Jeffrey R. Holt

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.

The molecular composition of the hair cell transduction channel has not been identified. Here we explore the novel hypothesis that hair cell transduction channels include HCN subunits. The HCN family of ion channels includes four members, HCN1-4. They were orginally identified as the molecular correlates of the hyperpolarization-activated, cyclic nucleotide gated ion channels that carry currents known as I_f, I_Q or I_h. However, based on recent evidence it has been suggested that HCN subunits may also be components of the elusive hair cell transduction channel. To investigate this hypothesis we examined expression of mRNA that encodes HCN1-4 in sensory epithelia of the mouse inner ear, immunolocalization of HCN subunits 1, 2 and 4, uptake of the transduction channel permeable dye, FM1-43 and electrophysiological measurement of mechanotransduction current. Dye uptake and transduction current were assayed in cochlear and vestibular hair cells of wildtype mice exposed to HCN channel blockers or a dominant-negative form of HCN2 that contained a pore mutation and in mutant mice that lacked HCN1, HCN2 or both. We found robust expression of HCNs 1, 2 and 4 but little evidence that localized HCN subunits in hair bundles, the site of mechanotransduction. Although high concentrations of the HCN antagonist, ZD7288, blocked 50–70% of the transduction current, we found no reduction of transduction current in either cochlear or vestibular hair cells of HCN1- or HCN2- deficient mice relative to wild-type mice. Furthermore, mice that lacked both HCN1 and HCN2 also had normal transduction currents. Lastly, we found that mice exposed to the dominant-negative mutant form of HCN2 had normal transduction currents as well. Taken together, the evidence suggests that HCN subunits are not required for mechanotransduction in hair cells of the mouse inner ear.

Auditory function in the mammalian inner ear is optimized by collaboration of two classes of sensory cells known as inner and outer hair cells. Outer hair cells amplify and tune sound stimuli that are transduced and transmitted by inner hair cells. Although they subserve distinct functions, they share a number of common properties. Here we compare the properties of mechanotransduction and adaptation recorded from inner and outer hair cells of the postnatal mouse cochlea. Rapid outer hair bundle deflections of about 0.5 micron evoked average maximal transduction currents of about 325 pA, whereas inner hair bundle deflections of about 0.9 micron were required to evoke average maximal currents of about 310 pA. The similar amplitude was surprising given the difference in the number of stereocilia, 81 for outer hair cells and 48 for inner hair cells, but may be reconciled by the difference in single-channel conductance. Step deflections of inner and outer hair bundles evoked adaptation that had two components: a fast component that consisted of about 60% of the response occurred over the first few milliseconds and a slow component that consisted of about 40% of the response followed over the subsequent 20–50 ms. The rate of the slow component in both inner and outer hair cells was similar to the rate of slow adaptation in vestibular hair cells. The rate of the fast component was similar to that of auditory hair cells in other organisms and several properties were consistent with a model that proposes calcium-dependent release of tension allows transduction channel closure.

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.

The confined fluid-filled labyrinth of the human inner ear presents an opportunity for introduction of gene therapy reagents designed to treat hearing and balance dysfunction. Here we present a novel model system derived from the sensory epithelia of human vestibular organs and show that the tissue can survive up to 5 days in vitro. We generated organotypic cultures from 26 human sensory epithelia excised at the time of labyrinthectomy for intractable Meniere's disease or vestibular schwannoma. We applied multiply deleted adenoviral vectors at titers between 10⁵ and 10⁸ viral particles/ml directly to the cultures for 4–24 h and examined the tissue 12–96 h post-transfection. We noted robust expression of the exogenous transgene, green fluorescent protein (GFP), in hair cells and supporting cells suggesting both were targets of adenoviral transfection. We also transfected cultures with a vector that carried the genes for GFP and KCNQ4, a potassium channel subunit that causes dominant-progressive hearing loss when mutated. We noted a positive correlation between GFP fluorescence and KCNQ4 immunolocalization. We conclude that our in vitro model system presents a novel and effective experimental paradigm for evaluation of gene therapy reagents designed to restore cellular function in patients who suffer from inner ear disorders.

How mechanical information is encoded in the vestibular periphery has not been clarified. To begin to address the issue we examined the intrinsic firing properties of postnatal mouse vestibular ganglion neurons using the whole cell, tight-seal technique in current-clamp mode. We categorized two populations of neurons based on the threshold required to evoke an action potential. Low-threshold neurons fired with an average minimum current injection of −43 pA, whereas high-threshold neurons required −176 pA. Using sine-wave stimuli, we found that the neurons were inherently tuned with best frequencies that ranged up to 40 Hz. To investigate the membrane properties that contributed to the variability in firing properties we examined the same neurons in voltage-clamp mode. High-threshold neurons had larger cell bodies and whole cell capacitances but a resting conductance density of 0.18 nS/pF, nearly identical to that of low-threshold neurons, suggesting that cell size was an important parameter determining threshold. We also found that vestibular ganglion neurons expressed a heterogeneous population of potassium conductances. TEA-sensitive conductances contributed to the position of the tuning curve in the frequency domain. A 4-AP–sensitive conductance was active at rest and hyperpolarized resting potential, limited spontaneous activity, raised threshold, and prevented repetitive firing. In response to sine-wave stimulation 4-AP–sensitive conductances prevented action potential generation at low frequencies and thus contributed to the high-pass corner of the tuning curve. The mean low-pass corner (about 29 Hz) was determined by the membrane time constant. Together these factors contributed to the sharply tuned, band-pass characteristics intrinsic to postnatal vestibular ganglion neurons.

Sensory transduction in hair cells requires assembly of membrane-bound transduction channels, extracellular tip-links and intracellular adaptation motors with sufficient precision to confer nanometer displacement sensitivity. Here we present evidence based on FM1-43 fluorescence, scanning electron microscopy and RT-PCR that these three essential elements are acquired concurrently between embryonic day 16 and 17, several days after the appearance of hair bundles¹, and that their acquisition coincides with the onset of mechanotransduction.

Several classes of viral vectors including adenovirus, adeno-associated virus, herpes simplex virus, lentivirus and vaccinia virus have been reported to infect cells of the inner ears of mammals and may be useful for protein manipulation and therapeutic purposes. We have screened a few of these for use as vectors to mediate gene transfer into the sensory hair cells of organotypic cultures from the neonatal mouse cochlea and utricle. Recombinant, replication-deficient adenovirus has emerged as a useful vector for several reasons: ease of vector generation at high titer; efficient hair cell specific infection; robust expression of reporter genes and minimal toxicity. Previously, we characterized adenovirus infected hair cells using a vector that carried the gene for green fluorescent protein (GFP). We screened GFP-positive cells electrophysiologically and found that although hair cells survive adenoviral vector infection, their mechanosensitivity was compromised. Until recently this has limited the scope of adenovirus application to the problems of inner ear physiology and pathophysiology. However, a modified adenoviral vector, now available, has been reported to have reduced ototoxicity in vivo. The modifications include the deletion of the adenoviral genes E1, E3, the viral polymerase, and the preterminal protein. We are currently working to characterize viral-mediated gene transfer into hair cells of the cultured mouse utricle using this new modified adenoviral vector. We have found that hair cells infected with the modified vector have intact hair bundles and robust mechanotransduction.