 °C with a perfusion rate of 5 ml min -1 . Positive mechanical pressure was applied to excised outside-out patches using a custom-built pressure application system consisting of a pressure pump (Linicon LV-435A, Nitto Kohki CO. LTD, Tokyo, Japan), a solenoid valve for rapid pressure switching (VY1D00-6M5, SMC, Tokyo, Japan), and a pressure monitor amplifier (PA-011, Nihon Koden, Tokyo, Japan). Molecular Modeling Molecular modeling was performed using UCSF Chimera v1.18 40 . Predicted three-dimensional (3D) structural data for full-length human PIEZO1 (hPIEZO1; AlphaFold DB entry AF-Q92508_F1-v6, 2521 AAs) were obtained from the AlphaFold database 41 , 42 . A homology model of chicken PIEZO1 (gPIEZO1; XP_015148150.2, 2492 AAs) was generated using the SWISS-MODEL server 43 , with the AlphaFold-predicted hPIEZO1 structure as the template. For construction of a trimeric gPIEZO1 model (Supplementary Fig.S3), the cryo-electron microscopy structure of mouse PIEZO1 (mPIEZO1; PDB ID: 6LQI) was used as a template. Structural visualization, alignment, and analysis were performed using UCSF Chimera, Clustal Omega 44 , and Jalview 45 .. RNA interference-mediated knockdown of hPIEZO1 Stealth small interfering RNA (siRNA) duplexes targeting human PIEZO1 (siPIEZO1; Invitrogen, Carlsbad, CA, USA) were used to suppress PIEZO1 expression. The siPIEZO1 sequences were as follows: sense strand, 5’- GCCUCGUGGUCUACAAGAUTT-3’; antisense strand, 5’-AUCUUGUAGACCACGAGGCTT-3’ 37 . A medium GC Stealth RNAi negative control duplex (ncRNA, Invitrogen) was used as a control. HaCaT cells were cultured in 35-mm dishes and washed with antibiotic-free medium 3 h before transfection. The siRNA or ncRNA was transfected using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions, yielding a final siRNA or ncRNA concentration of 50 nM. Cells were incubated for 72-96 h before electrophysiological experiments. Statistical analyses Data are presented as individual data points together with the mean ± s.d. and the interquartile ranges (25 th -75 th percentiles), as indicated. Comparisons between two groups were performed using paired or unpaired two-tailed Student’s t -tests, as appropriate. Comparisons among multiple groups were conducted using one-way ANOVA followed by Tukey’s post hoc test or two-way ANOVA, as specified. Statistical analyses were performed using Origin J9.1 (LightStone, Tokyo, Japan). A P value < 0.05 was considered statistically significant. Sample sizes are indicated in the figures or corresponding legends. Declarations Acknowledgments We thank Dr. J. Dempster (University of Strathclyde, UK) for developing the electrophysiology software (WinWCP and WinEDR) and Dr. Noriyuki Hatano (Aichi-Gakuin University) for technical assistance. We also acknowledge the Avian Bioscience Research Center at Nagoya University for providing chicken biological resources. Author Contributions KM conceived and designed the study, secured funding, coordinated the project, performed electrophysiological experiments, analyzed and interpreted the data, generated figures, and wrote the manuscript. AM and HS performed Western blot analyses. YM generated all plasmid constructs. All authors reviewed and approved the final manuscript. Competing interests The authors declare no competing interests. Additional information Use of AI-assisted technology During the preparation of this manuscript, the authors used ChatGPT to improve language clarify and readability. All content was critically reviewed and revised by the authors, who take full responsibility for the integrity and accuracy of the work. Funding This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS, 20K07599) to K.M. Data availability The data supporting the findings of this study are available from the corresponding author upon reasonable request. In addition, the datasets of gPIEZO1 gene generated and/or analyzed during the current study are available in the DDBJ repository, https://getentry.ddbj.nig.ac.jp/top-e.html, and Accession No. LC915090. Supplementary Information The online version contains supplementary material available. References Coste, B. et al. Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels. Science 330 , 55-60, doi:10.1126/science.1193270 (2010). Coste, B. et al. Piezo proteins are pore-forming subunits of mechanically activated channels. Nature 483 , 176-181, doi:10.1038/nature10812 (2012). Li, J. et al. Piezo1 integration of vascular architecture with physiological force. Nature 515 , 279-282, doi:10.1038/nature13701 (2014). Ranade, S. S. et al. 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Caenorhabditis elegans PIEZO channel coordinates multiple reproductive tissues to govern ovulation. Elife 9 , doi:10.7554/eLife.53603 (2020). Ergin, B. et al. De Novo Cloning and Functional Characterization of a Mechanosensitive Piezo-Like Ion Channel in the Crayfish. Cell Physiol Biochem 57 , 226-237, doi:10.33594/000000640 (2023). Botello-Smith, W. M. et al. A mechanism for the activation of the mechanosensitive Piezo1 channel by the small molecule Yoda1. Nat Commun 10 , 4503, doi:10.1038/s41467-019-12501-1 (2019). Zhao, Q. et al. Ion Permeation and Mechanotransduction Mechanisms of Mechanosensitive Piezo Channels. Neuron 89 , 1248-1263, doi:10.1016/j.neuron.2016.01.046 (2016). Zeitzschel, N. & Lechner, S. G. The activation thresholds and inactivation kinetics of poking-evoked PIEZO1 and PIEZO2 currents are sensitive to subtle variations in mechanical stimulation parameters. Channels (Austin) 18 , 2355123, doi:10.1080/19336950.2024.2355123 (2024). Knight, T. et al. Mild erythrocytosis as a presenting manifestation of PIEZO1 associated erythrocyte volume disorders. Pediatr Hematol Oncol 36 , 317-326, doi:10.1080/08880018.2019.1637984 (2019). Karamatic Crew, V. et al. Missense mutations in PIEZO1, which encodes the Piezo1 mechanosensor protein, define Er red blood cell antigens. Blood 141 , 135-146, doi:10.1182/blood.2022016504 (2023). Maciak, K. et al. Hereditary xerocytosis - spectrum and clinical manifestations of variants in the PIEZO1 gene, including co-occurrence with a novel beta-globin mutation. Blood Cells Mol Dis 80 , 102378, doi:10.1016/j.bcmd.2019.102378 (2020). Passini, F. S. et al. Shear-stress sensing by PIEZO1 regulates tendon stiffness in rodents and influences jumping performance in humans. Nat Biomed Eng 5 , 1457-1471, doi:10.1038/s41551-021-00716-x (2021). Nakamichi, R. et al. The mechanosensitive ion channel PIEZO1 is expressed in tendons and regulates physical performance. Sci Transl Med 14 , eabj5557, doi:10.1126/scitranslmed.abj5557 (2022). Li, W. et al. PIEZO1 promotes psoriasis-like skin inflammation in mice via NF-kappaB/IL-17 signaling pathway activation. Mol Med 31 , 225, doi:10.1186/s10020-025-01279-2 (2025). Li, Y. J. et al. Keratinocyte Piezo1 and CD39 initiated the acupuncture analgesic signals via Co-regulating extracellular ATP mobilization at acupoints. J Tradit Complement Med 15 , 668-677, doi:10.1016/j.jtcme.2024.09.006 (2025). Liu, X., Du, S., Jiang, Y. & Chen, Y. Piezo1 exacerbates psoriasis by promoting macrophage M1 polarization and inhibits autophagy via activating PI3K/AKT signaling pathway. Inflamm Res 74 , 149, doi:10.1007/s00011-025-02111-7 (2025). Suzuki, T., Muraki, Y., Hatano, N., Suzuki, H. & Muraki, K. PIEZO1 Channel Is a Potential Regulator of Synovial Sarcoma Cell-Viability. Int J Mol Sci 19 , doi:10.3390/ijms19051452 (2018). Suzuki, H. et al. The NADPH oxidase inhibitor diphenyleneiodonium activates the human TRPA1 nociceptor. Am J Physiol Cell Physiol 307 , C384-394, doi:10.1152/ajpcell.00182.2013 (2014). Muraki, K. et al. An environmental pollutant, 9,10-phenanthrenequinone, activates human TRPA1 via critical cysteines 621 and 665. Pharmacol Res Perspect 5 , doi:10.1002/prp2.342 (2017). Pettersen, E. F. et al. UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem 25 , 1605-1612, doi:10.1002/jcc.20084 (2004). Varadi, M. et al. AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Res 50 , D439-D444, doi:10.1093/nar/gkab1061 (2022). Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596 , 583-589, doi:10.1038/s41586-021-03819-2 (2021). Waterhouse, A. et al. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res 46 , W296-W303, doi:10.1093/nar/gky427 (2018). Sievers, F. & Higgins, D. G. Clustal Omega for making accurate alignments of many protein sequences. Protein Sci 27 , 135-145, doi:10.1002/pro.3290 (2018). Waterhouse, A. M., Procter, J. B., Martin, D. M., Clamp, M. & Barton, G. J. Jalview Version 2--a multiple sequence alignment editor and analysis workbench. Bioinformatics 25 , 1189-1191, doi:10.1093/bioinformatics/btp033 (2009). Additional Declarations The authors declare no competing interests. Supplementary Files SciRepoPIEZOSupplementaryinformationTiffversion.docx Functional conservation and species-specific tuning of PIEZO1 mechanotransduction in birds and humans Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8855948","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":589892740,"identity":"0e068c14-1f0b-46ae-898c-e40248a1ca36","order_by":0,"name":"Katsuhiko Muraki","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIiWNgGAWjYBACCTB5QEKO4QCSqAExWoxJ1sKQ2HAAnzJkIDntjNmHH2cs0vtuNz9g+PHrMAN/+wGG4gI8WqSlc4xn9tyQyJ1555gBY2/fYQaJMwkMxjPwaJEDamHg+SCRu+FGDgMDb89hBoYbDAzGPAS0MP75IJFuANTC+BeoRZ6QFpDDmHluSCSAtDDz/DjMYEBIi+TstGJmmTMShiC/HJZtSOcxPJPYgNcvEreTNzO+OVYnz3e7+eHDN3+s5eSOHz5mjC/EkDQDo4exjQHoJMY2Y6J0QCL1D5jJ/Jg4LaNgFIyCUTBCAAAre01NPfZeCAAAAABJRU5ErkJggg==","orcid":"","institution":"Aichi-Gakuin University","correspondingAuthor":true,"prefix":"","firstName":"Katsuhiko","middleName":"","lastName":"Muraki","suffix":""},{"id":589896171,"identity":"c5322c4f-6bdd-4dcb-9546-9f05f23fc7eb","order_by":1,"name":"Ayami Morita","email":"","orcid":"","institution":"Aichi-Gakuin University","correspondingAuthor":false,"prefix":"","firstName":"Ayami","middleName":"","lastName":"Morita","suffix":""},{"id":589896172,"identity":"3e7f84fd-6703-4d20-b48a-e89047305127","order_by":2,"name":"Hiroka Suzuki","email":"","orcid":"","institution":"Aichi-Gakuin University","correspondingAuthor":false,"prefix":"","firstName":"Hiroka","middleName":"","lastName":"Suzuki","suffix":""},{"id":589896173,"identity":"7f61539a-7cfd-4c8d-ab7e-811d07f10f49","order_by":3,"name":"Yukiko Muraki","email":"","orcid":"","institution":"Aichi-Gakuin University","correspondingAuthor":false,"prefix":"","firstName":"Yukiko","middleName":"","lastName":"Muraki","suffix":""}],"badges":[],"createdAt":"2026-02-11 22:51:26","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-8855948/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8855948/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102743879,"identity":"ff3e5fa1-4497-4459-933a-308c8fcca019","added_by":"auto","created_at":"2026-02-16 08:25:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5509698,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNative HEK cells lack functional PIEZO1 activity, whereas chicken and human PIEZO1 show conserved architecture despite sequence divergence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A, B) Whole-cell recordings from native HEK (nHEK) cells during application of 3 μM Yoda1. (A) Time course of peak currents measured at −90 and +90 mV during repeated voltage ramps (−110 to +90 mV, 300 ms). Arrows indicate acquisition of representative current–voltage (I–V) relationships shown in the right panel.(B) Summary of peak current amplitudes at -90 and +90 mV in the absence and presence of 3 μM Yoda1 (n=11 cells). (C) Mechanically evoked currents in excised outside-out patches from nHEK cells. Left, experimental protocol showing voltage ramps (−110 to +90 mV, 100 ms) applied before graded positive pressure (2.5–50 mmHg, digitally filtered at 100 Hz). Right, summary of peak current amplitudes measured before (0 mmHg) and 30 ms (red) or 200 ms (blue) after pressure application at −60 mV (n=3–6 patches). (D) Summary of peak currents evoked by 20 mmHg pressure in the absence and presence of 3 μM Yoda1. (E) Western blot (WB) analysis of PIEZO1 protein expression in nHEK, HEK cells expressing hPIEZO1 or gPIEZO1, and HaCaT cells. β-actin serves as a loading control. Blots cropped from different regions of the same gel and from identical exposure conditions are explicitly delineated by black dividing lines. The original uncropped Western blot images corresponding to Fig. 1E (Supplementary Fig. S1A) are provided in Supplementary Fig. S1. Data are representative of three independent experiments. (F) Partial AA sequence alignment of two structurally important regions of PIEZO1 (Beam-Central-Linker and Central Plug) from mouse, human, and chicken orthologs. Residues differing from mPIEZO1 are highlighted in red for hPIEZO1; residues differing between hPIEZO1 and gPIEZO1 are underlined. Full-length alignment is shown in Supplementary Fig. S2. (G) Structural comparison of hPIEZO1 and gPIEZO1. A predicted full-length protomer model of gPIEZO1 (orange) is superimposed on the AlphaFold2 model of hPIEZO1 (blue). A-trimeric homology model of gPIEZO1 is shown in Supplementary Fig.S3. Statistical significance was assessed using paired Student’s \u003cem\u003et\u003c/em\u003e-test (B) and Tukey’s post hoc test (C, D).\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-8855948/v1/a18cfffdac0f2e637c169387.png"},{"id":102743968,"identity":"4a1d71f2-b981-4519-b4f3-65b4d444ae60","added_by":"auto","created_at":"2026-02-16 08:25:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3731099,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHuman PIEZO1 is activated by Yoda1 and positive mechanical pressure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A, B) Whole-cell recordings from HEK cells expressing hPIEZO1 during application of Yoda1 (0.1-3 μM). (A) Time course of peak currents and representative I–V relationships before and after Yoda1 application. (B) Summary of peak current amplitudes at -90 and +90 mV (n=5, 6, and 15 cells for 0.1, 1, and 3 μM Yoda1, respectively). (C-D) Pressure-dependent currents recorded from excised outside-out patches. (C) Representative current traces evoked by graded positive pressures (2.5 to 50 mmHg). (D) Summary of peak current amplitudes measured before and 30 (red) or 200 ms (blue) after pressure application. (E) Representative holding current responses at -50 mV during application of 20 mmHg pressure, Yoda1 (3 μM), and Gd\u003csup\u003e3+\u003c/sup\u003e (10 μM). Vertical lines indicate voltage ramps. (F) Summary of peak currents evoked by 20 mmHg pressure at -50 mV. Statistical significance was assessed using paired Students’ \u003cem\u003et\u003c/em\u003e-test (B, F) and Tukey’s post hoc test (D, F).\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-8855948/v1/40f8a63d836d75d8ccec7382.png"},{"id":102743942,"identity":"bf5500ae-9412-49e5-b37c-a0ecce2ef748","added_by":"auto","created_at":"2026-02-16 08:25:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1760919,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMechanical stimulation and Yoda1 induce similar hPIEZO1 single-channel currents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative single-channel recordings from an excised outside-out patch at -50 mV during application of 20 mmHg pressure or 3 μMYoda1 (same patch as in Fig.2E). The dashed line indicates the closed state. Lower panels show amplitude histograms derived from 4 s of recording after stimulation. (B) Summary of unitary current amplitudes at -50 mV for mechanically and chemically evoked events. Channel states are indicated as closed and open levels (open1-open4).