Paralemmin-3 sustains the integrity of the lateral plasma membrane and subsurface cisternae of auditory hair cells

In the mammalian inner ear, cochlear inner hair cells (IHCs) enable accurate and faithful synaptic sound encoding, while outer hair cells (OHCs) perform frequency-specific sound amplification and fine-tuning through their intrinsic voltage-dependent somatic electromotility. This latter process is facilitated by the unique trilaminate structure of the OHC lateral wall, which consists of the plasma membrane that is densely occupied by the transmembrane motor protein Prestin, the submembrane actin- and spectrin-based cytoskeleton, and the endomembranous subsurface cisternae. This complex system provides mechanical resilience while allowing for cell expansion and contraction during electromotility. Whereas the ultrastructure of the lateral wall is well described, its molecular architecture remains largely elusive. Here, we identified Paralemmin-3 (Palm3) as a novel protein specifically localized to the lateral walls of auditory HCs to play a crucial role in connecting the plasma membrane to the underlying cytoskeleton and subsurface cisternae. Palm3-KO mice display early-onset and progressive hearing impairment that results from diminished cochlear amplification. Subsequent multiscale morphological analyses revealed structural collapse of OHCs that led to progressive and extensive OHC loss along the tonotopic axis. Palm3-KO OHCs exhibited disrupted expression and distribution of several membrane-associated proteins - including spectrin isoforms and Prestin - suggesting an essential role of Palm3 in plasma membrane scaffolding. Electron tomography of OHC lateral walls revealed significantly fewer and structurally perturbed subsurface cisternae. Finally, adeno-associated virus (AAV)-mediated rescue of Palm3 during early postnatal development partly restored hearing function, enhanced OHC survival, and restored OHC cell shape as well as membrane protein expression levels. In summary, Palm3 is a key component of the submembrane cytoskeleton in cochlear hair cells, playing a fundamental role in hair cell biology and hearing, and emerges as an attractive candidate for the long-elusive "pillar" component of the hair cell lateral wall ultrastructure.