Regulation of membrane homeostasis by TMC1 mechanoelectrical transduction channels is essential for hearing

Abstract

The mechanoelectrical transduction (MET) channel in auditory hair cells converts sound into electrical signals, enabling hearing. Transmembrane-like channel 1 and 2 (TMC1 and TMC2) are implicated in forming the pore of the MET channel. Here, we demonstrate that inhibition of MET channels, breakage of the tip links required for MET, or buffering of intracellular Ca^... induces pronounced phosphatidylserine externalization, membrane blebbing, and ectosome release at the hair cell sensory organelle, culminating in the loss of TMC1. Membrane homeostasis triggered by MET channel inhibition requires Tmc1 but not Tmc2, and three deafness-causing mutations in Tmc1 cause constitutive phosphatidylserine externalization that correlates with deafness phenotype. Our results suggest that, in addition to forming the pore of the MET channel, TMC1 is a critical regulator of membrane homeostasis in hair cells, and that Tmc1-related hearing loss may involve alterations in membrane homeostasis.

Fig. 1.. Blockade of the MET channel triggers PS externalization and membrane blebbing.

(A) Confocal images of P6 wild-type hair cells incubated with WGA (gray) and AnV in the absence (control) or presence of 100 μM neomycin, benzamil, or curare. (B) Quantification of AnV fluorescence intensity in OHCs (○) or IHCs (●) treated as in (A). (C) Confocal images of P6 mT/mG^Tg/+ hair cells expressing the mtdTomato membrane reporter (green) treated as in (A). Quantification of the AnV (D) or mtdTomato (E) fluorescence intensity in IHCs and OHCs treated as in (C). (F) Live imaging of wild-type hair cells labeled with CellMask (green) and AnV (magenta) in the absence (control) or presence of curare. (G) Confocal planes of OHCs treated with curare as in (F). (H) Diameter of AnV-positive vesicles (n = 20). In (B), (D), and (E), mean ± SD is shown for n = 70 to 85 OHCs and 22 to 27 IHCs from two cochleae. One-way analysis of variance (ANOVA) analysis was performed (ns P > 0.05, *P < 0.05, ***P < 0.001, and ****P < 0.0001). Scale bars, 10 μm in (A), (C), and (F) and 5 μm in (G). See also figs. S1 to S3.

Fig. 2.. PS externalization and membrane blebbing after tip link disruption and in biological extracellular solutions.

(A) Confocal images of P6 mT/mG^Tg/+ hair cells expressing mtdTomato (green) treated with HBSS (control) or 5 mM BAPTA before the addition of AnV (magenta). Quantification of the AnV (B) and mtdTomato (C) fluorescence intensity at the apical region of OHCs (○) and IHCs (●) treated as in (A). (D) Confocal images of wild-type hair cells treated with HBSS (control), HBSS cation-free medium (HBSS-CFM), or 5 mM BAPTA in HBSS-CFM containing WGA (gray) before the addition of AnV (Fire LUT). (E) AnV fluorescence intensity quantification as in (D). (F) Confocal images of P6 mT/mG^Tg/+ mice hair cells untreated or treated with 100 μM benzamil in perilymph-like buffer containing AnV. (G) Confocal images of P6 mT/mG^Tg/+ hair cells untreated or treated with 100 μM benzamil in endolymph-like buffer containing AnV. Quantification of AnV (H) and mtdTomato (I) fluorescence intensity as in (F) and (G). In (B), (C), (E), (H), and (I), mean fluorescence intensity ± SD is represented for n = 82 to 126 OHCs and 19 to 43 IHCs from two cochleae. Unpaired t test (B and C) or one-way ANOVA analysis (E, H, and I) was performed (ns P > 0.05, *P < 0.05, **P < 0.01, and ****P < 0.0001). Scale bar, 10 μm. See also fig. S4.

Fig. 3.. PS externalization and membrane blebbing in mice lacking MET.

(A) Confocal images of P6 mT/mG^Tg/+;Tmc1^−/−;Tmc2^−/− hair cells incubated with HBSS containing AnV in the absence (control) or presence of 100 μM neomycin, benzamil, or curare. Quantification of the AnV (B) and mtdTomato (C) fluorescence intensity at the apical region of the OHCs (○) and IHCs (●) treated as in (A). (D) Confocal images of hair cells from P6 wild-type, mT/mG^Tg/+, and mT/mG^Tg/Tg littermates incubated in HBSS containing AnV in the absence (control) or presence of 100 μM benzamil. Quantification of the AnV (E) and mtdTomato (F) fluorescence intensity as in (D). In (B), (C), (E), and (F), mean fluorescence intensity ± SD is represented for n = 57 to 126 OHCs and 21 to 43 IHCs from two cochleae. One-way ANOVA analysis was performed (ns P > 0.05, and ****P < 0.0001). Scale bar, 10 μm. See also fig. S5.