\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-8855948/v1/5fbecf3ce3367d6eea554572.png"},{"id":102743979,"identity":"e93187bf-6b3e-429c-b2ba-63005def43fc","added_by":"auto","created_at":"2026-02-16 08:25:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4355513,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChicken PIEZO1 is activated by Yoda1 and mechanical pressure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A, B) Whole-cell recordings from HEK cells expressing gPIEZO1 during application of Yoda1 (0.1-3 μM). (A) Representative time course of peak currents and I-V relationships. (B) Summary of peak current amplitudes at -90 and +90 mV (n=8, 9, 6, and 12 cells for 0.1, 0.3, 1, and 3 μM Yoda1, respectively). (C, D) Pressure-dependent currents recorded from excised outside-out patches. (C) Representative current traces evoked by graded positive pressures. (D) Summary of peak current amplitudes measured before and after pressure application. (E-H) Modulation of pressure-induced gPIEZO1 currents by Yoda1 and Gd\u003csup\u003e3+\u003c/sup\u003e. Representative current traces (E-G) and summary data (H) are shown. Statistical significance was assessed using paired Student’s \u003cem\u003et\u003c/em\u003e-test (B) and Tukey’s test (D, H).\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-8855948/v1/d47f9f4fc64f1ed034d46871.png"},{"id":102743992,"identity":"c0cab300-d4b9-4adf-ab18-1b372c3cf64f","added_by":"auto","created_at":"2026-02-16 08:26:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2082682,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSingle-channel properties of gPIEZO1 activated by mechanical pressure and Yoda1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative single-channel recordings from an excised outside-out patch at -50 mV during application of 20 mmHg pressure or 3 μM Yoda1 (same patch as in Fig.4E). The dashed line indicates the closed state. Lower panels show amplitude histograms derived from 3 s of recording. (B) Summary of unitary current amplitudes at -50 mV. Channel states are indicated as closed and open levels (open1-open5).\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-8855948/v1/f824d146634f683e4c5a067b.png"},{"id":102743965,"identity":"a26191a2-88d2-4e83-8a52-6143aecd70e9","added_by":"auto","created_at":"2026-02-16 08:25:50","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2521159,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChicken PIEZO1 exhibits enhanced mechanical and Yoda1 sensitivity compared with human PIEZO1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Voltage ramp protocols applied during mechanical stimulation in excised outside-out patches from HEK cells expressing gPIEZO1 (left) or hPIEZO1 (right). (B) Averaged I-V relationships for gPIEZO1 (n=8 patches) and hPIEZO1 (n=3 patches). (C) Comparison of pressure sensitivity using data from Fig. 2D and Fig.4D. (D) Comparison of Yoda1 sensitivity using data from Fig. 2B and Fig.4B. Statistical significance was assessed using two-way ANOVA (C, D).\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-8855948/v1/e42383f12dee0684ec248700.png"},{"id":102743983,"identity":"966f8778-82cf-42e6-8c8c-c4d68a0ca170","added_by":"auto","created_at":"2026-02-16 08:26:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3853772,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEndogenous PIEZO1 mediates mechanically evoked currents in human keratinocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A, B) Whole-cell recordings from HaCaT cells during application of 3 μM Yoda1. (A) Representative time course and I-V relationships. (B) Summary of peak current amplitudes at -90 and +90 mV (n=4 cells). (C, D) Pressure-dependent currents recorded from excised outside-out patches. (C) Representative traces evoked by graded pressure. (D) Summary of peak current amplitudes. (E, F) Effects of Yoda1 and the TRPV4 agonist GSK1016790A on pressure-induced currents. Representative traces (E) and summary data (F) are shown. Statistical significance was assessed using paired Student’s \u003cem\u003et\u003c/em\u003e-test (B) and Tukey’s test (D, F).\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-8855948/v1/f0fc74477391bd68eecae984.png"},{"id":102743945,"identity":"5d4270e1-3d64-4ecf-95f7-a6121d6f0676","added_by":"auto","created_at":"2026-02-16 08:25:43","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":3825047,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSingle-channel properties of endogenous hPIEZO1 and effects of PIEZO1 knockdown\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A, B) Representative single-channel recordings from HaCaT cells at -50 mV during application of mechanical pressure, Yoda1, or GSK (same patch as in Fig.7E). The dashed line indicates the closed state. Lower panels show amplitude histograms.(C) Summary of unitary current amplitudes. (D, E) Effects of siRNA-mediated PIEZO1 knockdown on pressure-induced currents. Representative traces (left) and summary data (right, n=9-17 patches except for 85 mmHg) are shown. Statistical significance was determined using two-way ANOVA (D, E).\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-8855948/v1/4a134fbcc939049477aba276.png"},{"id":102743984,"identity":"e6fca4cb-8826-4c6a-9fc7-2e1031c19d25","added_by":"auto","created_at":"2026-02-16 08:26:02","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":4082242,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHumanTRPV4 is insensitive to positive mechanical pressure under the present stimulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A, B) Whole-cell recordings from HEK cells expressing hTRPV4 during application of Yoda1 (3 μM) and GSK (10 nM). (A) Representative time course and I-V relationships. (B) Summary of peak current amplitudes (n=12 and 7 cells for Yoda1 and GSK, respectively). (C) Pressure-dependent responses recorded from excised outside-out patches. Representative current traces (left) and summary of data (right) are shown (n=4-7 patches except for 65 mmHg at 200 ms). (D-G) Effects of Yoda1 and GSK on pressure-induced currents. Representative current traces (D-F) and summary data (G) are shown. Statistical significance was assessed using paired Student’s \u003cem\u003et\u003c/em\u003e-test (B) and Tukey’s test (C, G).\u003c/p\u003e","description":"","filename":"Fig9.png","url":"https://assets-eu.researchsquare.com/files/rs-8855948/v1/d4f1924e1c42cbe08bc942d6.png"},{"id":102744124,"identity":"7c7ba880-f7ad-477a-aa7b-7ec8506cdc3c","added_by":"auto","created_at":"2026-02-16 08:26:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":29671138,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8855948/v1/1e3ca448-cc22-4c01-b8fd-077e5b4e9a98.pdf"},{"id":102743934,"identity":"4685ec6a-eb2d-4674-861f-5f93cbff5c5e","added_by":"auto","created_at":"2026-02-16 08:25:40","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":21165206,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional conservation and species-specific tuning of PIEZO1 mechanotransduction in birds and humans\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"SciRepoPIEZOSupplementaryinformationTiffversion.docx","url":"https://assets-eu.researchsquare.com/files/rs-8855948/v1/f88d75f839347ed81e7ae5e3.