ATP8B1–TMEM30B Flippase Activity Maintains Stereocilia Lipid Asymmetry Required for Hearing

Reviewer #1 (Public review):

Sensory hair cells of the inner ear convert mechanical sound vibrations into electrical signals through mechano-electrical transduction (MET). While the protein components of the MET machinery have been studied extensively, much less is known about how the surrounding membrane lipid environment contributes to hair cell function. The recent discovery that TMC1 and TMC2 also function as lipid scramblases has brought renewed attention to the importance of membrane lipid asymmetry and the mechanisms that maintain it in sensory hair cells.

In this study, the authors identify the P4-ATPase ATP8B1 and its partner TMEM30B as key regulators of membrane lipid asymmetry in outer hair cells. Using complementary genetic models, HA-tagged knock-in mice, localization analyses, and functional experiments, they show that ATP8B1-TMEM30B is enriched in stereocilia and the apical membrane of outer hair cells and is required to maintain phosphatidylserine asymmetry, support hair cell survival, and preserve normal hearing. The parallels between the ATP8B1/TMEM30B loss-of-function phenotypes and TMC1 deafness-associated mutants with constitutive scrambling support a model in which ATP8B1-TMEM30B flippase activity maintains membrane lipid asymmetry and homeostasis, whereas constitutive TMC1-mediated phospholipid scrambling disrupts this balance and contributes to membrane instability.

The authors have addressed the points raised during the initial review thoroughly. The revised manuscript includes clearer methodological details, additional physiological characterization, improved presentation and quantification of several datasets, and a more balanced interpretation of the localization and mechanistic findings. These changes improve both the clarity and rigor of the study while leaving its main conclusions unchanged.

As with any study that opens a new area of investigation, important mechanistic questions remain. In particular, it will be interesting to determine how disruption of membrane lipid asymmetry ultimately impairs MET function and triggers hair cell degeneration, how flippase and scramblase activities are coordinated in vivo, and how these pathways are integrated with the broader molecular machinery underlying mechanotransduction. These questions highlight the exciting directions that this study opens for the field.

Overall, this work provides evidence that ATP8B1-TMEM30B is a critical regulator of stereocilia membrane lipid asymmetry and represents an important contribution to our understanding of membrane homeostasis in auditory hair cells. I have no further major concerns and support publication.

Reviewer #2 (Public review):

Summary:

Prior work identified TMEM30B (knockout mice) as well as ATP8B1 (human genetics and mouse model), ATP8A2 (knockout mice), and ATP811A (human genetics) as relevant for hearing. The authors also reasoned that given the recent discovery of TMC1 and TMC2's dual function as mechanotransduction channels of the inner ear and as lipid scramblases, a counterpart flippase should be in the sensory hair-cell stereocilia bundle where mechanotransduction happens. They use CRISPR/CAS to modify the endogenous mouse genes and add an HA tag at the N-terminus of the ATP8B1, ATP8A1, ATP8A2, and ATP11A proteins. Their experiments with these mice unambiguously localized ATP8B1 at the base of outer hair cell stereocilia bundles. Knockout of ATP8B1 results in loss of outer hair cells, deficient auditory function (ABR), and degeneration of outer hair cell stereocilia bundles. Similarly, hair cells from genetically modified mice with endogenous HA-tagged TMEM30B proteins show localization of this protein to outer hair cell stereocilia bundles. TMEM30B knock out mice phenocopy the ATP8B1 knock out model. Interestingly, the authors show that annexing V staining precedes hair cell loss in ATP8B1 and TMEM30B knockout mice and that proper localization of these proteins is lost in mice that lack CIB2, a protein essential for hair cell mechanotransduction.

Strengths:

(1) Use of knock-in HA-tagged proteins to unambiguously localize ATP8B1 and TMEM30B

(2) Systematic characterization of auditory function (ABR), hair cell loss, and hair-cell stereocilia bundle morphology.

(3) Advances our understanding of the role played by lipid homeostasis in auditory function.

(4) Reports on mouse models that will be helpful to further understand the mechanistic role played by ATP8B1 and TMEM30B in normal hearing and hereditary deafness.

Weaknesses:

(1) Are the HA tags causing any functional issues? Function and localization of tagged proteins can sometimes be compromised. This is checked for TMEM30B and ATP8B1, but not for ATP8A1, ATP8A2, and ATP11A.

