The selectivity of the hair cell’s mechanoelectrical-transduction channel promotes Ca2+ flux at low Ca2+ concentrations

The mechanoelectrical-transduction channel of the hair cell is permeable to both monovalent and divalent cations. Because Ca2+ entering through the transduction channel serves as a feedback signal in the adaptation process that sets the channel’s open probability, an understanding of adaptation requires estimation of the magnitude of Ca2+ influx. To determine the Ca2+ current through the transduction channel, we measured extracellular receptor currents with transepithelial voltage-clamp recordings while the apical surface of a saccular macula was bathed with solutions containing various concentrations of K+, Na+, or Ca2+. For modest concentrations of a single permeant cation, Ca2+ carried much more receptor current than did either K+ or Na+. For higher cation concentrations, however, the flux of Na+ or K+ through the transduction channel exceeded that of Ca2+. For mixtures of Ca2+ and monovalent cations, the receptor current displayed an anomalous mole-fraction effect, which indicates that ions interact while traversing the channel’s pore. These results demonstrate not only that the hair cell’s transduction channel is selective for Ca2+ over monovalent cations but also that Ca2+ carries substantial current even at low Ca2+ concentrations. At physiological cation concentrations, Ca2+ flux through transduction channels can change the local Ca2+ concentration in stereocilia in a range relevant for the control of adaptation.

Effects of extracellular Ca2+ concentration on hair-bundle stiffness and gating-spring integrity in hair cells

When a hair cell is stimulated by positive deflection of its hair bundle, increased tension in gating springs opens transduction channels, permitting cations to enter stereocilia and depolarize the cell. Ca2+ is thought to be required in mechanoelectrical transduction, for exposure of hair bundles to Ca2+ chelators eliminates responsiveness by disrupting tip links, filamentous interstereociliary connections that probably are the gating springs. Ca2+ also participates in adaptation to stimuli by controlling the activity of a molecular motor that sets gating-spring tension. Using a flexible glass fiber to measure hair-bundle stiffness, we investigated the effect of Ca2+ concentration on stiffness before and after the disruption of gating springs. The stiffness of intact hair bundles depended nonmonotonically on the extracellular Ca2+ concentration; the maximal stiffness of ≈1200 μN⋅m−1 occurred when bundles were bathed in solutions containing 250 μM Ca2+, approximately the concentration found in frog endolymph. For cells exposed to solutions with sufficient chelator capacity to reduce the Ca2+ concentration below ≈100 nM, hair-bundle stiffness fell to ≈200 μN⋅m−1 and no longer exhibited Ca2+-dependent changes. Because cells so treated lost mechanoelectrical transduction, we attribute the reduction in bundle stiffness to tip-link disruption. The results indicate that gating springs are not linearly elastic; instead, they stiffen with increased strain, which rises with adaptation-motor activity at the physiological extracellular Ca2+ concentration.

Predominance of the α1D subunit in L-type voltage-gated Ca2+ channels of hair cells in the chicken’s cochlea

The voltage-gated Ca2+ channels that effect tonic release of neurotransmitter from hair cells have unusual pharmacological properties: unlike most presynaptic Ca2+ channels, they are sensitive to dihydropyridines and therefore are L-type. To characterize these Ca2+ channels, we investigated the expression of L-type α1 subunits in hair cells of the chicken’s cochlea. In PCRs with five different pairs of degenerate primers, we always obtained α1D products, but only once an α1C product and never an α1S product. A full-length α1D mRNA sequence was assembled from overlapping PCR products; the predicted amino acid sequence of the α1D subunit was about 90% identical to those of the mammalian α1D subunits. In situ hybridization confirmed that the α1D mRNA is present in hair cells. By using a quantitative PCR assay, we determined that the α1D mRNA is 100–500 times more abundant than the α1C mRNA. We conclude that most, if not all, voltage-gated Ca2+ channels in hair cells contain an α1D subunit. Furthermore, we propose that the α1D subunit plays a hitherto undocumented role at tonic synapses.