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eFunctional conservation and species-specific tuning of PIEZO1 mechanotransduction in birds and humans\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMechanical forces are fundamental regulators of cellular and tissue physiology across diverse biological systems, contributing to processes such as touch sensation, audition, tissue morphogenesis, and vascular homeostasis. In multicellular organisms, cells continuously experience mechanical stimuli arising from membrane tension, fluid flow, and changes in extracellular matrix stiffness, and convert these forces into biochemical and electrical signals through mechanotransduction mechanisms. Identifying how these mechanisms are conserved and diversified across species is essential for understanding the evolutionary principles underlying mechanosensory biology. Birds experience unique and extreme mechanical demands associated with powered flight, rapid locomotion, and specialized sensory behaviors, making them an informative vertebrate model for exploring how mechanotransduction pathways are evolutionarily tuned to meet distinct biomechanical challenges.\u003c/p\u003e \u003cp\u003ePIEZO proteins constitute a family of mechanically activated cation channels that play central roles in mechanotransduction. Since their initial identification, PIEZO1 and PIEZO2 have been shown to mediate mechanically evoked ionic currents in a wide range of cell types and organisms\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. PIEZO1, originally characterized in neuronal cells, is now known to be broadly expressed in both neuronal and non-neuronal tissues, including vascular endothelial cells, where it regulates vascular development, blood flow\u0026ndash;dependent signaling, and myogenic tone\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Consistent with these diverse functions, PIEZO channels are evolutionarily conserved across vertebrates and invertebrates, and homologs have also been identified in plants and protozoa, indicating that PIEZO-mediated mechanotransduction represents a fundamental biological process\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePIEZO channels are directly activated by multiple forms of mechanical force, including membrane stretch, cell-poking by a mechanical probe, shear stress generated by fluid flow, and alterations in membrane tension or stiffness\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The functional roles of PIEZO channels have been extensively investigated in several animal models, including mouse\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, zebrafish\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, drosophila\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, and duck\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, revealing their involvement in sensory transduction, development, and tissue homeostasis. The discovery of the synthetic PIEZO1 agonist Yoda1\u003csup\u003e11\u003c/sup\u003e has further accelerated functional studies by enabling chemical activation of PIEZO1 independent of mechanical stimulation, thereby uncovering its contributions to diverse physiological processes such as bladder distention sensing\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, mechanically induced osteoclastogenesis\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, and red blood cell volume regulation\u003csup\u003e\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Despite major advances in the structural, biophysical, and physiological characterization of PIEZO channels, comparative analyses of their electrophysiological properties across species remain limited. Studies of other ion channel families highlight the importance of such cross-species comparisons, as channel gating, pharmacological sensitivity, and ion permeability can vary substantially among orthologs. For example, species-dependent differences in agonist sensitivity and ion permeation have been reported for transient receptor potential channels, illustrating how evolutionary divergence can fine-tune channel function while preserving overall mechanistic frameworks\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Such differences not only provide insight into channel structure\u0026ndash;function relationships but also inform the development of species-selective pharmacological tools.\u003c/p\u003e \u003cp\u003eAmong avian species, mechanosensory function of PIEZO channels has been only partially explored. PIEZO2 is functionally expressed in trigeminal ganglia of tactile-foraging ducks, where it responds to mechanical stimulation in a manner comparable to human PIEZO2\u003csup\u003e10\u003c/sup\u003e. In contrast, the expression patterns and functional properties of avian PIEZO1 channel remain largely unexplored, and direct electrophysiological characterization of avian PIEZO1 has not been reported. Here, we compare the electrophysiological characteristics of chicken PIEZO1 (gPIEZO1) and human PIEZO1 (hPIEZO1) using heterologous expression in HEK cells, and we further examine endogenous hPIEZO1 in human keratinocytes as a native cellular model. We show that gPIEZO1 mediates mechanically activated currents with biophysical properties similar to those in hPIEZO1, yet exhibits significantly greater sensitivity to both mechanical stimulation and Yoda1. These findings establish PIEZO1 as a functional mechanosensor in birds and reveal quantitative, species-dependent tuning of PIEZO1 mechanosensitivity, providing new insight into the evolutionary diversification of mechanically gated ion channels.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eNative HEK cells show negligible PIEZO1 activity and the undetectable protein expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrior to comparing the electrophysiological properties of gPIEZO1 and hPIEZO1 heterologously expressed in HEK cells, we first tested the effects of Yoda1 and positive mechanical pressure in native HEK cells without exogenous PIEZO1 expression (nHEK). As shown in Figures 1A and 1B, application of 3 \u0026mu;M Yoda1 failed to evoke PIEZO1-like currents in nHEK cells. Moreover, when positive mechanical pressures ranging from 2.5 to 100 mmHg were applied to excised outside-out membrane patches from nHEK cell, pressure-induced current activation was minimal and inconsistent (Figure 1C). Even in the presence of 3 \u0026mu;M Yoda1, currents elicited by 20 mmHg were not significantly different from those recorded at 0 mmHg (Figure 1D), suggesting that negligible endogenous PIEZO1 activity in nHEK cells. Consistent with these functional observations, PIEZO1 protein was undetectable in nHEK cells by western blot (WB) analysis (Figure 1E). In contrast, robust hPIEZO1 expression was detected in HEK cells transiently expressing hPIEZO1 (HEK-hPIEZO1) as well as in HaCaT cells, a human keratinocyte cell line (Figure 1E). Together, these data indicate that endogenous hPIEZO1 expression in nHEK cells is minimal, justifying the use of HEK cells as a heterologous expression system for direct comparison of gPIEZO1 and hPIEZO1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChicken PIEZO1 shows sequence divergence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe full-length amino acid (AA) sequence of gPIEZO1 was aligned with that of hPIEZO1 (Supplementary Figure S1). Two structurally important regions of PIEZO1\u0026mdash;the Beam-central plug linker and the central plug \u0026mdash;were further compared among mouse, human, and chicken orthologs (Figure 1F). While hPIEZO1 exhibited relatively high sequence identity with mouse PIEZO1 (mPIEZO1), gPIEZO1 displayed greater divergence within these regions. Consistent with this sequence divergence, gPIEZO1 expressed in HEK cells (HEK-gPIEZO1) was not detected by an antibody recognizing an hPIEZO1 epitope spanning residues 1275-1540, whereas both heterologously expressed hPIEZO1 in HEK-hPIEZO1 and endogenous hPIEZO1 in HaCaT cells were readily detected (Figure 1F). To assess whether this sequence divergence affects overall channel architecture, we generated a three-dimensional (3D) full-length protomer model of gPIEZO1 (XP_015148150.2, 2492 AAs; orange, Figure 1G) and superimposed it onto the AlphaFold2-predicted structure of hPIEZO1 (AF-Q92508-F1-v4, 2521 AAs; blue). In addition, a trimeric homology model of full-length gPIEZO1 was also constructed (Supplementary FigureS2). These structural analyses propose strong overall conservation of PIEZO channel architecture despite localized sequence divergence.