Mammalian TMC1 or 2 are necessary for scramblase activity in auditory hair cells

Sensory transduction in auditory hair cells gates mechanosensitive ion channels, converting sound information into electrical signals (Zheng and Holt, 2021). Previously, we found that Transmembrane channel (TMC) proteins 1 and 2 form the pore of hair cell transduction channels (Pan et al., 2013; 2018). The structure of C. elegans TMC proteins (Jeong et al., 2022; Clark et al., 2024) and predicted mammalian TMC structures (Hahn et al., 2009; Ballesteros et al., 2018; Pan et al., 2018) are reminiscent of TMEM16 proteins, which function as Ca²⁺-activated ion channels and lipid scramblases. Here, we investigated lipid scramblase activity in live auditory hair cells with pharmacologic or genetic disruption of TMC1, extending work reported by Ballesteros and Swartz (2022). We used annexin-V to label phosphatidylserine (PS) localized in the outer leaflet of hair cell stereocilia membranes. PS externalization was triggered by disruption of sensory transduction using the blocker, benzamil, or by genetic mutations that affect TMC1 permeation properties. We found that expression of either TMC1 or TMC2, was essential for PS externalization. Tmc1/Tmc2 knockout mice and Tmie mutant mice lacked PS externalization completely. We also determined that expression of exogenous human TMCs (hTMC1 or hTMC2) in Tmc1/Tmc2 knockout mice induced PS externalization. Lastly, we demonstrated that expression of a dominant mutation in Tmc1 evoked constitutive PS externalization, while a recessive mutation eliminated PS externalization. Our data suggest that disruption of sensory transduction may lead to dysregulation of membrane homeostasis in hair cells and thus may contribute to auditory dysfunction in mice and humans.