(2) Following on the point above, is it possible that ATP8B1-HA is well localized, but localization for the other three flippases (ATP8A1-HA, ATP8A2-HA, and ATP11A-HA) is compromised by the tag? Is this potential miss-localization causing any functional phenotypes? I find surprising that there are flippases only in outer hair cells and only formed by ATP8B1. A possible explanation is that the tag is interfering with trafficking. If so, there should be a phenotype (ABRs), although this might be masked by redundancy among these flippases or caused by systemic issues (admittedly difficult to sort out).

Author response:

The following is the authors’ response to the original reviews.

Summary of Revisions Performed:

We have clarified the qPCR methodology in the methods section and stated the housekeeping gene GAPDH to address potential misunderstandings.

We have assessed hearing in the generated HA-tagged mouse lines and included an adequately powered ABR measurements analysis in the revised manuscript as a supplemental figure.

We have included powered DPOAE experiments in both ATP8B1 and TMEM30B KO mice to strengthen the findings of the ABRs.

We have clarified the presentation of the z-stack in Figure 1F.

We have elaborated on the analysis for Figure 7B to strengthen comprehension by readers.

We have revised the statement to read: “No IHC stereocilia-enriched P4-ATPases were detected under the conditions examined.”

While we appreciate the suggestion to examine TMEM30B localization on the ATP8B1 KO background, this is not feasible within a reasonable timeframe; we have clarified this limitation in the manuscript.

We have incorporated relevant prior work (e.g., George and Ricci, 2026) demonstrating minimal Annexin V labeling prior to P6 and lack of PS externalization in TMC1/2 double knockout models.

We have clarified that hearing thresholds for TMEM30B-HA and ATP8B1-HA lines were addressed in this study, while additional HA-tagged flippase lines (ATP8A1, ATP8A2, ATP11A) are part of ongoing work to be reported separately.

We have softened statements regarding HA-tag insertion and clarified that, to our knowledge, localization and function are not disrupted, while acknowledging this as a potential limitation.

We have revised the Methods section to clarify differences in fluorescence measurements across experiments.

Public Reviews:

Reviewer #1 (Public review):

Figure1D.

The authors should clarify how the qPCR data were normalized and specify the reference (housekeeping) genes used. This information is necessary to evaluate the robustness and comparability of the gene expression data.

We thank the reviewer for this comment. qPCR data were normalized to GAPDH as the reference (housekeeping) gene. We have clarified this in the Methods section to ensure transparency and reproducibility.

(2) Figure 1F.

The lack of F-actin staining at the hair cell base raises the possibility that the permeabilization conditions may have limited antibody access to certain membrane regions. This is especially important given that the authors used a gentle permeabilization agent such as saponin to preserve membrane integrity. Because the authors conclude that ATP8B1 and TMEM30B are localized "almost exclusively to OHC bundles and the apical membrane, with minimal staining in the remaining plasma membrane," (line 128). Including co-labeling with a plasma membrane marker or more comprehensive F-actin visualization of lateral and basal regions would help ensure that the restricted localization is biological rather than technical. In the absence of such controls, the localization claim may be somewhat overstated and should be tempered accordingly.

We thank the reviewer for this important point. The image shown represents a single z-slice from a larger stack, and the hair cell body lies outside the plane of this section. To clarify this, we revised the accompanying text.

(3) Figure 7B.

Although quantification of ATP8B1-HA intensity at the bundle appears similar between WT and Cib2 KO samples, the representative image suggests that some bundles lack detectable labeling. To better capture phenotype variability, it would be helpful to include an additional quantification showing the fraction or number of bundles with detectable ATP8B1-HA signal in Cib2 KO mice.

We thank the reviewer for this suggestion. We have clarified the quantification of the fraction of hair cell bundles with detectable ATP8B1-HA and TMEM30B-HA signal per field of view. Although the representative images may give the impression that some hair bundles lack staining, this is due to changes in ATP8B1-HA and TMEM30B-HA distribution within the cell body. In all cases, detectable ATP8B1-HA and TMEM30B-HA signal remained present in the hair bundles.

(4) Lines 346-349

The manuscript suggests that IHCs lack stereocilia-enriched P4-ATPases. However, this conclusion is not directly supported by the presented data. The authors should either provide supporting localization or expression data for other P4-ATPases or soften the statement to indicate that no stereocilia-enriched P4-ATPases were detected under the conditions examined.

We agree with the reviewer and have revised this statement to read: “No IHC stereocilia-enriched P4-ATPases were detected under the conditions examined.”

