Functional Characterization of TMEM63 in Cochlear Hair Cells Reveals a Role in Calcium Signaling, Proliferation, Apoptosis, and Migration

TMEM63 is a mechanosensitive transmembrane protein implicated in calcium regulation; however, its specific function in cochlear auditory cells remains undefined. Calcium signaling is essential for cochlear hair cell proliferation, apoptosis, and migration—processes fundamental to hearing preservation and potential regeneration. This research sought to examine TMEM63's regulatory function in proliferation, apoptosis, and migration within HEI‐OC1 cochlear hair cell systems, aimed at delineating the involvement of calcium‐dependent signaling pathways, particularly the CDC42/AMPK axis. HEI‐OC1 cells were subjected to TMEM63 overexpression or knockdown via plasmid transfection or RNA interference. Calcium chelation was performed using BAPTA‐AM. Cell proliferation was assessed by CCK‐8 screen, apoptosis via Annexin V‐fluorescein isothiocyanate/propidium iodide analysis, as well as cellular cycling by flow cytometric evaluation. Wound repair combined with transwell analysis was conducted toward evaluate motility and invasiveness. Intracellular calcium levels were measured using ELISA, and signaling pathway activation was analyzed via Western blot and immunofluorescence. TMEM63 overexpression significantly promoted cell proliferation, enhanced migration, and suppressed apoptosis, while knockdown had the opposite effects. These phenotypes were reversed by BAPTA‐AM, indicating a calcium‐dependent mechanism. TMEM63 modulated intracellular Ca²⁺ levels and activated CDC42 and CaMKII while suppressing AMPK phosphorylation. Immunofluorescence confirmed consistent changes in CDC42, β‐catenin, AMPK, and E‐cadherin expression. TMEM63 regulates cochlear hair cell proliferation, apoptosis, and migration through calcium‐dependent activation of the CDC42/AMPK signaling pathway. These findings highlight TMEM63 as a potential modulator of cochlear cell function and a novel target for hearing preservation strategies.