Regeneration of broken tip links and restoration of mechanical transduction in hair cells

A hair cell’s tip links are thought to gate mechanoelectrical transduction channels. The susceptibility of tip links to acoustic trauma raises questions as to whether these fragile structures can be regenerated. We broke tip links with the calcium chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid and found that they can regenerate, albeit imperfectly, over several hours. The time course of tip-link regeneration suggests that this process may underlie recovery from temporary threshold shifts induced by noise exposure. Cycloheximide does not block tip-link regeneration, indicating that new protein synthesis is not required. The calcium ionophore ionomycin prevents regeneration, suggesting regeneration normally may be stimulated by the reduction in stereociliary Ca2+ when gating springs rupture and transduction channels close. Supporting the equivalence of tip links with gating springs, mechanoelectrical transduction returns over the same time period as tip links; strikingly, adaptation is substantially reduced, even 24 hr after breaking tip links.

α10: A determinant of nicotinic cholinergic receptor function in mammalian vestibular and cochlear mechanosensory hair cells

We report the cloning and characterization of rat α10, a previously unidentified member of the nicotinic acetylcholine receptor (nAChR) subunit gene family. The protein encoded by the α10 nAChR subunit gene is most similar to the rat α9 nAChR, and both α9 and α10 subunit genes are transcribed in adult rat mechanosensory hair cells. Injection of Xenopus laevis oocytes with α10 cRNA alone or in pairwise combinations with either α2-α6 or β2-β4 subunit cRNAs yielded no detectable ACh-gated currents. However, coinjection of α9 and α10 cRNAs resulted in the appearance of an unusual nAChR subtype. Compared with homomeric α9 channels, the α9α10 nAChR subtype displays faster and more extensive agonist-mediated desensitization, a distinct current–voltage relationship, and a biphasic response to changes in extracellular Ca2+ ions. The pharmacological profiles of homomeric α9 and heteromeric α9α10 nAChRs are essentially indistinguishable and closely resemble those reported for endogenous cholinergic eceptors found in vertebrate hair cells. Our data suggest that efferent modulation of hair cell function occurs, at least in part, through heteromeric nAChRs assembled from both α9 and α10 subunits.

Two mechanisms for transducer adaptation in vertebrate hair cells

Deflection of the hair bundle atop a sensory hair cell modulates the open probability of mechanosensitive ion channels. In response to sustained deflections, hair cells adapt. Two fundamentally distinct models have been proposed to explain transducer adaptation. Both models support the notion that channel open probability is modulated by calcium that enters via the transduction channels. Both also suggest that the primary effect of adaptation is to shift the deflection-response [I(X)] relationship in the direction of the applied stimulus, thus maintaining hair bundle sensitivity. The models differ in several respects. They operate on different time scales: the faster on the order of a few milliseconds or less and the slower on the order of 10 ms or more. The model proposed to explain fast adaptation suggests that calcium enters and binds at or near the transduction channels to stabilize a closed conformation. The model proposed to explain the slower adaptation suggests that adaptation is mediated by an active, force-generating process that regulates the effective stimulus applied to the transduction channels. Here we discuss the evidence in support of each model and consider the possibility that both may function to varying degrees in hair cells of different species and sensory organs.

Putting ion channels to work: Mechanoelectrical transduction, adaptation, and amplification by hair cells

As in other excitable cells, the ion channels of sensory receptors produce electrical signals that constitute the cellular response to stimulation. In photoreceptors, olfactory neurons, and some gustatory receptors, these channels essentially report the results of antecedent events in a cascade of chemical reactions. The mechanoelectrical transduction channels of hair cells, by contrast, are coupled directly to the stimulus. As a consequence, the mechanical properties of these channels shape our hearing process from the outset of transduction. Channel gating introduces nonlinearities prominent enough to be measured and even heard. Channels provide a feedback signal that controls the transducer's adaptation to large stimuli. Finally, transduction channels participate in an amplificatory process that sensitizes and sharpens hearing.

The entry and clearance of Ca2+ at individual presynaptic active zones of hair cells from the bullfrog's sacculus.

