Cadherin-23 mutations cause calcium-dependent, allele-sensitive mechanosensory defects.

Point mutations in tip-link proteins, molecular filaments that transmit mechanical tension from sound stimuli to sensory transduction channels, are abundantly associated with hereditary hearing loss. Intriguingly, many of these mutations lie far from the protein binding interface and do not affect balance or vision. Here, we explore two such distal mutations that cause congenital deafness in homozygous individuals and progressive hearing loss in compound heterozygotes, while sparing vestibular and retinal function. Using a combination of protein engineering, single-molecule force spectroscopy, and molecular dynamics simulations, we reconstructed wild-type and mutant tip-link complexes to examine how these mutations alter their mechanical structure. Our experiments reveal that the mutations subtly change the folding kinetics and force-dependent rupture behavior of the tip-link complexes, particularly under low calcium conditions that mimic the cochlear environment. These mechanical alterations were significantly attenuated at higher calcium concentrations, consistent with the calcium-rich milieu of the vestibular and retinal tissues. Together, our findings suggest that distal mutations can compromise tip-link function in a calcium-sensitive manner, offering a mechanistic explanation for how the same mutations selectively impair hearing while leaving balance and vision intact.