Transmembrane channel-like protein 1 (TMC1), a pore-forming component of the mechano-electrical transducer (MET) channel in cochlear outer hair cells, is subject to numerous mutations causing deafness and hair cell death. We studied mice harboring semidominant mutations Tmc1 p.T416K, p.M412K, and p.D569N, which all display functional MET channels at postnatal day (P)6 but become deaf by P21. Early signs of concomitant hair cell apoptosis were assayed in neonatal Tmc1 mutants by labeling with Calcein-acetomethyl ester (AM), MitoTracker, and Annexin V, the latter labeling scramblase externalization of phosphatidyl serine. Reduced labeling with Calcein-AM was correlated with reduced MitoTracker, the targeting of mitochondria being confirmed with the uncoupling agent carbonylcyanide p -trifluoromethoxyphenylhydrazone, and use of MitoLight to monitor mitochondrial membrane potential. These markers demonstrated mitochondrial dysfunction in Tmc1 mutants, even at P6 when MET currents were still present. Acoustic brainstem responses established that Tmc1 p.D569N and Tmc1 p.M412K mice were deaf by P15 and Tmc1 p.T416K by P21. Two methods of blocking the stereociliary PMCA2 Ca 2+ pump both elicited scramblase activity, suggesting that apoptosis is promoted by elevation of hair bundle [Ca 2+ ]. Reduced PMCA2 density was found in the stereocilia of Tmc1 mutants and was correlated with a decrease in MET channel Ca 2+ permeability. Cre-Lox excision of the mutant M412K exon at P1 fully preserved hearing across all frequencies by P19 and promoted recovery to wild type of PMCA2 density. These results demonstrate that hair cells in Tmc1 mutants have embarked on apoptosis at P6 and argue for connections between stereociliary PMCA2 density, hair cell apoptosis, and deafness.

中文翻译：

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Tmc1 突变中的毛细胞凋亡和耳聋


跨膜通道样蛋白 1 （TMC1） 是耳蜗外毛细胞中机械电换能器 （MET） 通道的成孔成分，会受到许多突变的影响，导致耳聋和毛细胞死亡。我们研究了携带半显性突变 Tmc1 p.T416K、p.M412K 和 p.D569N 的小鼠，它们在出生后 （P）6 都显示功能性 MET 通道，但在 P21 时变得耳聋。通过用钙黄绿素-乙酰甲酯 （AM） 、 MitoTracker 和 Annexin V 标记，在新生儿 Tmc1 突变体中检测伴随毛细胞凋亡的早期迹象，后者标记磷脂酰丝氨酸的扰乱酶外化。用钙黄绿素-AM 标记的减少与 MitoTracker 的减少相关，线粒体的靶向性通过解偶联剂羰基氰化物 p-三氟甲氧基苯腙得到证实，以及使用 MitoLight 监测线粒体膜电位。这些标志物在 Tmc1 突变体中显示出线粒体功能障碍，即使在 MET 电流仍然存在时在 P6 也是如此。脑干听觉反应确定 Tmc1 p.D569N 和 Tmc1 p.M412K 小鼠在 P15 时耳聋，Tmc1 p.T416K 在 P21 中耳聋。两种阻断立体纤毛 PMCA2 Ca 2+ 泵的方法都引起了扰乱酶活性，表明发束升高促进了细胞凋亡 [Ca 2+]。在 Tmc1 突变体的立体纤毛中发现 PMCA2 密度降低，并且与 MET 通道 Ca 2+ 通透性降低相关。P1 突变体 M412K 外显子的 Cre-Lox 切除完全保留了 P19 在所有频率上的听力，并促进了 PMCA2 密度野生型的恢复。这些结果表明，Tmc1 突变体中的毛细胞在 P6 开始凋亡，并争论立体纤毛 PMCA2 密度、毛细胞凋亡和耳聋之间的联系。