Fig. 4.. TMC1, but not TMC2, is required for PS externalization and membrane blebbing.

(A) Confocal images of P6 mT/mG^Tg/+;Tmc2^−/− hair cells incubated with HBSS containing AnV (magenta) in the absence (control) or presence of 100 μM neomycin, benzamil, or curare. Quantification of AnV (B) and mtdTomato (C) fluorescence intensity at the apical region of OHCs (○) and IHCs (●) treated as in (A). (D) Confocal images of P6 mT/mG^Tg/+;Tmc1^−/− hair cells treated as in (A). Quantification of AnV (E) and mtdTomato (F) fluorescence in mT/mG^Tg/+;Tmc1^−/− as in (D). (G) Confocal images of hair cells from P6 mosaic mice expressing TMC1-cherry untreated (control) or treated with 100 μM benzamil. F-actin (blue) is included to visualize the stereocilia. In (B), (C), (E), and (F), mean ± SD is shown for n = 45 to 81 OHCs and 17 to 24 IHCs from two cochleae. One-way ANOVA analysis was performed (ns P > 0.05, *P > 0.05, and ****P < 0.0001). Scale bar, 10 μm. See also fig. S5.

Fig. 5.. Buffering of intracellular Ca²⁺ triggers PS externalization and membrane blebbing when TMC1 is present.

Confocal images of P6 mT/mG^Tg/+ (A) or mT/mG^Tg/+;Tmc1^−/− (B) hair cells incubated with HBSS containing AnV (magenta) in the presence of BAPTA-AM or the vehicle (Control). Scale bar, 10 μm. Quantification of AnV (C) and mtdTomato (D) fluorescence intensity at the apical region of OHCs (○) and IHCs (●) treated as in (A) and (B). Mean fluorescence intensity ± SD is represented for n = 70 to 100 OHCs and 15 to 30 IHCs from two cochleae. One-way ANOVA analysis was performed (ns P > 0.05, *P < 0.05, and ****P < 0.0001). See also fig. S4F and G.

Fig. 6.. Constitutive externalization of PS in TMC1 deafness–causing mutant mice.

Confocal images of P6 Tmc1^M412K/+ and Tmc1^M412K/M412K (A), Tmc1^D569N/+ and Tmc1^D569N/D569N (B), or Tmc1^D528N/+ and Tmc1^D528N/D528N (C) OHCs and IHCs labeled with AnV in the absence (control) or presence of 100 μM benzamil. Scale bar, 5 μm. Quantification of AnV fluorescence intensity in OHCs (D) or IHCs (E). Mean ± SD from three to five mice is shown. Each dot represents the mean fluorescence intensity of >100 OHCs or >30 IHCs from one cochlea. One-way ANOVA analysis was performed (ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). See also fig. S7.

Fig. 7.. MET blockage and PS externalization alter TMC1 localization at the stereocilia tips.

Confocal images of OHCs (A) and IHCs (B) from P6 TMC1-cherry mice untreated (control) or treated with 100 μM benzamil for 10, 120, and 240 min. TMC1-cherry (green), AnV (red), and WGA (gray) channels are shown. Scale bar, 5 μm. Yellow arrows indicate protruding TMC1- and AnV-positive vesicles. Quantification of TMC1 (C) and AnV (D) fluorescence intensity at the apical region of OHCs (○) and IHCs (●) treated as in (A) and (B). Mean ± SD is shown for n = 79 to 125 OHCs and 23 to 45 IHCs from two cochleae. One-way ANOVA analysis was performed (ns P > 0.05, *P < 0.05, **P < 0.01, and ****P < 0.0001). See also fig. S8.

Fig. 8.. TMC1-dependent regulation of the apical hair cell membrane homeostasis.

Plasma membrane asymmetry is preserved in wild-type murine auditory hair cells under physiological conditions. However, pharmacological blockade of the MET channel, disruption of the tip links, or intracellular Ca²⁺ buffering triggers the externalization of PS and membrane blebbing at the stereocilia and apical region of hair cells expressing TMC1. In addition, two autosomal dominant (M412K and D569N) and one recessive (D528N) Tmc1 deafness–causing mutations cause constitutive externalization of PS that correlates with deafness phenotype in these mutant mice, highlighting the relevance of membrane homeostasis in hearing.