ATP8B1 is a P4-ATPase flippase selectively expressed in outer hair cells.

(A) Schematic illustrating ATP8B1 and its obligate chaperone TMEM30B embedded in the plasma membrane (adopted from PDB, 8OX4) (Dieudonné et al. 2023). (B) Cladogram of the 14 P4-ATPase family members expressed in mice. (C) Of the 14 P4-ATPases, four were detected in hair cells, and HA knock-in mouse lines were generated for each. ATP8B1-HA (bold) was the only flippase found to be expressed in outer hair cells. (D) qPCR analysis of Atp8b1 expression at P0, P7, and P20. Four to five cochleae were analyzed per age group. (E) ATP8B1-HA Immunofluorescence across development and along the cochlear tonotopic axis in the organ of Corti. Organ of Corti were stained for anti-HA and phalloidin for F-actin. (F) Orthogonal view of ATP8B1-HA immunofluorescence of a P17 organ of Corti (G) ATP8B1-HA immunofluorescence in P12 utricle showing minimal expression. Scale bars 10 μm.

ATP8B1 is enriched toward the base of stereocilia.

(A) ATP8B1-HA immunofluorescence showing OHC specific expression. (B,C) ATP8B1-HA IF is enriched towards the stereocilia base and the apical hair cell membrane. (D) Average normalized fluorescence intensity profiles of actin and ATP8B1 along short (row 2) and long (row 1) stereocilia. Position is normalized from 0 (base) to 1 (tip), and intensities are normalized to total signal per stereocilium. Shaded regions indicate SEM. (Scale bar: 5 μm and 0.5 μm in stereocilia inset)

Loss of ATP8B1 causes progressive OHC bundle degeneration, hair cell loss, and elevated auditory thresholds

(A,B) Representative immunofluorescence images of WT and Atp8b1 KO cochleae at P17 and P20 stained for MYO7A (hair cells) and phalloidin (F-actin). OHC loss is first observed in the basal turn of Atp8b1 KO cochleae. Scale bar, 10 μm. (C) Quantification of OHC numbers in WT and Atp8b1 KO cochleae at P17 and P20 across apical, middle, and basal turns (n = 3–5 cochleae per group). At P20, OHC loss was significant in all tonotopic regions in KO mice compared with WT (Welch’s t-test, p < 0.001), whereas no significant differences were observed at P17. (D) Quantification of IHC survival in WT and Atp8b1 KO cochleae at P17 and P20 across apical, middle, and basal turns (n = 3–5 cochleae per group). No significant differences were detected between genotypes at either age (Welch’s t-test). (E) Auditory brainstem response (ABR) thresholds at P30 are significantly elevated in Atp8b1 KO mice compared with littermate controls (KO, n = 12; WT, n = 9). Thresholds were increased at all tested frequencies (8, 11, 16, 22, and 32 kHz; Mann–Whitney U test, p < 0.001). (F) Scanning electron microscopy (SEM) images of WT and Atp8b1 KO organs of Corti at P20. OHCs are pseudo-colored orange and IHCs yellow. (G) High-magnification SEM images at P20 reveal stereocilia fusion and OHC loss in Atp8b1 KO mice, features absent in WT. Arrows point out areas of stereocilia fusion and loss. IHCs remain morphologically intact. Scale bars: 1 μm (left), 0.5 μm (middle and right).

The P4-ATPase chaperone TMEM30B is enriched in OHC bundles.

(A) Immunofluorescence images of TMEM30B-HA across development and along the cochlear tonotopic axis in the organ of Corti. Organ of Corti were stained for anti-HA and phalloidin for F-actin. Scale bars 10 μm. (B) Orthogonal view of TMEM30B-HA immunofluorescence of a P17 organ of Corti. (C) Immunofluorescence images of a P17 TMEM30B-HA utricle showing moderate expression in utricular hair cells.

Loss of TMEM30B causes progressive OHC degeneration, hair cell loss, and elevated auditory thresholds.

