Disruption of tmc1/2a/2b Genes in Zebrafish Reveals Subunit Requirements in Subtypes of Inner Ear Hair Cells.

Detection of sound and head movement requires mechanoelectrical transduction (MET) channels at tips of hair-cell stereocilia. In vertebrates, the transmembrane channel-like (TMC) proteins TMC1 and TMC2 fulfill critical roles in MET, and substantial evidence implicates these TMCs as subunits of the MET channel. To identify developmental and functional roles of this Tmc subfamily in the zebrafish inner ear, we tested the effects of truncating mutations in tmc1, tmc2a, and tmc2b on in vivo mechanosensation at the onset of hearing and balance, before gender differentiation. We find that tmc1/2a/2b triple-mutant larvae cannot detect sound or orient with respect to gravity. They lack acoustic-evoked behavioral responses, vestibular-induced eye movements, and hair-cell activity as assessed with FM dye labeling and microphonic potentials. Despite complete loss of hair-cell function, tmc triple-mutant larvae retain normal gross morphology of hair bundles and proper trafficking of known MET components Protocadherin 15a (Pcdh15a), Lipoma HMGIC fusion partner-like 5 (Lhfpl5), and Transmembrane inner ear protein (Tmie). Transgenic, hair cell-specific expression of Tmc2b-mEGFP rescues the behavioral and physiological deficits in tmc triple mutants. Results from tmc single and double mutants evince a principle role for Tmc2a and Tmc2b in hearing and balance, respectively, whereas Tmc1 has lower overall impact. Our experiments reveal that, in developing cristae, hair cells stratify into an upper, Tmc2a-dependent layer of teardrop-shaped cells and a lower, Tmc1/2b-dependent tier of gourd-shaped cells. Collectively, our genetic evidence indicates that auditory/vestibular end organs and subsets of hair cells therein rely on distinct combinations of Tmc1/2a/2b.SIGNIFICANCE STATEMENT We assessed the effects of tmc1/2a/2b truncation mutations on mechanoelectrical transduction (MET) in the inner-ear hair cells of larval zebrafish. tmc triple mutants lacked behavioral responses to sound and head movements, while further assays demonstrated no observable mechanosensitivity in the tmc1/2a/2b triple mutant inner ear. Examination of tmc double mutants revealed major contributions from Tmc2a and Tmc2b to macular function; however, Tmc1 had less overall impact. FM labeling of lateral cristae in tmc double mutants revealed the presence of two distinct cell types, an upper layer of teardrop-shaped cells that rely on Tmc2a, and a lower layer of gourd-shaped cells that rely on Tmc1/2b.

The development, distribution and density of the plasma membrane calcium ATPase 2 calcium pump in rat cochlear hair cells.

Calcium is tightly regulated in cochlear outer hair cells (OHCs). It enters mainly via mechanotransducer (MT) channels and is extruded by the plasma membrane calcium ATPase (PMCA)2 isoform of the PMCA, mutations in which cause hearing loss. To assess how pump expression matches the demands of Ca(2+) homeostasis, the distribution of PMCA2 at different cochlear locations during development was quantified using immunofluorescence and post-embedding immunogold labeling. The PMCA2 isoform was confined to stereociliary bundles, first appearing at the base of the cochlea around post-natal day (P)0 followed by the middle and then the apex by P3, and was unchanged after P8. The developmental appearance matched the maturation of the MT channels in rat OHCs. High-resolution immunogold labeling in adult rats showed that PMCA2 was distributed along the membranes of all three rows of OHC stereocilia at similar densities and at about a quarter of the density in inner hair cell stereocilia. The difference between OHCs and inner hair cells was similar to the ratio of their MT channel resting open probabilities. Gold particle counts revealed no difference in PMCA2 density between low- and high-frequency OHC bundles despite larger MT currents in high-frequency OHCs. The PMCA2 density in OHC stereocilia was determined in low- and high-frequency regions from calibration of immunogold particle counts as 2200/µm(2) from which an extrusion rate of ∼200 ions/s per pump was inferred. The limited ability of PMCA2 to extrude the Ca(2+) load through MT channels may constitute a major cause of OHC vulnerability and high-frequency hearing loss.

A DNA variant within the MYO7A promoter regulates YY1 transcription factor binding and gene expression serving as a potential dominant DFNA11 auditory genetic modifier.