\u003c/p\u003e\n\u003cp\u003e[Figure 1, here]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman PIEZO1 is robustly activated by Yoda1 and positive pressure in heterologous expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo establish a reference for cross-species comparison, we first characterized the electrophysiological properties of hPIEZO1 (Figures. 2, 3, and 6). Under whole-cell patch-clamp conditions, Yoda1 elicited PIEZO1-like currents in HEK-hPIEZO1 cells in a concentration-dependent manner (Figures 2A and 2B). In excised outside-out patch recordings, positive pressure applied to the membrane patch induced PIEZO1-like currents that increased with pressure magnitude (Figures 2C and 2D). Moreover, current activity evoked by 20 mmHg was significantly potentiated by 3 \u0026mu;M Yoda1 and effectively inhibited by 10 \u0026mu;M Gd\u003csup\u003e3+\u003c/sup\u003e, a non-selective cation channel blocker (Figures 2E and 2F). In some outside-out patches, discrete single-channel events were observed at -50 mV following mechanical stimulation or in the presence of Yoda1 (Figure 3A). The unitary current amplitudes induced by mechanical stimulation were comparable to those evoked by Yoda1 (Figure 3B), suggesting that both stimuli activate the same hPIEZO1 channel population.\u003c/p\u003e\n\u003cp\u003e[Figure 2, here], [Figure 3, here]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChicken PIEZO1 is robustly activated by Yoda1 and positive pressure in heterologous expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe next examined the electrophysiological properties of gPIEZO1 using an experimental paradigm analogous to that used for hPIEZO1 (Figures 4, 5, and 6). In whole-cell recordings, Yoda1 induced PIEZO1-like currents in HEK-gPIEZO1 cells in a concentration-dependent manner (Figures 4A and 4B). Similarly, in excised outside-out patches, positive pressure induced PIEZO1-like currents in a pressure-dependent manner (Figures 4C and 4D). Currents evoked by 20 mmHg were significantly potentiated by Yoda1 at concentrations ranging from 0.1 to 3 \u0026mu;M and were inhibited by 3 \u0026mu;M Gd\u003csup\u003e3+\u003c/sup\u003e (Figures 4E and 4F). Single-channel activity was detected at -50 mV in some excised patches following mechanical stimulation or in the presence of Yoda1 (Figure 5A). As observed for hPIEZO1, the unitary current amplitudes induced by positive pressure were comparable to those induced by Yoda1 (Figure 5B), indicating that both stimuli activate gPIEZO1 channels.\u003c/p\u003e\n\u003cp\u003e[Figure 4, here], [Figure 5, here]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChicken PIEZO1 exhibits enhanced mechanical and Yoda1 sensitivity \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo directly compare gPIEZO1 and hPIEZO1, we analyzed current-voltage (I-V) relationships and stimulus sensitivities (Figure 6). During the application of graded positive pressures, voltage ramp protocols were applied to excised outside-out patches from HEK cells expressing gPIEZO1 or hPIEZO1 (Figure 6A). The resulting I-V relationships were highly similar between the two channels (Figure 6B), indicating conserved permeation properties. In contrast, both mechanical sensitivity (Figure 6C) and Yoda1 sensitivity (Figure 6D) were significantly greater for gPIEZO1 than for hPIEZO1. These results demonstrate that, while gPIEZO1 shares fundamental electrophysiological properties with hPIEZO1, it exhibits enhanced responsiveness to both mechanical stimulation and Yoda1. To explore the evolutionary conservation of gain-of-function (GOF)-associated substitutions, we performed AA sequence alignments across 100 avian PIEZO1 orthologs (Supplementary Table S2), This analysis revealed that six residues corresponding to known GOF-associated positions in hPIEZO1\u0026mdash;706E (G718S), 1086V (S1117L), 1554I (T1589I), 2212N (R2245Q), 2351D (G2384S), and 2377R (E2407K) \u0026mdash;are recurrently substituted in multiple avian species (Supplementary Fig.S4), suggesting that enhanced PIEZO1 mechanosensitivity may represent an evolutionarily selected feature in birds.\u003c/p\u003e\n\u003cp\u003e[Figure 6, here]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEndogenous PIEZO1 mediates mechanically and chemically evoked currents in human keratinocytes\u003c/strong\u003e \u003c/p\u003e\n\u003cp\u003eWe next investigated endogenous hPIEZO1 activity in HaCaT cells (Figures 7 and 8). In whole-cell recordings, application of 3 \u0026mu;M Yoda1 induced PIEZO1-like currents in HaCaT cells (Figures7A and 7B). In excised outside-out patches, positive pressure elicited PIEZO1-like currents in a pressure-dependent manner (Figures 7C and 7D). Currents evoked by 20 mmHg were significantly potentiated by 3 \u0026mu;M Yoda1 (Figures 7E and 7F). Because HaCaT cells also express TRPV4, a mechanoregulated cation channel \u003csup\u003e20\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e21\u003c/sup\u003e, we examined the effect of the TRPV4 agonist GSK1016790A (GSK) and positive pressure in this system. Although 10 nM GSK robustly activated TRPV4-like currents (Figure 7E), applying 20 mmHg did not alter these currents (Figures 7E and 7F). \u003c/p\u003e\n\u003cp\u003e[Figure 7, here]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003esiRNA knockdown confirms that keratinocyte mechanosensitive currents require PIEZO1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn some excised patches, single-channel activity was detected at -50 mV following mechanical stimulation or in the presence of Yoda1 and GSK (Figure 8A). The unitary current amplitudes induced by pressure were comparable to those evoked by Yoda1 (Figure 8B), indicating that mechanical stimulation and Yoda1 activate endogenous hPIEZO1 in HaCaT cells. To further confirm the functional contribution of hPIEZO1, we knocked down \u003cem\u003ePIEZO1\u003c/em\u003eexpression in HaCaT cells using Stealth siRNA. After transfection with PIEZO1-trageting siRNA (siRNA), we tested the mechanosensitivity of HaCaT cells compared with negative control RNA (ncRNA). In excised outside-out patches, mechanically evoked responses were significantly attenuated in PIEZO1-knockdown cells relative to ncRNA-treated cells (Figures 8C and 8D), demonstrating that endogenous hPIEZO1 functions as a mechanosensor in HaCaT cells.