ATP8B1–TMEM30B Flippase Activity Maintains Stereocilia Lipid Asymmetry Required for Hearing
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eLife Assessment
Mechanical transduction channels of sensory hair cells possess lipid scramblase activity. Membrane lipid disruption resulting from mechanical transduction is thought to be restored by flippase activities. This fundamental study provides compelling evidence that ATP8B1, a P4-ATP flippase and its subunit TMEM30B, are key in mediating this restorative function in outer hair cells of the mammalian cochlea.
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Abstract
Sensory hair cells convert sound-induced vibrations into electrical signals through a process called mechano-electrical transduction (MET). While the protein components of the MET complex are well studied, increasing evidence indicates that MET channel properties are significantly modulated by the surrounding lipid bilayer. The asymmetric distribution of membrane lipids between the inner and outer membrane leaflets is well established to shape membrane mechanics. The recent discovery that the core MET components TMC1 and TMC2 also act as lipid scramblases suggests a direct role for membrane lipid asymmetry in the dynamic shaping of auditory transduction. Because scramblase activity of TMC1/2 disrupts lipid asymmetry, we hypothesized that an opposing flippase may be required to restore and maintain lipid asymmetry. Here, we identify the P4-ATPase ATP8B1 and its chaperone TMEM30B as selectively expressed in outer hair cells (OHCs), enriched in stereocilia, and upregulated following the onset of MET and hearing. Loss of either protein results in elevated auditory brainstem response (ABR) thresholds, phosphatidylserine (PS) externalization, and rapid hair-cell degeneration, demonstrating that lipid homeostasis is crucial for OHC survival. Together, these findings establish ATP8B1 and TMEM30B as key regulators of membrane lipid asymmetry in sensory hair cells and establish TMEM30B as a novel deafness gene.
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eLife Assessment
Mechanical transduction channels of sensory hair cells possess lipid scramblase activity. Membrane lipid disruption resulting from mechanical transduction is thought to be restored by flippase activities. This fundamental study provides compelling evidence that ATP8B1, a P4-ATP flippase and its subunit TMEM30B, are key in mediating this restorative function in outer hair cells of the mammalian cochlea.
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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 …
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.
Comments on second revision:
I appreciate the authors' efforts to address the issue and perform the additional experiment, and I agree that removing these data is reasonable and does not affect the main conclusions of the manuscript.
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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 …
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.
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.
Comments on second revision:
It is interesting that in the images selected as representative of the new experiments the effect seems to be opposite to what was reported before - more ATP8B1-HA in the bundle with transduction blockage. However, this does not seem to be supported by the quantification, and further exploration at different ages, blocker concentrations, effective transduction blockage, etc. might be needed to really have a solid result. Thus, I agree that leaving out these results, and the previous sections linking mechanotransduction to localization of ATP8B1, is the best option.
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Author response:
The following is the authors’ response to the previous reviews.
Public Reviews:
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 …
Author response:
The following is the authors’ response to the previous reviews.
Public Reviews:
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).
We would like to apologize for the oversight on our end that caused the delay and repeat consultation with you, and would like to thank you for your patience.
As a reminder, after our manuscript underwent the full review, we discovered an issue with the final figure of the paper. This experiment is ancillary to the main story, and its removal does not alter the paper’s central findings or conclusions. This Figure 7 asked whether genetic loss of mechanotransduction (MET) alters ATP8B1/TMEM30B distribution in hair cells. After we discovered a problem with the MET-negative mouse model, we agreed with the reviewers to instead test whether pharmacological MET block has the same effect. We found that a 30-minute incubation with benzamil (MET blocker) did not alter ATP8B1 distribution (see uploaded PDF named “ATP8B1-HA_Benzamil”).
Because this experiment was limited in scope and is difficult to compare directly with genetic deletion of MET, we think it is best to proceed with the manuscript version that removes this line of inquiry.