Mutations within MYO7A can lead to recessive and dominant forms of inherited hearing loss. We previously identified a large pedigree (referred to as the HL2 family) with hearing loss that first impacts the low and mid frequencies segregating a dominant MYO7A mutation in exon 17 at DNA residue G2164C. The MYO7A(G2164C) mutation predicts a nonconservative glycine-to-arginine (G722R) amino acid substitution at a highly conserved glycine residue. The degree of low and mid frequency hearing loss varies markedly in the family, suggesting the presence of a genetic modifier that either rescues or exacerbates the primary MYO7A(G2164C) mutation. Here we describe a single nucleotide polymorphism (SNP) T/C at position -4128 in the wild-type MYO7A promoter allele that sorts with the degree of hearing loss severity in the pedigree. Electrophoretic mobility shift assay analysis indicates that the SNP differentially regulates the binding of the YY1 transcription factor with the T(-4128) allele creating an YY1 binding site. Immunocytochemistry demonstrates that Yy1 is expressed in hair cell nuclei within the cochlea. Given that Myo7a is also expressed in cochlear hair cells, Yy1 shows the appropriate localization to regulate Myo7a transcription within the inner ear. YY1 appears to be acting as a transcriptional repressor as the MYO7A promoter allele containing the T(-4128) SNP drives 41 and 46% less reporter gene expression compared with the C(-4128) SNP in the ARPE-19 and HeLa cell lines, respectively. The T(-4128) SNP may be contributing to the severe hearing loss phenotype in the HL2 pedigree by reducing expression of the wild-type MYO7A allele.

A Myo6 mutation destroys coordination between the myosin heads, revealing new functions of myosin VI in the stereocilia of mammalian inner ear hair cells.

Myosin VI, found in organisms from Caenorhabditis elegans to humans, is essential for auditory and vestibular function in mammals, since genetic mutations lead to hearing impairment and vestibular dysfunction in both humans and mice. Here, we show that a missense mutation in this molecular motor in an ENU-generated mouse model, Tailchaser, disrupts myosin VI function. Structural changes in the Tailchaser hair bundles include mislocalization of the kinocilia and branching of stereocilia. Transfection of GFP-labeled myosin VI into epithelial cells and delivery of endocytic vesicles to the early endosome revealed that the mutant phenotype displays disrupted motor function. The actin-activated ATPase rates measured for the D179Y mutation are decreased, and indicate loss of coordination of the myosin VI heads or 'gating' in the dimer form. Proper coordination is required for walking processively along, or anchoring to, actin filaments, and is apparently destroyed by the proximity of the mutation to the nucleotide-binding pocket. This loss of myosin VI function may not allow myosin VI to transport its cargoes appropriately at the base and within the stereocilia, or to anchor the membrane of stereocilia to actin filaments via its cargos, both of which lead to structural changes in the stereocilia of myosin VI-impaired hair cells, and ultimately leading to deafness.

Vestibular hair bundles control pH with (Na+, K+)/H+ exchangers NHE6 and NHE9.

In hair cells of the inner ear, robust Ca2+/H+ exchange mediated by plasma-membrane Ca2+-ATPase would rapidly acidify mechanically sensitive hair bundles without efficient removal of H+. We found that, whereas the basolateral membrane of vestibular hair cells from the frog saccule extrudes H+ via an Na+-dependent mechanism, bundles rapidly remove H+ in the absence of Na+ and HCO3(-), even when the soma is acidified. K+ was fully effective and sufficient for H+ removal; in contrast, Rb+ failed to support pH recovery. Na+/H+-exchanger isoform 1 (NHE1) was present on hair-cell soma membranes and was likely responsible for Na+-dependent H+ extrusion. NHE6 and NHE9 are organellar isoforms that can appear transiently on plasma membranes and have been proposed to mediate K+/H+ exchange. We identified NHE6 in a subset of hair bundles; NHE9 was present in all bundles. Heterologous expression of these isoforms in yeast strains lacking endogenous exchangers conferred pH-dependent tolerance to high levels of KCl and NaCl. NHE9 preferred cations in the order K+, Na+ >> Rb+, consistent with the relative efficacies of these ions in promoting pH recovery in hair bundles. Electroneutral K+/H+ exchange, which we propose is performed by NHE9 in hair bundles, exploits the high-K+ endolymph, responds only to pH imbalance across the bundle membrane, is unaffected by the +80 mV endocochlear potential, and uses mechanisms already present in the ear for K+ recycling. This mechanism allows the hair cell to remove H+ generated by Ca2+ pumping without ATP hydrolysis in the cell.

PHR1, an integral membrane protein of the inner ear sensory cells, directly interacts with myosin 1c and myosin VIIa.

By using the yeast two-hybrid technique, we identified a candidate protein ligand of the myosin 1c tail, PHR1, and found that this protein can also bind to the myosin VIIa tail. PHR1 is an integral membrane protein that contains a pleckstrin homology (PH) domain. Myosin 1c and myosin VIIa are two unconventional myosins present in the inner ear sensory cells. We showed that PHR1 immunoprecipitates with either myosin tail by using protein extracts from cotransfected HEK293 cells. In vitro binding assays confirmed that PHR1 directly interacts with these two myosins. In both cases the binding involves the PH domain. In vitro interactions between PHR1 and the myosin tails were not affected by the presence or absence of Ca2+ and calmodulin. Finally, we found that PHR1 is able to dimerise. As PHR1 is expressed in the vestibular and cochlear sensory cells, its direct interactions with the myosin 1c and VIIa tails are likely to play a role in anchoring the actin cytoskeleton to the plasma membrane of these cells. Moreover, as both myosins have been implicated in the mechanotransduction slow adaptation process that takes place in the hair bundles, we propose that PHR1 is also involved in this process.

