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Tip links are extracellular filaments that connect pairs of hair cell stereocilia and convey tension to mechanosensitive channels. Recent evidence suggests that tip links are formed by calcium-dependent interactions between the N-terminal domains of cadherin-23 (CDH23) and protocadherin-15 (PCDH15). Mutations in either CDH23 or PCDH15 cause deafness in mice and humans, indicating the molecules are required for normal inner ear function. However, there is little physiological evidence to support a direct role for CDH23 and PCDH15 in hair cell mechanotransduction. To investigate the contributions of CDH23 and PCDH15 to mechanotransduction and tip-link formation, we examined outer hair cells of mouse cochleas during development and after chemical disruption of tip links. We found that tip links and mechanotransduction with all the qualitative properties of mature transduction recovered within 24 h after disruption. To probe tip-link formation, we measured transduction currents after extracellular application of recombinant CDH23 and PCDH15 fragments, which included putative interaction domains (EC1). Both fragments inhibited development and regeneration of transduction but did not disrupt transduction in mature cells. PCDH15 fragments that carried a mutation in EC1 that causes deafness in humans did not inhibit transduction development or regeneration. Immunolocalization revealed wild-type fragments bound near the tips of hair cell stereocilia. Scanning electron micrographs revealed that hair bundles exposed to fragments had a reduced number of linkages aligned along the morphological axis of sensitivity of the bundle. Together, the data provide direct evidence implicating CDH23 and PCDH15 proteins in the formation of tip links during development and regeneration of mechanotransduction.

Sensory hair cells of the inner ear express multiple physiologically defined conductances, including mechanotransduction, Ca2+, Na+, and several distinct K+conductances, all of which are critical for normal hearing and balance function. Yet, the molecular underpinnings and their specific contributions to sensory signaling in the inner ear remain obscure. We sought to identify hair-cell conductances mediated by KCNQ4, which, when mutated, causes the dominant progressive hearing loss DFNA2. We used the dominant-negative pore mutation G285S and packaged the coding sequence of KCNQ4 into adenoviral vectors. We transfected auditory and vestibular hair cells of organotypic cultures generated from the postnatal mouse inner ear. Cochlear outer hair cells and vestibular type I cells that expressed the transfection marker, green fluorescent protein, and the dominant-negative KCNQ4 construct lacked the M-like conductances that typify nontransfected control hair cells. As such, we conclude that the M-like conductances in mouse auditory and vestibular hair cells can include KCNQ4 subunits and may also include KCNQ4 coassembly partners. To examine the function of M-like conductances in hair cells, we recorded from cells transfected with mutant KCNQ4 and injected transduction current waveforms in current-clamp mode. Because the M-like conductances were active at rest, they contributed to the very low potassium-selective input resistance, which in turn hyperpolarized the resting potential and significantly attenuated the amplitude of the receptor potential. Modulation of M-like conductances may allow hair cells the ability to control the amplitude of their response to sensory stimuli.

How and when sensory hair cells acquire the remarkable ability to detect and transmit mechanical information carried by sound and head movements has not been illuminated. Previously, we defined the onset of mechanotransduction in embryonic hair cells of mouse vestibular organs to be at approximately embryonic day 16 (E16). Here we examine the functional maturation of hair cells in intact sensory epithelia excised from the inner ears of embryonic mice. Hair cells were studied at stages between E14 and postnatal day 2 using the whole-cell, tight-seal recording technique. We tracked the developmental acquisition of four voltage-dependent conductances. We found a delayed rectifier potassium conductance that appeared as early as E14 and grew in amplitude over the subsequent prenatal week. Interestingly, we also found a low-voltage-activated potassium conductance present at E18, ∼1 week earlier than reported previously. An inward rectifier conductance appeared at approximately E15 and doubled in size over the next few days. We also noted transient expression of a voltage-gated sodium conductance that peaked between E16 and E18 and then declined to near zero at birth. We propose that hair cells undergo a stereotyped developmental pattern of ion channel acquisition and that the precise pattern may underlie other developmental processes such as synaptogenesis and functional differentiation into type I and type II hair cells. In addition, we find that the developmental acquisition of basolateral conductances shapes the hair cell receptor potential and therefore comprises an important step in the signal cascade from mechanotransduction to neurotransmission.

Hair cells of inner ear organs sensitive to frequencies above 10 Hz adapt to maintained hair bundle deflections at rates that reduce their responses to lower frequencies. Mammalian vestibular organs detect head movements at frequencies well below 10 Hz. We asked whether hair cells of the mouse utricle adapt, and if so, whether the adaptation was similar to that in higher frequency organs such as the frog saccule.Whole-cell transduction currents were recorded from hair cells in the epithelium of the mouse utricle. Hair bundles were deflected by a fluid jet or a stiff probe. The transduction currents evoked by step deflections adapted over 10–100 msec. The mean operating range was 1.5 μm (deflection of the tip of the bundle), approximately threefold larger than in frog saccule. Taller and more compact bundles of the mouse utricle account for this difference. As in frog saccular hair cells, adaptation shifted the current–deflection (I(X)) relation along the deflection axis. These adaptive shifts had time constants of 10–20 msec and reached 60–80% of stimulus amplitude. The adaptive shift and voltage-dependent bundle movement are consistent with the motor model of adaptation. When the fluid jet was used, adaptation also broadened theI(X) relation and reduced the maximum current.Adaptation attenuated the transduction currents evoked by sinusoidal bundle deflections below 5 Hz, within the frequency range of the utricle, but because it was incomplete, substantial responses remained. Moreover, the adaptive shift mechanism preserves sensitivity even in the presence of large stimuli that would otherwise saturate transduction.