Data from: Lipid composition and mechanical force underlie multi-modal regulation of Piezo1 gating

Published Apr 21, 2026 on Dryad. https://doi.org/10.5061/dryad.xgxd254wg

Data files

Apr 21, 2026 version files 35.48 KB

README.md

3.75 KB
Source_Data_File_SciAdv2026_.xlsx

31.74 KB

Abstract

Piezo1 ion channels are widely expressed cellular mechanosensors. They adopt an intrinsically curved shape when closed and are thought to open when mechanical forces applied to the membrane favor a more flattened conformation. By reconstituting Piezo1 channels into lipid vesicles, a flattened conformation has been determined, however, the ion conduction pore remained closed. In line with this observation, Piezo1 channels do not exhibit mechanical activation in the kind of lipids used in the structural studies. Here we show first that Piezo1 channels in cell-derived membranes retain functional mechanical gating, and second, that in cell-derived membranes they adopt a completely flattened disk shape associated with large conformational changes within and around the ion conduction pathway. These conformational changes occurring in cell-derived lipid membranes, suggest that mechanical force is necessary but insufficient, and that a specific membrane-derived cofactor apparently complements mechanical force to activate Piezo1.

Dataset DOI: 10.5061/dryad.xgxd254wg

Description of the data and file structure

Data from electrophysiology experiments as described in the results and methods section of the manuscript.

Files and variables

File: Source_Data_File_SciAdv2026_.xlsx

Description: One Excel spreadsheet, with separate tabs for each figure, listed in the order the appear in the manuscript. Details of data pertaining to each figure below:

Fig1D: Single channel patch-clamp recording of a patch excised from a GPMV overexpressing mPiezo1.

Count = number of data points falling into given amplitude bin

pA = amplitude of current recorded

Fig1F: Comparison of peak current measured in membrane patches excised from GPMVs which are untransfected or overexpressing mPiezo1. The measurement was made holding at +60 mV and -60 mmHg.

Mean and standard deviation are given and an Unpaired t test with Welch's correction was used to assess significant difference across the two conditions.

Fig 3G: Outside-out pressure clamp recordings of cells overexpressing WT, S1330A or S1330V Piezo1. Currents were held at +60mV and pressurized from 0 to -90 mmHg. Peak currents at each pressure step were normalized to the peak current for that patch. Normalized values are shown.

Fig 4C: Variables are Piezo1 radius and Vesicle radius. "Reconstituted Tomography Data" refers to value points taken from DOI: 10.1073/pnas.2208034119

"Piezo1 GV PMV dataset Outside-in" is data collected in this study. Piezo1 radius measurements (nm) were measured from 3D reconstructions of Piezo particles after 2D classification to separate into different vesicle size classes. The measurement was made by fitting a circle to the midplane of the Piezo-membrane dome and measuring its radius. Vesicle radius values (nm) were measured by taking a random subset of 30 Piezo1 particles in this class and measuring the vesicle size manually from the micrograph image in cryosparc.

Fig S2C-D: Conductance measurements of Piezo1 in varying potassium chloride concentrations. Excised patches from cells or GPMVs. Variables given are the voltage the patch is held at and the measured amplitude (pA) corresponding to a single-channel opening.

Fig S2E: The conductance values are measured from the slope of a line fit to measurements made in fig S2C-D and are plotted against the potassium chloride concentration for Cell and GPMV patches across n=3 repeats. The data points are fit to a Michaelis-Menten equation where the Vmax gives the estimated maximum single channel conductance.

Fig S3D: Comparison of inactivation of mPiezo1 across 3 conditions: cells without N-ethylmaleimide, GPMVs or cells treated with 7.5 mM N-ethylmaleimide in the bath solution for 1 hour. Values are given as % mechanosensitive current after 100ms which is measured as the current remaining 100ms into the pressure pulse divided by the peak pressure-activated current.

Fig S3G: Pressure-dependent activity of Piezo1 for cell-attached and GPMV recordings. Patches were held at 0mV before going to -60mV under increasing negative pressures from 0 to -80 mmHG. In each patch the current at each pressure is normalized to the maximal pressure-activated current.

Fig S11. Single channel amplitudes in GUVs under different conditions. The conditions are listed.