Extrasynaptic localization of inactivating calcium-activated potassium channels in mouse inner hair cells.

Auditory hair cells from nonmammalian vertebrates are electrically tuned to specific sound frequencies primarily by the interactions of voltage-gated calcium channels and calcium-activated potassium (BK) channels colocalized at synaptic active zones. Mammalian inner hair cells are not electrically tuned and, yet, BK channels are also thought to reside at active zones. Using patch-clamp recordings and immunofluorescence, we characterized BK channel expression in mouse inner hair cells. Unexpectedly, these channels have inactivating currents and are clustered near the apex of the cell away from synaptic sites near the base. These results indicate a novel function of BK channels in mammalian inner hair cells and provide a framework for future research.

Two adaptation processes in auditory hair cells together can provide an active amplifier.

The hair cells of the vertebrate inner ear convert mechanical stimuli to electrical signals. Two adaptation mechanisms are known to modify the ionic current flowing through the transduction channels of the hair bundles: a rapid process involves Ca(2+) ions binding to the channels; and a slower adaptation is associated with the movement of myosin motors. We present a mathematical model of the hair cell which demonstrates that the combination of these two mechanisms can produce "self-tuned critical oscillations", i.e., maintain the hair bundle at the threshold of an oscillatory instability. The characteristic frequency depends on the geometry of the bundle and on the Ca(2+) dynamics, but is independent of channel kinetics. Poised on the verge of vibrating, the hair bundle acts as an active amplifier. However, if the hair cell is sufficiently perturbed, other dynamical regimes can occur. These include slow relaxation oscillations which resemble the hair bundle motion observed in some experimental preparations.

Contribution of BK Ca2+-activated K+ channels to auditory neurotransmission in the Guinea pig cochlea.

Large-conductance calcium-activated potassium (BK) channels are known to play a prominent role in the hair cell function of lower vertebrates where these channels determine electrical tuning and regulation of neurotransmitter release. Very little is known, by contrast, about the role of BK channels in the mammalian cochlea. In the current study, we perfused specific toxins in the guinea pig cochlea to characterize the role of BK channels in cochlear neurotransmission. Intracochlear perfusion of charybdotoxin (ChTX) or iberiotoxin (IbTX) reversibly reduced the compound action potential (CAP) of the auditory nerve within minutes. The cochlear microphonics (CM at f1 = 8 kHz and f2 = 9.68 kHz) and their distortion product (DPCM at 2f1-f2) were essentially not affected, suggesting that the BK specific toxins do not alter the active cochlear amplification at the outer hair cells (OHCs). We also tested the effects of these toxins on the whole cell voltage-dependent membrane current of isolated guinea pig inner hair cells (IHCs). ChTX and IbTX reversibly reduced a fast outward current (activating above -40 mV, peaking at 0 mV with a mean activation time constant tau ranging between 0.5 and 1 ms). A similar block of a fast outward current was also observed with the extracellular application of barium ions, which we believe permeate through Ca2+ channels and block BK channels. In situ hybridization of Slo antisense riboprobes and immunocytochemistry demonstrated a strong expression of BK channels in IHCs and spiral ganglion and to a lesser extent in OHCs. Overall, our results clearly revealed the importance of BK channels in mammalian cochlear neurotransmission and demonstrated that at the presynaptic level, fast BK channels are a significant component of the repolarizing current of IHCs.

Efferent neurotransmitters and intracellular Ca2+ concentrations in inner hair cells of guinea pig.

To elucidate efferent inhibitory mechanisms of the cochlear inner hair cells (IHCs), the effects of candidate efferent neurotransmitters on intracellular Ca2+ concentrations ([Ca2+]i) in the isolated IHCs of guinea pig were determined using the Ca2+ sensitive dye fura-2 and digital imaging microscopy. In the presence of 1 mM acetylcholine (ACh), there was a gradual and reversible increase in [Ca2+]i. In the presence of 10 microM ATP, there was a rapid and marked rise in [Ca2+]i. Thus, both ACh and ATP seem to be efferent neurotransmitters in IHCs of guinea pig. The ATP-induced elevation of [Ca2+]i in IHCs was suppressed when ACh was concomitantly applied; therefore, ACh may act as an antagonist of ATP-induced Ca2+ entry. With the independent addition of 10 microM calcitonin gene-related peptide and 10 microM enkephalin, there were no significant increases in [Ca2+]i. These neuropeptides are putative candidates for the efferent neuromodulators in IHCs of the guinea pig.