Yoda molecules agonize PIEZO2

Reviewer #1 (Public review):

Summary:

In this work, T. Wijerathne et al. investigated and reported the agonistic effect of Yoda1 and Yoda2 over PIEZO2 function using patch clamp electrophysiology, Ca2+ imaging, and molecular dynamics. They find that Yoda1 sensitizes PIEZO2 to membrane tension, can induce Ca2+ influx, and decreases its inactivation to a lesser degree than it does to PIEZO1 channels. Additionally, their data shows that Yoda2 sensitizes PIEZO2 channels to membrane indentation to a greater extent, but it has a weaker effect on channel inactivation than Yoda1. Interestingly, they report that a mutation in a conserved arginine between PIEZO channels can be used to abolish PIEZO1-mediated Ca2+ flux in response to Yoda molecules. As a whole, the results presented here should be put into perspective against previous and future works involving systems where both PIEZO1 and PIEZO2 might be expressed. This is especially true for works where Yoda1 has been used as a basis for determining the absence of PIEZO2.

Strengths:

The authors use multiple techniques to investigate how Yoda molecules affect the three most important biophysical aspects of PIEZO channels that, when changed, result in pathophysiological responses: a) sensitivity to mechanical stimuli, b) Ca2+ entry, and c) channel inactivation. Lastly, they find a specific amino acid/region that could be exploited for drug design and/or development.

Weaknesses:

The methods and discussion sections are lacking enough detail to fully evaluate the findings and put them into perspective, respectively.

Reviewer #2 (Public review):

Summary:

This manuscript challenges the long-standing assumption that Yoda1 and Yoda2 are PIEZO1-selective activators. Using patch-clamp electrophysiology and calcium imaging in HEK293TΔPZ1 cells overexpressing PIEZO2, the authors demonstrate that Yoda1 potentiates PIEZO2 stretch-activated currents to a similar extent as PIEZO1 and slows PIEZO2 poking-current inactivation (albeit with lower efficacy). They further show that the more potent analog Yoda2 affects PIEZO2 at nanomolar concentrations and use mutagenesis and molecular dynamics simulations to propose that Yoda2's benzoic acid group forms a transient salt bridge with R1724 in the putative Yoda binding pocket, explaining its enhanced potency.

Strengths:

The authors are established Piezo/biophysics experts; the study is highly important, technically competent, and carries significant implications for the reinterpretation of prior work that used Yoda compounds as PIEZO1-selective probes.

The core finding that Yoda1 modulates PIEZO2 stretch currents is convincing and important. However, several conceptual, methodological, and presentational issues need to be addressed before acceptance, as detailed below.

Weaknesses:

(1) The abstract states that Yoda1 potentiates PIEZO2 "as efficaciously as PIEZO1." This claim is accurate only for stretch currents and single-channel open probability, but the paper itself demonstrates important asymmetries: i) Yoda molecules slow PIEZO2 poking-current inactivation ~2-fold, versus ~5-10 fold for PIEZO1 (Figure 3b and ref #60). ii) Spontaneous Ca²⁺ entry via PIEZO2 requires non-physiological conditions (high extracellular Ca²⁺, hypertonic solutions) that are unlikely to occur in native cells.

The abstract should be revised to clearly qualify where equivalence holds and where efficacy differences exist. IMO, the current wording risks overcorrecting the historical bias (PIEZO1-only) by going too far in the other direction.

(2) Related concern: the PIEZO2 Ca²⁺ signal in Figure 2 is only detectable using a Ca²⁺-boosted solution (CBS ie 30 mM Ca²⁺). Physiological extracellular Ca²⁺ and cells normally do not experience sustained hypertonicity at these magnitudes. The authors should explicitly clarify that the practical implication of their findings is primarily for electrophysiological (patch-clamp) experiments and that the Ca²⁺ imaging caveat applies only under amplified conditions. Specifically, the authors should state that in standard Ca²⁺ imaging assays with physiological buffers, PIEZO2 is unlikely to confound Yoda1 results.

Related point: Can cytochalasin D (CytoD) restore a Yoda1-dependent Ca²⁺ signal in physiological saline? This would help determine whether the weak PIEZO2 response is primarily a membrane tension issue (cytoskeletal tethering) versus intrinsically lower channel expression or permeability. The authors already have tagged PIEZO1/2 constructs and could, in principle, normalize by surface expression.

(3) The mean inactivation tau values for wild-type PIEZO2 poking currents in both DMSO and Yoda1 conditions (Figure 3b, approximately 15-40 ms range) appear substantially higher than values reported in published literature (typically 5-10 ms; eg, PMID: 20813920). This discrepancy needs to be addressed.

(4) The authors perform all MD simulations on a truncated PIEZO1 model and justify this choice by noting that the Yoda binding region is highly conserved between homologs. This is a reasonable and defensible starting point given the availability of well-validated PIEZO1 simulation set ups in their lab. A few points are nonetheless worth addressing: While PIEZO2 simulations are not strictly required, the authors are encouraged to briefly discuss whether any long-range structural differences between PIEZO1 and PIEZO2 (outside the binding site itself) could influence Yoda2 binding dynamics, particularly in light of the chimera data showing that PIEZO2 sequence in repeat A abolishes Yoda1 sensitivity. This reviewer still doesn't understand the reason behind this discrepancy despite it being acknowledged in the text.

