1. Evaluation Summary:

    The work, when completed, will provide important mechanistic detail on how Piezo channels, the most important and versatile mechanoreceptor molecules, functionally interact in the plane of the plasma membrane. It will be of interest to the field of mechanobiology and sensory mechanotransduction.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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  2. Joint Public Review:

    Lewis and Grandl propose that Piezo1 channels density does not have an effect on pressure sensitivity and that these channels do not cooperate in the nominal absence of membrane tension. To arrive at this conclusion the authors combined single channel measurements along with stochastic simulations of Piezo1 spatial distributions. An important element of this study is the use of two types of cells, one with intrinsically low level of Piezo1, and another with overexpressed channel. An interesting technical aspect is the use of a ramp pressure protocol, which overcomes the drawbacks of the standard step pressure method and help better estimate mechanosensitivity.

    The topic of this manuscript is timely and relevant as the study of this family of ion channels is still very new and the details of their gating mechanism are unknown, and the central conclusion is important for understanding the physiological role of Piezo1 is various types of mechanically sensitive cells.

    Although the manuscript is a tour de force, there are some concerns that should be addressed to validate the conclusions.

    1. Patch imaging to validate conclusions. We note that the authors departed from their previous strategy of patch visualization and direct tension determination (Lewis and Grandl, elife 2015). We do understand how tedious these experiments are, but the use of this approach in some of the trials would make this particularly study cleaner and more conclusive. The authors never mention tension as the parameter driving gating, and they work exclusively in the units of pressure. They postulated that their pipettes ranging between 3 and 6 MOhm in resistance provide 'standard' patches of given size and curvature. In fact, this range of resistances is large and roughly translates into a 1.4-fold difference in pipette diameters. If we look at Supplemental Fig 2D, we see that the largest pipettes (2-2.5 MOhm) produce 5 times the current recorded by 4-5 MOhm pipettes. These patches are unquestionably larger and no one can expect that the midpoint of channel activation by pressure is the same. The authors stated that only pipettes > 3MOhms were included into analysis, but this does not exclude actual size variation among patches. Fig. 4F,G clearly shows that patches containing 0-20 channels have higher P50. It is very likely that these patches are smaller and therefore will require higher pressure for activation. We expect that imaging can help here. The authors do not need to cover the entire range of densities; only two sets of measurements with low (native) and high (overexpression) densities would be sufficient. We suggest that you choose reasonably large pipettes (2.5-3 MOhm) so you can reliably image cell-attached patches. Two sets of tension-Po curves, their tension midpoints and slope factors, recorded from HEK cells transfected at low and high expression levels will provide a clinching result.

    2. Inactivated and silent channels. Because Piezo1 channels inactivate and require the removal of the stimulus to be reactivated, the presence of basal tension in the patch will render some Piezo1 inactivated. Given that the basal pressure differs between electrodes, different patches will always contain some number of 'silent' channels, which are physically present in the membrane, but do not manifest themselves functionally. This makes the estimation of a true number of channels in the patch virtually impossible using electrophysiology alone. The reviewers discussed this point at length. It was agreed that a few additional experiments (outlined below) would be ideal for mitigating all concerns on this issue. However, it was also agreed that the patch-imaging experiments requested above should be sufficient for supporting the paper's main conclusions, and therefore the experiments suggested below are suggested but not required. If the suggested experiments are not performed, we request that the authors mention / discuss the 'silent channel' point and indicate that their experiment provide a good estimation of the total number of channels, rather than the exact number.
      Suggested experiments:

    a) The authors claim that their novel stimulation protocol for single channel analysis allows them to measure the number of channels in each patch with high accuracy. The pressure ramps, used in this manuscript,¬ last about 3,000 ms at + 60mV. However, according to the same group (Wu et al, 2017, Cell Reports) Piezo1 inactivates within 90-100 ms at +60 mV. Hence, it is quite possible that in those patches the authors are underestimating the number of channels. It is not clear why the authors did not measure macroscopic currents using similar conditions as the ones used for single channel measurements. Particularly, macroscopic currents should be measured at positive potentials (to slow down inactivation), instead of at -80 mV, where Piezo1 tends to inactivate more rapidly than at positive potentials.

    b) The authors should consider perfusing Yoda1 (Piezo1 specific agonist) at the end of the pressure ramp experiments to reveal that there are indeed no more channels in the patch membrane than those visible ones with pressure.

    c) The representative traces on Figure 1B and 1D suggest that -60 mmHg does not saturate the response of Piezo1. The authors should add a control square pulse at the end of the experiment (equal to or larger than -60 mmHg) to ensure the currents are saturated.

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