We have made the following changes (compared to the most recent submission):
- Removed Figure 7
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eLife Assessment
Mechanical transduction channels of sensory hair cells possess lipid scramblase activity. Membrane lipid disruption resulting from mechanical transduction is thought to be restored by flippase activities. This fundamental study provides compelling evidence that ATP8B1, a P4-ATP flippase and its subunit TMEM30B, are key in mediating this restorative function in outer hair cells of the mammalian cochlea.
[Editors’ note, September 9, 2026: There is a potential confound with the findings shown in Figure 7 suggesting that specific targeting of ATP8B1 and TMEM30B subunits to the hair cell stereocilia is dependent on mechanoelectrical transduction channel activity. This does not affect the central findings or conclusions, but readers should be aware that a revised version of the paper is being prepared.]
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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 …
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.
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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 …
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).
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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 …
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.
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The authors included RRIDs in their in bioRxiv paper! Thanks for making your methods matter! #OpenResearch #OpenScience #RRID
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eLife Assessment
Mechanical transduction channels of sensory hair cells possess lipid scramblase activity. Membrane lipid disruption resulting from mechanical transduction is thought to be restored by flippase activities. This fundamental study provides compelling evidence that ATP8B1, a P4-ATP flippase and its subunit TMEM30B, are key in mediating this restorative function in outer hair cells of the mammalian cochlea.
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Reviewer #1 (Public review):
Sensory hair cells of the inner ear convert mechanical sound vibrations into electrical signals through mechano-electrical transduction (MET), a process critically dependent on the specialized organization and lipid composition of their plasma membrane. Although the protein components of the MET complex are relatively well characterized, the role of the lipid environment remains poorly understood and often overlooked. Recent discoveries that core MET proteins TMC1 and TMC2 function as lipid scramblases, disrupting membrane lipid asymmetry, expose a significant gap in our understanding of how lipid homeostasis is regulated in hair cells and how membrane dynamics influence MET function.
In this study, the authors address this gap by identifying the P4-ATPase ATP8B1 and its chaperone TMEM30B as essential …
Reviewer #1 (Public review):
Sensory hair cells of the inner ear convert mechanical sound vibrations into electrical signals through mechano-electrical transduction (MET), a process critically dependent on the specialized organization and lipid composition of their plasma membrane. Although the protein components of the MET complex are relatively well characterized, the role of the lipid environment remains poorly understood and often overlooked. Recent discoveries that core MET proteins TMC1 and TMC2 function as lipid scramblases, disrupting membrane lipid asymmetry, expose a significant gap in our understanding of how lipid homeostasis is regulated in hair cells and how membrane dynamics influence MET function.
In this study, the authors address this gap by identifying the P4-ATPase ATP8B1 and its chaperone TMEM30B as essential regulators of membrane lipid asymmetry in outer hair cells. They also generated HA-tagged knock-in mice to precisely localize the P4-ATPase ATP8B1 and its chaperone TMEM30B within outer hair cells, demonstrating their enrichment in stereocilia, and convincingly demonstrate that loss of these proteins causes phosphatidylserine externalization, hair cell degeneration, and hearing loss in mouse models, phenocopying defects observed in TMC1 mutant mice with constitutive scrambling activity. While these findings establish lipid flippase pathways as critical for hair cell survival and auditory function, they also raise important questions about the precise mechanisms linking lipid asymmetry disruption to MET dysfunction and hair cell pathology.
Overall, the data convincingly support the conclusion that ATP8B1-TMEM30B flippase activity is required to maintain stereocilia lipid asymmetry and auditory function. The study substantially advances understanding of how lipid homeostasis intersects with MET. However, several points require clarification to ensure that localization claims and mechanistic interpretations are fully supported by the presented data.
Revisions considered essential by this reviewer are:
(1) Figure 1D.
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.(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.(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.(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.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.
(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.(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. -
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), …
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, rather than antibody staining, 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. 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.
(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.
(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?
(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?