\u003c/p\u003e\n\u003cp\u003e[Figure 8, here]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTRPV4 does not account for pressure-evoked currents under the present stimulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFinally, to assess the mechanosensitivity of hTRPV4 under our stimulation paradigm, we examined HEK cells heterologously expressing hTRPV4 (HEK-hTRPV4, Figure 9). In whole-cell recordings, application of 3 \u0026mu;M Yoda1 failed to evoke a current response, whereas 10 nM GSK robustly activated TRPV4-like currents (Figures 9A and 9B). Consistently, in excised outside-out patches, positive pressures did not induce pressure-sensitive currents in HEK-hTRPV4 cells (Figures 9C and 9D). Even in the presence of 3 \u0026mu;M Yoda1, 20 mmHg failed to evoke mechanically sensitive current components (Figs 9C and 9E). In contrast, 10 nM GSK reliably induced TRPV4-like currents (Figures 9C and 9E), suggesting that hTRPV4 is insensitive to positive pressure under these conditions. \u003c/p\u003e\n\u003cp\u003e[Figure 9, here]\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we provide the first direct electrophysiological characterization of avian PIEZO1 and demonstrate that chicken PIEZO1 (gPIEZO1) functions as a mechanically activated ion channel with properties broadly conserved with human PIEZO1 (hPIEZO1). Using whole-cell and excised patch recordings, we show that gPIEZO1 is robustly activated by both mechanical stimulation and the synthetic PIEZO1 agonist Yoda1. Importantly, while the core electrophysiological features of gPIEZO1 closely resemble those of hPIEZO1, gPIEZO1 exhibits significantly greater sensitivity to both mechanical force and chemical activation. These findings establish PIEZO1 as a functional mechanosensor in birds and reveal species-dependent tuning of PIEZO1 mechanosensitivity. \u003c/p\u003e\n\u003cp\u003eA critical prerequisite for cross-species comparison is an expression system with minimal endogenous PIEZO1 activity. We therefore first assessed whether native HEK cells express functional PIEZO1. Although occasional mechanically evoked currents were observed in excised patches from native HEK cells, these responses were inconsistent and not potentiated by Yoda1. Consistent with these functional observations, hPIEZO1 protein was undetectable by western blotting. These results indicate that endogenous PIEZO1 expression in native HEK cells is negligible, validating HEK cells as a suitable platform for comparative analysis of heterologously expressed human and chicken PIEZO1 channels. \u003c/p\u003e\n\u003cp\u003eYoda1 has been widely used as a chemical tool to probe PIEZO1 function across species\u003csup\u003e11\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e22-25\u003c/sup\u003e. In the present study, Yoda1 reversibly activated gPIEZO1 in both whole-cell and excised patch configurations, suggesting that Yoda1-responsive structural elements are largely conserved. Two of the three residues previously implicated in Yoda1 sensitivity in mouse PIEZO1 (A1719, A2091, and A2094\u003csup\u003e26\u003c/sup\u003e) are conserved in gPIEZO1 (A1684 and A2045), whereas the third position is substituted by serine (S2042). Despite this substitution, gPIEZO1 exhibited robust activation at low Yoda1 concentrations, suggesting that this residue contributes only modestly to Yoda1 binding or efficacy. Notably, both gPIEZO1 and hPIEZO1 were significantly potentiated by submicromolar Yoda1 under our recording conditions, whereas higher EC\u003csub\u003e50\u003c/sub\u003e values have been reported in other systems\u003csup\u003e11\u003c/sup\u003e. Differences in experimental conditions, including solution flow-induced shear stress and the high lipophilicity of Yoda1, may contribute to these apparent differences in potency. Despite differences in sensitivity, unitary currents activated by Yoda1 or mechanical stimulation were comparable between gPIEZO1 and hPIEZO1. This similarity is consistent with the conservation of negatively charged residues within the inner helix region that determine unitary conductance, Ca\u003csup\u003e2+\u003c/sup\u003e permeability, and blocker sensitivity\u003csup\u003e27\u003c/sup\u003e. In contrast, residues surrounding the extracellular fenestration site\u0026mdash;previously implicated in ion conduction efficiency\u0026mdash;are partially diverged between species\u003csup\u003e27\u003c/sup\u003e. Such sequence differences may subtly modulate permeation without altering overall conductance, thereby preserving fundamental channel function while enabling species-specific fine-tuning. \u003c/p\u003e\n\u003cp\u003eIn addition to chemical activation, gPIEZO1 displayed greater sensitivity to positive pressure than hPIEZO1 in excised patch recordings. Although variability in mechanically evoked responses was observed across patches, likely reflecting differences in membrane geometry or local mechanical conditions\u003csup\u003e28\u003c/sup\u003e, several key features were conserved: mechanically evoked gPIEZO1 currents were potentiated by Yoda1, inhibited by Gd\u003csup\u003e3+\u003c/sup\u003e, and exhibited unitary currents indistinguishable from those of hPIEZO1. These findings support the conclusion that the core mechanogating mechanism of PIEZO1 is conserved, whereas quantitative differences in force sensitivity reflect evolutionary tuning rather than fundamental mechanistic divergence. Interestingly, several AA substitutions in gPIEZO1 map to positions corresponding to GOF-associated SNPs in hPIEZO1, including residues 706E (G718S\u003csup\u003e14\u003c/sup\u003e), 1086V (S1117L\u003csup\u003e14\u003c/sup\u003e), 1554I (T1589I\u003csup\u003e29\u003c/sup\u003e), 2212N (R2245Q\u003csup\u003e30\u003c/sup\u003e), 2246S (R2279C\u003csup\u003e29\u003c/sup\u003e), and 2377R (E2407K\u003csup\u003e30\u003c/sup\u003e). These substitutions align with mutations previously shown to potentiate PIEZO1 activity in humans (Supplementary Fig.S4). In addition, the polyglutamine-rich region spanning residues 739-750, which contains the mild GOF-associated Q745 deletion in hPIEZO1 (Supplementary Fig.S2), is absent in avian\u003csup\u003e31\u003c/sup\u003e. Together, these sequence features suggest that avian PIEZO1 may have evolved intrinsic structural determinants that favor enhanced channel activity. In mammalian systems, GOF mutations in PIEZO1 have been linked to increased physical performance\u003csup\u003e32\u003c/sup\u003e and altered mechanosensory phenotypes\u003csup\u003e33\u003c/sup\u003e. Although direct causal relationships remain to be established, the enrichment of GOF\u0026ndash;associated substitutions in gPIEZO1 raises the possibility that enhanced PIEZO1 mechanosensitivity may contribute to biomechanical adaptations characteristic of birds. This hypothesis provides a testable framework for future studies linking PIEZO1 function to avian physiology and biomechanics. \u003c/p\u003e\n\u003cp\u003eTo assess physiological relevance beyond heterologous expression systems, we examined endogenous mechanotransduction in human keratinocytes. In these cells, both Yoda1 and mechanical stimulation elicited inward currents that were abolished by siRNA-mediated knockdown of PIEZO1, demonstrating that endogenous hPIEZO1 mediates these responses. Although keratinocytes also express other mechanosensitive channels such as TRPV4\u003csup\u003e20\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e21\u003c/sup\u003e, TRPV4-mediated currents were insensitive to the positive-pressure paradigm used here, indicating that PIEZO1 is the primary mediator of mechanically evoked currents under these conditions. These findings are consistent with previous reports of functional PIEZO1 expression in keratinocytes and support the relevance of our comparative analysis to a native cellular context\u003csup\u003e34-36\u003c/sup\u003e. \u003c/p\u003e\n\u003cp\u003eIn summary, our study establishes avian PIEZO1 as a functional mechanosensor and reveals that PIEZO1 mechanotransduction is both evolutionarily conserved and quantitatively tuned across species. By directly comparing chicken and human PIEZO1 electrophysiology and validating endogenous PIEZO1 function in human cells, we provide insight into how conserved ion channel architectures support diverse mechanosensory demands. These findings lay the groundwork for future studies exploring the evolutionary, structural, and physiological determinants of mechanosensitivity across vertebrates.