Hair cell apoptosis and deafness in mutations.

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 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 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-trifluoromethoxyphenylhydrazone, and use of MitoLight to monitor mitochondrial membrane potential. These markers demonstrated mitochondrial dysfunction in mutants, even at P6 when MET currents were still present. Acoustic brainstem responses established that p.D569N and p.M412K mice were deaf by P15 and p.T416K by P21. Two methods of blocking the stereociliary PMCA2 Ca pump both elicited scramblase activity, suggesting that apoptosis is promoted by elevation of hair bundle [Ca]. Reduced PMCA2 density was found in the stereocilia of mutants and was correlated with a decrease in MET channel Ca 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 mutants have embarked on apoptosis at P6 and argue for connections between stereociliary PMCA2 density, hair cell apoptosis, and deafness.