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Author Response:
Summary of Planned Revisions:
We will clarify the qPCR methodology and interpretation to address potential misunderstandings.
We will assess hearing in the generated HA-tagged mouse lines and, where appropriate, include a properly powered ABR analysis in the revised manuscript.
We will address concerns regarding the z-stack in Figure 1f.
We will include additional quantification for Figure 7B to strengthen the analysis.
We will revise the relevant 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 will clarify this limitation in the manuscript.
We will incorporate relevant prior work (e.g., George and Ricci, 2026) …
Author Response:
Summary of Planned Revisions:
We will clarify the qPCR methodology and interpretation to address potential misunderstandings.
We will assess hearing in the generated HA-tagged mouse lines and, where appropriate, include a properly powered ABR analysis in the revised manuscript.
We will address concerns regarding the z-stack in Figure 1f.
We will include additional quantification for Figure 7B to strengthen the analysis.
We will revise the relevant 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 will clarify this limitation in the manuscript.
We will incorporate 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 will clarify that hearing thresholds for TMEM30B-HA and ATP8B1-HA lines will be addressed in this study, while additional HA-tagged flippase lines (ATP8A1, ATP8A2, ATP11A) are part of ongoing work to be reported separately.
We will soften statements regarding HA-tag insertion and clarify that, to our knowledge, localization and function are not disrupted, while acknowledging this as a potential limitation.
We will revise the Methods section to clarify differences in fluorescence measurements across experiments.
In addition to the experiments in response to reviewer’s suggestions, we will add the following data that we have generated while the paper was in review:
Distortion product otoacoustic emission (DPOAEs) of the Atp8b1 KO and Tmem30b KO mice. Consistent with OHC function, their DPOAEs thresholds were elevated.
Public Reviews:
Reviewer #1 (Public review):
(1) 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 will clarify 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 appreciate 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 will revise the figure presentation. Specifically, we can provide the full z-stack (already available via OSF) and/or replace the image with a resliced whole-mount view to better visualize the full cellular context.
In terms of the possibility that the lack of staining in the hair cell’s plasma membrane might be due to insufficient antibody penetrance, we routinely perform Prestin (located in OHC plasma membrane) staining after saponin-mediated permeabilization and have never experienced antibody accessibility issues. Nevertheless, we will perform co-labeling for Prestin and include in the new submission.
(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. To better capture variability, we will include an additional quantification measuring the fraction of hair cell bundles with detectable ATP8B1-HA and TMEM30B-HA signal per field of view. This analysis will complement the existing intensity-based quantification.
(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 will revise 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.
We appreciate this insightful suggestion. However, performing this experiment would require generating a compound mouse line (crossing TMEM30B-HA into the ATP8B1 knockout background), which is not feasible within the revision timeframe. Additionally, the lack of a robust commercial antibody for TMEM30B further complicates this approach. We will note this as a future direction in the revised manuscript.
(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 will incorporate 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 will expand the discussion 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 will 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 aging. Preliminary (pilot) ABR measurements indicate wild-type–like hearing thresholds. We agree that this is important and will attempt to raise sufficient mouse numbers (in the time given) for a properly powered ABR analysis in the revised manuscript.
(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 will therefore soften our conclusions about IHC flippases and clarify 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 signal at P0 as reflecting transcription preceding detectable protein expression. While expression in other cochlear cell types is possible, we have not observed detectable ATP8B1 localization outside hair cells using the HA-tagged model. We will clarify this point in the manuscript.
(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 will revise the Methods section to explicitly state this and clarify that comparisons are intended only within panels, not across experiments.
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ATP8B1-TMEM30B Flippase Activity Maintains Stereocilia Lipid Asymmetry Required for Hearing https://www.biorxiv.org/content/10.64898/2026.02.12.705475v1
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ATP8B1-TMEM30B Flippase Activity Maintains Stereocilia Lipid Asymmetry Required for Hearing https://www.biorxiv.org/content/10.64898/2026.02.12.705475v1
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