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eReagents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe following reagents were used: \u003cstrong\u003e2-[5-[[(2,6-Dichlorophenyl)methyl]thio]-1,3,4-thiadiazol-2-yl]-pyrazine\u003c/strong\u003e (Yoda1, Tocris Bioscience, Bristol, UK), GSK1016790A (GSK, Sigma/Aldrich, St. Louis, MO), and gadolinium chloride (Gd\u003csup\u003e3+\u003c/sup\u003e, Fujifilm, Osaka, Japan). Each reagent was dissolved in the vehicle recommended by the manufacturer.\u003cem\u003e \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman keratinocytes (HaCaT, American Type Culture Collection, Manassas, VA) and Human embryonic kidney (HEK) cells (Health Science Research Resources Bank, HSRRB, Osaka, Japan) were maintained in Dulbecco\u0026rsquo;s modified Minimum Essential Medium (D-MEM; Sigma-Aldrich, Tokyo, Japan) supplemented with 10% heat-inactivated fetal calf serum (FCS; Sigma-Aldrich), penicillin G (100 U/mL, Meiji Seika Pharma Co., Ltd., Tokyo, Japan), and streptomycin (100 mg/mL, Meiji Seika Pharma Co., Ltd.). Cells were cultured at 37 \u0026deg;C in a humidified atmosphere containing 5% CO₂.\u003cem\u003e \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRecombinant expression of PIEZO1and hTRPV4 in HEK cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHEK cells at 40-60% confluency were transiently transfected with pIRES2-AcGFP1 expression plasmids (Takara Bio INC., Shiga, Japan) encoding human PIEZO1 (hPIEZO1, NP_001136336.2, 2521AAs, \u003csup\u003e37\u003c/sup\u003e), chicken PIEZO1 (gPIEZO1, XP_015148150.2, 2492AAs), or human TRPV4 (hTRPV4, NP_067638, 871AAs\u003csup\u003e38\u003c/sup\u003e). Transfections were performed using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer\u0026rsquo;s instructions. The full-length gPIEZO1 coding sequence was amplified from cDNA prepared from total RNA isolated from whole chicken brain tissue (G. gallus domesticus (GSP (Fayoumi breed)). Tissue samples were provided by Avian Bioscience Research Center at Nagoya University through the National Bio-Resource Project of the MEXT, Japan\u003csup\u003e19\u003c/sup\u003e). For RT-PCR amplification of the gPIEZO1 coding region, six sets of gene-specific primers were designed (primer sequences are listed in Supplementary Table S1). The initial assembly of the gPIEZO1 coding sequence (284 bp), in which identical AA residues are conserved, was performed by a commercial service (Supplementary Table S1, Integrated DNA Technologies, Tokyo, Japan). All constructs were verified by DNA sequencing. The cloned gPIEZO1 construct contained nine amino acid changes: I103V, I541V, S705A, R1150S, R1305H, S1450V, deletion of A1468, H1532R, and deletion of E1791 deletion (2490 AAs). In an alignment across 100 avian PIEZO1 orthologs (Supplementary Table S2), 103V (5%), 541V (67%), 705A (50%), 1305H (100%), deletion of A1468 (57%), and deletion of E1791 deletion (16%) were conserved. Electrophysiological and biochemical experiments were performed within 48 h after transfection. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHEK cells both with or without PIEZO1 expression and HaCaT cells were lysed in 50 \u0026mu;l of lysis buffer containing (in mM) 50 Tris-HCl (pH 8.0), 150 NaCl, 5 EDTA supplemented with 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS, as well as a protease inhibitor cocktail. Lysates were incubated on ice for 30 min with intermittent vortexing at 5-min intervals and subsequently centrifugated at 20000 \u0026times; g for 30 min at 4 \u0026deg;C. Protein concentrations were determined, and equal amounts of protein (50 \u0026micro;g per lane) were separated on 8% polyacrylamide gels and electro-transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked for 2 h in Tris-buffered saline (TBS) containing 5% skim milk and 0.1% Tween-20 to prevent nonspecific antibody binding. For immunodetection, membranes were incubated overnight at 4 \u0026deg;C with a monoclonal antibody against human PIEZO1 recognizing AAs 1275-1540 (MA5-32876, Invitrogen, 1:1000 dilution). After three washes with TBS containing 0.1% Tween-20, membranes were incubated with a horseradish peroxidase-conjugated IgG antibody (1:5000 dilution). Immunoreactive bands were visualized using enhanced chemiluminescence reagents (Millipore Japan, Tokyo, Japan). To normalize PIEZO1 protein expression, membranes were re-probed with a monoclonal antibody against \u0026beta;-actin (A5441, Sigma-Aldrich, 1:5000 dilution) as a loading control. Chemiluminescent signals were detected using a FUSION SOLO. 7S. EDGE (M\u0026amp;S Instruments Inc., Osaka, Japan).\u003cem\u003e \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatch-clamp recordings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhole-cell and excised outside-out patch-clamp recordings were performed as described previously\u003csup\u003e38\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e39\u003c/sup\u003e. Patch pipettes had a resistance of 3\u0026ndash;5 MW when filled with the pipette solution. The Cs\u003csup\u003e+\u003c/sup\u003e-based pipette solution contained (in mM): 110 Cs-aspartate, 30 CsCl, 1 MgCl\u003csub\u003e2\u003c/sub\u003e, 10 HEPES, 1 EGTA, and 2 Na\u003csub\u003e2\u003c/sub\u003eATP (pH 7.2, adjusted with CsOH). The extracellular solution contained (in mM): 137 NaCl, 5.9 KCl, 2.2 CaCl\u003csub\u003e2\u003c/sub\u003e, 1.2 MgCl\u003csub\u003e2\u003c/sub\u003e, 14 glucose, 10 HEPES (pH7.4, adjusted with NaOH). Membrane currents were amplified using an EPC-800 patch-clamp amplifier (HEKA, Lambrechit, Germany) and digitized at 10 KHz with a 5 kHz low-pass filter using a PCI6229 A/D converter (National Instruments Japan, Tokyo, Japan). Data acquisition and analysis were performed using WinWCPV4.5 for whole-cell recordings and WINEDR3.38 for excised patch single-channel recordings (software developed by Dr. John Dempster, University of Strathclyde, UK). The liquid junction potential (\u0026minus;10 mV) was corrected. For whole-cell recordings, voltage ramp commands from \u0026minus;110 to +90 mV (300 ms) were applied every 5 s from a holding potential of \u0026minus;10 mV. For excised outside-out patch recordings, voltage ramps (300 or 100 ms) were applied every 5 or 10 s from holding potentials of \u0026minus;50 mV (\u0026minus;100 to +100 mV) or -60 mV (\u0026minus;110 to +90 mV), as indicated. Single-channel recordings were digitally filtered at 1 kHz for amplitude histogram analysis. Leak current components were not subtracted. All recordings were performed at 25 \u0026plusmn; 1 \u0026deg;C with a perfusion rate of 5 ml min\u003csup\u003e-1\u003c/sup\u003e. Positive mechanical pressure was applied to excised outside-out patches using a custom-built pressure application system consisting of a pressure pump (Linicon LV-435A, Nitto Kohki CO. LTD, Tokyo, Japan), a solenoid valve for rapid pressure switching (VY1D00-6M5, SMC, Tokyo, Japan), and a pressure monitor amplifier (PA-011, Nihon Koden, Tokyo, Japan).\u003cem\u003e \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular Modeling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMolecular modeling was performed using UCSF Chimera v1.18\u003csup\u003e40\u003c/sup\u003e. Predicted three-dimensional (3D) structural data for full-length human PIEZO1 (hPIEZO1; AlphaFold DB entry AF-Q92508_F1-v6, 2521 AAs) were obtained from the AlphaFold database\u003csup\u003e41\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e42\u003c/sup\u003e. A homology model of chicken PIEZO1 (gPIEZO1; XP_015148150.2, 2492 AAs) was generated using the SWISS-MODEL server\u003csup\u003e43\u003c/sup\u003e, with the AlphaFold-predicted hPIEZO1 structure as the template. For construction of a trimeric gPIEZO1 model (Supplementary Fig.S3), the cryo-electron microscopy structure of mouse PIEZO1 (mPIEZO1; PDB ID: 6LQI) was used as a template. Structural visualization, alignment, and analysis were performed using UCSF Chimera, Clustal Omega\u003csup\u003e44\u003c/sup\u003e, and Jalview\u003csup\u003e45\u003c/sup\u003e..