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), 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), 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?

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.