Recommendations:

(5) The authors convincingly demonstrate that TMEM30B loss results in ATP8B1 mislocalization. While not essential to the central conclusions, examining TMEM30B localization in ATP8B1 KO hair cells would clarify whether this interdependence is reciprocal, as described for other P4-ATPase-CDC50 complexes.

While we agree that this experiment would provide valuable information, performing it would require generation of a compound mouse line carrying both the TMEM30B-HA allele and the ATP8B1 knockout allele. This work is beyond the scope of the current revision and cannot be completed within a reasonable timeframe.

(6) Lines 359-374. The discussion of Annexin V labeling is careful and balanced. This paragraph would benefit from referencing other studies that showed minimal Annexin V labeling in healthy P6 organ of Corti, reinforcing that robust PS externalization in the present study is pathological rather than developmental.

We thank the reviewer for this suggestion and have incorporated relevant prior work, including George and Ricci (2026), which demonstrates minimal Annexin V labeling prior to P6 and further supports our interpretation.

(7) Lines 392-399.

The proposed feedback model linking MET activity and ATP8B1-TMEM30B localization is compelling. The discussion could be strengthened by noting that in TMC1/2 double knockout hair cells, PS externalization is not observed, consistent with the idea that flippase activity becomes critical specifically when scrambling occurs. The mislocalization observed in Cib2 KO hair cells further supports the coupling between TMC-mediated scrambling and flippase-mediated membrane restoration.

We agree and have revised the text to include that TMC1/2 double knockout hair cells do not exhibit phosphatidylserine externalization, supporting the idea that flippase activity becomes critical in the context of scrambling.

Reviewer #2 (Public review):

Weaknesses:

(1) Are the HA tags causing any functional issues? Function and localization of tagged proteins can sometimes be compromised. It would be good to know, for each knock-in model (TMEM30B, ATP8B1, ATP8A1, ATP8A2, and ATP11A), whether the HA-tagged protein is causing any issues with the mice and particularly with hearing (ABRs). Are these mice normal? Can they hear? These data are missing.

We thank the reviewer for raising this important point. In this study, we focus on TMEM30B-HA and ATP8B1-HA mouse lines, while additional HA-tagged flippase lines (ATP8A1, ATP8A2, ATP11A) are part of ongoing work to be reported separately.

Both TMEM30B-HA and ATP8B1-HA mice are viable and exhibit normal breeding and ageing. We have included adequately powered ABR measurements of both TMEM30B-HA and ATP8B1-HA which indicate wild-type–like hearing thresholds.

(2) Following on the point above, is it possible that ATP8B1-HA is well localized, but localization for the other three flippases (ATP8A1-HA, ATP8A2-HA, and ATP11A-HA) is compromised by the tag? Is this potential mislocalization causing any functional phenotypes? (ABRs of point 1). I find it surprising that there are flippases only in outer hair cells and only formed by ATP8B1. A possible explanation is that the tag is interfering with trafficking. If so, there should be a phenotype (ABRs), although this might be masked by redundancy among these flippases or caused by systemic issues (admittedly difficult to sort out). Given that this manuscript will likely become foundational, and that there is evidence that at least two of the other flippases are involved in hearing loss, it would be good to provide more information about the mice and HA-tagged proteins in the other knock-ins (ATP8A1-HA, ATP8A2-HA, and ATP11A-HA). Depending on the data available for the knock-ins, the authors may want to discuss these scenarios and soften the statement indicating that inner-hair cells may lack flippase activity altogether.

We appreciate this concern. To our knowledge, the HA tag does not appear to disrupt localization or function of the tagged proteins. However, we agree that this cannot be fully excluded. We have therefore softened our conclusions about IHC flippases and clarified that additional flippases (ATP8A1, ATP8A2, ATP11A) are under investigation and will be described in a separate study.

(3) Expression of ATP8B1 at P0 (Figure 1D), when there should not be protein in outer hair cells yet seems high. Does this mean that other cells in the cochlea also express ATP8B1? Is this a concern?

We thank the reviewer for this observation. We interpret the elevated ATP8B1 transcript levels at P0 as reflecting transcription that precedes detectable protein accumulation in OHC stereocilia. While expression in other cochlear cell types cannot be excluded, we did not detect ATP8B1-HA immunolabeling outside hair cells in the knock-in model.

(4) Fluorescence scales in Figure 6 B and D and Figure 7 B and D are very different. So are the values for WT. One would expect that the WT would be similar in all cases (at least within the same compartments), given that the methods section indicates that "All images were collected using identical acquisition parameters, including zoom and laser power, across genotypes". If WT shows such variability, how can we compare?