\u003cem\u003e \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA interference-mediated knockdown of hPIEZO1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStealth small interfering RNA (siRNA) duplexes targeting human \u003cem\u003ePIEZO1 \u003c/em\u003e(siPIEZO1; Invitrogen, Carlsbad, CA, USA) were used to suppress PIEZO1 expression. The siPIEZO1 sequences were as follows: sense strand, 5\u0026rsquo;- GCCUCGUGGUCUACAAGAUTT-3\u0026rsquo;; antisense strand, 5\u0026rsquo;-AUCUUGUAGACCACGAGGCTT-3\u0026rsquo;\u003csup\u003e \u003c/sup\u003e\u003csup\u003e37\u003c/sup\u003e. A medium GC Stealth RNAi negative control duplex (ncRNA, Invitrogen) was used as a control. HaCaT cells were cultured in 35-mm dishes and washed\u003csup\u003e \u003c/sup\u003ewith antibiotic-free medium 3 h before transfection. The siRNA or ncRNA was transfected using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer\u0026rsquo;s instructions, yielding a final siRNA or ncRNA concentration\u003csup\u003e \u003c/sup\u003eof 50 nM. Cells were incubated for 72-96 h before electrophysiological experiments.\u003cem\u003e \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are presented as individual data points together with the mean \u0026plusmn; s.d. and the interquartile ranges (25\u003csup\u003eth\u003c/sup\u003e-75\u003csup\u003eth\u003c/sup\u003e percentiles), as indicated. Comparisons between two groups were performed using paired or unpaired two-tailed Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-tests, as appropriate. Comparisons among multiple groups were conducted using one-way ANOVA followed by Tukey\u0026rsquo;s post hoc test or two-way ANOVA, as specified. Statistical analyses were performed using Origin J9.1 (LightStone, Tokyo, Japan). A \u003cem\u003eP\u003c/em\u003e value \u0026lt; 0.05 was considered statistically significant. Sample sizes are indicated in the figures or corresponding legends.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. J. Dempster (University of Strathclyde, UK) for developing the electrophysiology software (WinWCP and WinEDR) and Dr. Noriyuki Hatano (Aichi-Gakuin University) for technical assistance. We also acknowledge the Avian Bioscience Research Center at Nagoya University for providing chicken biological resources.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKM conceived and designed the study, secured funding, coordinated the project, performed electrophysiological experiments, analyzed and interpreted the data, generated figures, and wrote the manuscript. AM and HS performed Western blot analyses. YM generated all plasmid constructs. All authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUse of AI-assisted technology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the preparation of this manuscript, the authors used ChatGPT to improve language clarify and readability. All content was critically reviewed and revised by the authors, who take full responsibility for the integrity and accuracy of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS, 20K07599) to K.M.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are available from the corresponding author upon reasonable request. In addition, the datasets of gPIEZO1 gene generated and/or analyzed during the current study are available in the DDBJ repository, https://getentry.ddbj.nig.ac.jp/top-e.html, and Accession No. LC915090.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e The online version contains supplementary material available.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCoste, B.\u003cem\u003e et al.\u003c/em\u003e Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e330\u003c/strong\u003e, 55-60, doi:10.1126/science.1193270 (2010).\u003c/li\u003e\n\u003cli\u003eCoste, B.\u003cem\u003e et al.\u003c/em\u003e Piezo proteins are pore-forming subunits of mechanically activated channels. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e483\u003c/strong\u003e, 176-181, doi:10.1038/nature10812 (2012).\u003c/li\u003e\n\u003cli\u003eLi, J.\u003cem\u003e et al.\u003c/em\u003e Piezo1 integration of vascular architecture with physiological force. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e515\u003c/strong\u003e, 279-282, doi:10.1038/nature13701 (2014).\u003c/li\u003e\n\u003cli\u003eRanade, S. 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Jalview Version 2--a multiple sequence alignment editor and analysis workbench. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 1189-1191, doi:10.1093/bioinformatics/btp033 (2009). \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"f409a647-689e-48f8-91c1-1f581e2d6364","identifier":"10.13039/501100001691","name":"Japan Society for the Promotion of Science","awardNumber":"20K07599","order_by":0}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Aichi Gakuin University","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"PIEZO1, birds, mechanosensor, human skin keratinocytes","lastPublishedDoi":"10.21203/rs.3.rs-8855948/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8855948/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBirds experience distinct mechanical environments and performance demands, yet how mechanotransduction mechanisms are conserved and quantitatively tuned in avian species remains poorly understood. PIEZO1 is a mechanosensitive cation channel that mediates diverse physiological processes in mammals; however, its functional role in birds has not been directly established. Here, we present a comprehensive electrophysiological comparison of chicken PIEZO1 (gPIEZO1) and human PIEZO1 (hPIEZO1) using heterologous expression systems and native human cells. Using whole-cell and excised outside-out patch-clamp recordings, we show that both gPIEZO1 and hPIEZO1 are robustly activated by membrane stretch and by the synthetic PIEZO1 agonist Yoda1, generating characteristic mechanically activated currents that are potentiated by Yoda1 and inhibited by gadolinium. Single-channel analyses revealed comparable unitary currents for mechanically and chemically evoked currents in both orthologs, indicating conservation of core permeation properties. Notably, gPIEZO1 exhibited significantly greater sensitivity to both mechanical stimulation and Yoda1 than hPIEZO1, revealing species-dependent tuning of PIEZO1 mechanosensitivity. Importantly, in human skin keratinocytes, mechanical stimulation and Yoda1 elicited PIEZO1-like currents that were abolished by siRNA-mediated knockdown of PIEZO1, confirming physiological relevance in a native cellular context. Together, these findings establish PIEZO1 as a functional mechanosensor in birds and illustrate conserved yet quantitatively tuned mechanotransduction across vertebrates.\u003c/p\u003e","manuscriptTitle":"Functional conservation and species-specific tuning of PIEZO1 mechanotransduction in birds and humans","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-16 08:23:29","doi":"10.21203/rs.3.rs-8855948/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dcde0f57-f3aa-4d2b-b1e5-f46cd54c8a0f","owner":[],"postedDate":"February 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":62771285,"name":"General Cell Biology \u0026 Physiology"}],"tags":[],"updatedAt":"2026-02-16T08:23:29+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-16 08:23:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8855948","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8855948","identity":"rs-8855948","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}