The vertebrate senses of hearing and balance rely on the molecular machinery of the inner ear to convert sound into electrical signals that the brain can process. In the inner ear, mechanotransduction is mediated by hair cells. Tension in their tip links conveys force to mechanosensitive ion channels. Each tip link comprises two helical filaments of atypical cadherins bound at their N-termini through two unique adhesion bonds. The tip links must be connected to convey mechanical tension to the transduction channels, but the dynamics of the connection and how deafness mutations impair adhesion and strength are unknown. We developed molecular and biophysical tools to describe the strength and dynamics of the tip-link connection at the resolution of individual molecules. We find that the tip-link bond is more mechanically stable than classic cadherins and less sensitive to the Ca2+-poor endolymph that bathes tip links in the inner ear. The double-stranded tip link connection has cis-dimerization interfaces that keep binding interfaces close together, which extends the lifetime of the connection through single-stranded rebinding. This single-stranded rebinding persists under relevant forces to extend the lifetime of the connection. We also find that Ca2+ modulates the elasticity of the tip-link complex through both alteration of intrinsic single-bond kinetics and the effective concentration for rebinding. A deafness mutation within the bond interface was discovered to increase the force sensitivity of the complex, giving insights into the etiology of the mutation. In a physiological context, the lifetime of the tip link is equal to the resting tension within the normal range of auditory stimuli. We propose that the tip-link connection is several thousand times more dynamic than previously thought, challenging current assumptions about tip-link stability and turnover rate. We further provide insight into how the mechanotransduction apparatus conveys mechanical information.
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