We appreciate the need for clarification. Identical acquisition parameters were maintained within each experiment used for direct comparison (e.g., within a given panel). However, different panels (e.g., Figures 6B vs. 6D) were acquired on different days using different imaging settings.

We have revised the Methods section to explicitly state this and clarify that comparisons are intended only within panels, not across experiments.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) Line 42: When discussing TMC similarity to TMEM16 scramblases, it may be helpful to mention that some TMEM16 family members (TMEM16A and B) function as ion channels, highlighting the dual ion/lipid functionality within the superfamily. The similarity to TMEM63/OSCA ion channels and lipid scramblases could also be noted. The fact that TMC, TMEM16, and TMEM63/OSCA belong to the same superfamily would provide a broader context. I also suggest referencing the work that initially suggested this relationship: (https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0192851)

We have included this citation and expanded on this discussion in the revised version.

(2) Line 45: Consider including the recent Cryo-EM structure of CeTMC2 (PNAS, 2023), which provides structural insight into TMC-lipid interactions.

We have included this citation in the revised version.

(3) Line 62: For precision, consider removing "calcium-activated," as caspase-activated scramblases also disrupt membrane asymmetry.

For precision, we have removed “calcium-activated” in the revised version.

(4) Line 71: Clarify whether this refers to "fusion of membranes" or "cell fusion."

We have clarified this statement to mean cell-cell fusion.

(5) Line 85: Consider citing studies showing constitutive PS externalization in TMC1 mutant mouse models linked to deafness.

We have added a citation to show that constitutive PS externalization is linked to deafness (Ballesteros and Swartz, 2022, and Beurg et al. 2025).

(6) Line 93: TMEM30C is not discussed. A brief comment on its expression or relevance in hair cells would provide completeness.

We have added a brief statement regarding TMEM30C and cited prior work describing its expression pattern (Osada et al. 2007).

(7) Figure 1A: Use distinct colors for the P4-ATPase and CDC50 subunit rather than a rainbow scheme to improve clarity.

We have retained the original color scheme in this panel.

(8) Figures 3C-D and 5C-D: Increase legend symbol size for clarity. Update Y-axis labels to "Number of OHCs/100 μm" and "Number of IHCs/100 μm." Correct "um" to "μm."

We changed the legend to improve the presentation of these panels to be more legible and changed the measurement to μm.

(9) Figures 3F, 5F, 5H: Add scale bars.

We have added scale bars to these figures.

(10) Figure 7: The confocal images (A, C) show the bundle on top and cell body below, but the quantification (B, D) is in the opposite order. Reorganizing the panels for consistent orientation would improve clarity.

We have reorganized the panels to improve clarity.

(11) ABR measurements: Please specify the sex of the mice tested or clarify whether both sexes were included.

We have included both male and female mice in this study as there were no differences in hearing function. We have added this clarification to the methods section under hearing tests.

Reviewer #2 (Recommendations for the authors):

(1) In Figure 1A, the panels show CDC50. I would either change to TMEM30B or mention in the caption that TMEM30B is also known as CDC50 as labeled in the figure.

We have changed CDC50 to TMEM30B.

(2) Figures 1F and 1G are missing scale bars.

We have added scale bars to these figures.

(3) Figures 2 C, D, and 5 C, D - difficult to tell what's what in the legend. Perhaps make symbols larger in front of WT P17, KO P17, etc.?

We changed the legend to improve the presentation of these panels.

(4) Text under "TMEM30B is required for hearing and OHC maintenance". There is a difference in phenotype between the TMEM30B (Figure 5C) and ATP8B1 (Figure 3C) knockouts that is not discussed, as apical and middle cells seem to be okay. Should this be discussed?

We appreciate this observation. We have elected not to expand the discussion of these regional differences because apical and middle hair cells also undergo degeneration at later ages (after P30), suggesting that the observed differences primarily reflect the timing of degeneration rather than distinct underlying mechanisms.

(5) In the discussion text, under "Why do ATP8B1/TMEM30B-deficient OHCs die?", "Tmc1/2 or Cib2" should probably be "TMC1/2 or CIB2" or "Tmc1/2 or Cib2"

We have changed this to read TMC1/2 or CIB2.

(6) The methods section states "..., whereas non-significant comparisons are not shown." However, non-significant p values are shown in Figures 7B and D (bottom panels).

We have removed the nonsignificant comparisons from Fig 7B and D to be consistent with the rest of the paper.