(A, B) Representative Immunofluorescence images of WT and Tmem30b KO cochleae at P17 and P20, stained for MYO7A (hair cells) and phalloidin (F-actin). OHC loss is first detected in the basal turn of Tmem30b KO cochleae at P17, and pronounced OHC loss in the basal turn of KO cochleae at P20.. Scale bar, 10 μm. (C) Quantification of OHC numbers in WT and Tmem30b KO cochleae at P17 and P20 across apical, middle, and basal turns (n = 3–5 cochleae per group). At P20, OHC loss was significant in the basal region of KO mice compared with WT (Welch’s t-test, p < 0.001), whereas no significant differences were observed at P17. (D) Quantification of IHC numbers in WT and Tmem30b KO cochleae at P17 and P20 across apical, middle, and basal turns (n = 3–5 cochleae per group). No significant differences were detected between genotypes at either age (Welch’s t-test). (E) Auditory brainstem response (ABR) thresholds at P30 are significantly elevated in Tmem30b KO mice compared with littermate controls (KO, n = 21; WT, n = 17). Thresholds were increased at all tested frequencies (8, 11, 16, 22, and 32 kHz; Mann–Whitney U test, p < 0.001). (F) Scanning electron microscopy (SEM) images of WT and Tmem30b KO organs of Corti at P20. OHCs are pseudo-colored orange and IHCs yellow. (G) High-magnification SEM images at P20 reveal stereocilia misorientation and OHC loss in Tmem30b KO mice, features absent in WT. Arrows point out areas of stereocilia misorientation. IHCs remain morphologically intact. Scale bars: 1 μm (left), 0.5 μm (middle and right). (H) Immunofluorescence images of organs of Corti from Tmem30b KO / ATP8B1-HA mice at P17 showing aberrant localization of ATP8B1-HA to the hair cell soma in the absence of TMEM30B (magenta).

Loss of plasma membrane asymmetry precedes hair cell loss in Atp8b1 and Tmem30b knockout mice.

(A) Representative confocal images of WT and Atp8b1 KO cochleae at P15 stained with Annexin V to label externalized phosphatidylserine (PS) and phalloidin to visualize F-actin. Increased Annexin V labeling is observed in Atp8b1 KO hair cells prior to overt cell loss. Scale bar, 10 μm. (B) Quantification of Annexin V fluorescence intensity in WT and Atp8b1 KO cochleae (n = 5–7 cochleae per group). Atp8b1 KO hair cells show significantly increased PS externalization compared with WT (Welch’s t-test, p < 0.0001). (C) Representative confocal images of WT and Tmem30b KO cochleae at P15 stained with Annexin V and phalloidin. Increased Annexin V labeling is similarly observed in Tmem30b KO hair cells. In Atp8b1 and Tmem30b KO organs, weak AnV labeling was occasionally observed also in IHCs, at the apical cell junctions and the hair bundle. Scale bar, 10 μm. (D) Quantification of Annexin V fluorescence intensity in WT and Tmem30b KO cochleae (n = 5–7 cochleae per group), demonstrating significantly elevated PS externalization in KO hair cells (Welch’s t-test, p < 0.01).

ATP8B1 and TMEM30B require mechanotransduction activity for proper subcellular localization.

(A, C) Representative ATP8B1-HA and TMEM30B-HA immunofluorescence in P15 cochleae (whole mount and profile view), on WT or Cib2 KO background, counterstained with phalloidin to visualize F-actin. In the absence of MET activity (Cib2 KO), ATP8B1-HA and TMEM30B-HA are aberrantly enriched in the hair cell soma of Cib2 KO mice. (B) Quantification of ATP8B1-HA immunofluorescence intensity in WT and Cib2 KO cochleae in the hair bundle and cell body (n = 4-6 cochleae per group). ATP8B1-HA intensity was significantly increased in the cell body of Cib2 KO hair cells (p < 0.01, Welch’s t-test), with no significant difference detected in the hair bundle. (D) Quantification of TMEM30B-HA fluorescence intensity in WT and Cib2 KO cochleae in the hair bundle and cell body (n = 4-6 cochleae per group) (whole mount and profile view). TMEM30B-HA intensity was significantly increased in the cell body of Cib2 KO hair cells (p < 0.05, Welch’s t-test), with no significant difference observed in the hair bundle. Scale bar 10 μm