Piezo1 ion channels are capable of conformational signaling

Piezo1 is a mechanically activated ion channel that senses forces with short latency and high sensitivity. Piezos undergo large conformational changes, induce far-reaching deformation onto the membrane, and modulate the function of two-pore potassium (K2P) channels. Taken together, this led us to hypothesize that Piezos may be able to signal their conformational state to other nearby proteins. Here, we use chemical control to acutely restrict Piezo1 conformational flexibility and show that Piezo1 conformational changes, but not ion permeation through it, are required for modulating the K2P channel TREK1. Super-resolution imaging and stochastic simulations further reveal that both channels do not co-localize, which implies that modulation is not mediated through direct binding interactions; however, at high Piezo1 densities, most TREK1 channels are within the predicted Piezo1 membrane footprint, suggesting the footprint may underlie conformational signaling. We speculate that physiologic..., Microscopy sample preparation
Cells were transiently transfected with TREK1HA and/or Piezo1Myc plasmids and plated on No 1.5 coverslips (Warner Instruments: CS-12R15, Catalog # 64-0712) 48 hours before staining, fixed in 2% formaldehyde, blocked with 10% normal goat serum, and stained with 1:100 chicken anti-Myc (Novus) primary antibody followed by 1:500 Alexa Fluor plus 594 goat anti-chicken (Thermo Fisher Scientific) secondary antibody to label Piezo1Myc channels and 1:500 rabbit anti-HA (Cell Signaling Technology) primary antibody followed by 1:200 Atto 647N goat anti-rabbit (Rockland Instruments) secondary antibody to label TREK1HA channels. Coverslips were then mounted with ProLong Glass Antifade Mountant (ThermoFisher Scientific) and cured at room temperature overnight before image acquisition. STED images were collected from at least three coverslips per condition, with n=3 independent transfections to generate biological replicates.
STED image acquisition and deconvolution
Two-..., , # Piezo1 ion channels are capable of long-range conformational signaling

## Description of the data and file structure

There are two files: One contains data for Neuro2A-Piezo1Myc cells overexpressing TREK1-HA (\"Endogenous\"); the other contains data for Neuro2a-Piezo1ko cells overexpressing both Piezo1-Myc and TREK1-HA (\"Overexpression\"). Within each folder are labelled cells (\"Cell B\", \"Cell C\", etc.) that correspond to Table 1 (Endogenous) and Table 2 (Overexpression) in the manuscript.

For the endogenous dataset, the PDF for each cell contains a STED image (scale bar: 1 micron) and associated cumulative frequency distribution, as in Figure 5A and 5B of the main manuscript.

For the overexpression dataset, the PDF for each cell contains a STED image and corresponding plot showing NNDT1-P1 as a function of local Piezo density, as in Figure 7A and 7C of the main manuscript.

## Code/Software

Python code used to generate random simulations and analyze nearest-neighbor distances ...