Lee, H.; Park, Y. C.; Wen, H.; Smith, H. E.; Balaraman, J.; Cui, R.; Sotomayor, M.; Ballesteros, A.

TMC1 and TMC2, the pore-forming subunits of the mechanoelectrical transduction (MET) complex in inner ear sensory hair cells, are essential for auditory and vestibular function. Pathogenic mutations in TMC1 are a leading cause of genetic hearing loss, but their underlying cellular mechanisms remain poorly understood. Here, we reveal that TMC1 and TMC2 are cholesterol-regulated lipid scramblases whose activity modulates plasma membrane asymmetry. Using reconstituted proteoliposomes and molecular dynamics simulations, we demonstrate that both proteins facilitate phospholipid translocation across membrane bilayers, a process tuned by cholesterol and enhanced by deafness-causing TMC1 mutations. We show that this scramblase activity correlates with TMC1-dependent externalization of phosphatidylserine and membrane blebbing in murine auditory hair cells, linking TMC1-dependent membrane homeostasis dysregulation to auditory sensory cell pathology. These findings identify TMCs as a novel family of lipid scramblases, advancing our understanding of MET complex biology and offering mechanistic insight into membrane-driven forms of hereditary deafness.