Another MD-related comment is that three simulation replicas (which is impressive for such a big system) show markedly different salt bridge occupancy (82.6%, 49.7%, 99.8%; stated in the text). This wide variation suggests incomplete sampling in at least one replica. The authors should provide RMSD plots for ligand and protein backbone to assess convergence and possibly discuss whether the 49.7% replica represents a genuinely distinct binding mode or incomplete equilibration.

(5) The Discussion proposes that PIEZO2's weaker Ca²⁺ response to Yoda1 could partly reflect lower membrane expression. Since the authors already have fluorescently tagged PIEZO1 and 2 constructs, a simple fluorescence intensity comparison between the two (acknowledging it would reflect total rather than surface expression) could provide at least indirect support for this claim. Alternatively, if such a comparison is not feasible, the authors may consider removing membrane expression from the list of proposed explanations or explicitly acknowledging that this remains unsubstantiated speculation. The max poking currents may somewhat and roughly indicate the level expression difference too, if done exactly side by side.

(6) The abstract or concluding remarks should highlight that Dooku1 is not PIEZO1-selective in its agonist-like action on PIEZO2, and that Cmpd15/Cmpd64 appear to be better PIEZO1-selective tools. This nuance is buried in the Results section.

(7) The authors should not cite PMID 31015490. Clearly, any work on MCC13 is confounded by the overwhelming expression of PIEZO1 (PMID: 42084270). Instead, the authors should also cite the literature from others who have clearly recorded stretch currents from PIEZO2 before the cited studies (eg, PMID: 37590348).

Reviewer #3 (Public review):

Summary:

The manuscript reports that Yoda1 and Yoda2 agonize PIEZO2 in a manner similar to PIEZO1, increasing open probability and stretch sensitivity, but the mechanism underlying this sensitivity is incomplete. Mutagenesis was shown exclusively in PIEZO1, with no corresponding mutagenesis in PIEZO2, so the proposed mechanism in PIEZO2 is inferred by homology rather than directly tested. All experiments use mouse PIEZO2, and the human ortholog should be used before generalizing the proposed reinterpretation of the field.

Strengths:

The pressure-clamp electrophysiology demonstrating a shift in half-activation pressure for PIEZO2 is compelling evidence in support of the central claim.

Weaknesses:

(1) In the single-channel recordings (Figure 1a), it's unclear how many channels were present in those patches. After applying -60 mmHg pressure, multiple channels would be activated (as seen in Figure 1e). The number of channels in the patch and their inactivation rate could significantly influence the open probability in such experiments. To overcome this, in the original Yoda1 article (Syeda, Ruhma, et al. eLife 2015), no additional pressure was used. Additionally, the reported open probability comparison (n=7 Yoda1 vs n=17 DMSO patches) has an SEM nearly as large as the effect itself (0.30 {plus minus} 0.11), consistent with a small number of outliers driving this. The underlying mean open and shut times are reported without any statistical test; only the derived open probability receives a p-value. Additionally, in Figure 1a, the Yoda1 condition noise is different from the control. This should be stated if noise filtering was applied and how, given that this could affect open probability analysis.

(2) The calcium imaging data in Figure 2 raise significant concerns regarding the chemical activation claim. The calcium-boosted solution (30 mM Ca2+) is not physiological and appears to be generally stressing cells rather than specifically activating PIEZO2: the control condition under CBS already shows an elevated signal, consistent with cells being unwell at this calcium concentration, and adding Yoda1 on top of this shifted baseline raises further questions about specificity rather than confirming it. Separately, it is unclear why DMSO alone produces measurable PIEZO2-associated calcium influx in HBSS, a result that is not addressed in the text. Figure 2 should clearly indicate when DMSO/Yoda1 perfusion was initiated, and y-axis labels are missing from panels A and B.

(3) In the poke experiments, an activation threshold should be calculated and reported, and amplitude data (e.g., peak current versus indentation depth) should be shown rather than only inactivation tau values. It is also unclear why mClover3- and N-GFP-tagged constructs were used in these experiments, since electrophysiological recording already confirms channel expression without requiring a fluorescent tag.

(4) For inactivation kinetics (Figure 3b), the authors use unpaired comparisons across separate cells, whereas the deactivation experiments (Figure 3c) use paired; it should be applied to the inactivation experiments as well. Deactivation kinetics for PIEZO2 itself should be shown. If the claim is that Yoda1 acts on PIEZO2 through the same mechanism proposed for PIEZO1, then a PIEZO1/2 chimera should be expected to show a corresponding effect on deactivation tau; instead, this chimera is reported as completely Yoda1-insensitive despite both parental channels being Yoda1-sensitive, as shown in this study.

(5) Given that this reflects a different experimental paradigm for Yoda EC50, PIEZO1 should be included within Figure 4b. Additionally, EC50 bar plots should be present on this figure. The inactivation time constant for PIEZO2 without Yoda1 is inconsistent across figures, below 20 ms in Figure 3b but above 20 ms in Figure 4c.

(6) Finally, the modeling is performed exclusively on PIEZO1, whereas the manuscript's central focus is PIEZO2. It is therefore unclear whether the proposed structural mechanism, including the basis for Yoda2's reduced efficacy on PIEZO2, can be directly extrapolated to PIEZO2.