Neurotransmitter is released when Ca2+ triggers the fusion of synaptic vesicles with the plasmalemma. To study factors that regulate Ca2+ concentration at the presynaptic active zones of hair cells, we used laser-scanning confocal microscopy with the fluorescent Ca2+ indicator fluo 3. The experimental results were compared with the predictions of a model of presynaptic Ca2+ concentration in which Ca2+ enters a cell through a point source, diffuses from the entry site, and binds to fixed or mobile Ca2+ buffers. The observed time course and magnitude of fluorescence changes under a variety of conditions were well fit when the model included mobile molecules as the only Ca2+ buffer. The results confirm the localized entry of Ca2+ underlying neurotransmitter release and suggest that Ca2+ is cleared from an active zone almost exclusively by mobile buffer.

Calmodulin controls adaptation of mechanoelectrical transduction by hair cells of the bullfrog's sacculus.

Deflection of the mechanically sensitive hair bundle atop a hair cell opens transduction channels, some of which subsequently reclose during a Ca2+-dependent adaptation process. Myosin I in the hair bundle is thought to mediate this adaptation; in the bullfrog's hair cell, the relevant isozyme may be the 119-kDa amphibian myosin I beta. Because this molecule resembles other forms of myosin I, we hypothesized that calmodulin, a cytoplasmic receptor for Ca2+, regulates the ATPase activity of myosin. We identified an approximately 120-kDa calmodulin-binding protein that shares with hair-bundle myosin I the properties of being photolabeled by vanadate-trapped uridine nucleotides and immunoreactive with a monoclonal antibody raised against mammalian myosin I beta. To investigate the possibility that calmodulin mediates Ca2+-dependent adaptation, we inhibited calmodulin action and measured the results with two distinct assays. Calmodulin antagonists increased photolabeling of hair-bundle myosin I by nucleotides. In addition, when introduced into hair cells through recording electrodes, calmodulin antagonists abolished adaptation to sustained mechanical stimuli. Our evidence indicates that calmodulin binds to and controls the activity of hair-bundle myosin I, the putative adaptation motor.

Detection of Ca2+ entry through mechanosensitive channels localizes the site of mechanoelectrical transduction in hair cells.

A hair cell, the sensory receptor of the internal ear, transduces mechanical stimuli into electrical responses. Transduction results from displacement of the hair bundle, a cluster of rod-shaped stereocilia extending from the cell's apical surface. Biophysical experiments indicate that, by producing shear between abutting stereocilia, a bundle displacement directly opens cation-selective transduction channels. Specific models of gating depend on the location of these channels, which has been controversial: although some physiological and immunocytochemical experiments have situated the transduction channels at the hair bundle's top, monitoring of fluorescence signals from the Ca2+ indicator fura-2 has instead suggested that Ca2+ traverses channels at the bundle's base. To examine the site of Ca2+ entry through transduction channels, we used laser-scanning confocal microscopy, with a spatial resolution of < 1 micron and a temporal resolution of < 2 ms, to observe hair cells filled with the indicator fluo-3. An unstimulated hair cell showed a "tip blush" of enhanced fluorescence at the hair bundle's top, which we attribute to Ca2+ permeation through transduction channels open at rest. Upon mechanical stimulation, individual stereocilia displayed increased fluorescence that originated near their tips, then spread toward their bases. Our results confirm that mechanoelectrical transduction occurs near stereociliary tips.

Postnatal developmental expression of the PDZ scaffolds Na+-H+ exchanger regulatory factors 1 and 2 in the rat cochlea

Sensory transduction in the mammalian cochlea requires the maintenance of specialized fluid compartments with distinct ionic compositions. This is achieved by the concerted action of diverse ion channels and transporters, some of which can interact with the PDZ scaffolds, Na+-H+ exchanger regulatory factors 1 and 2 (NHERF-1, NHERF-2). Here, we report that NHERF-1 and NHERF-2 are widely expressed in the rat cochlea, and that their expression is developmentally regulated. Reverse transcription/polymerase chain reaction (RT-PCR) and Western blotting initially confirmed the RNA and protein expression of NHERFs. We then performed immunohistochemistry on cochlea during various stages of postnatal development. Prior to the onset of hearing (P8), NHERF-1 immunolabeling was prominently polarized to the apical membrane of cells lining the endolymphatic compartment, including the stereocilia and cuticular plates of the inner and outer hair cells, marginal cells of the stria vascularis, Reissner's epithelia, and tectorial membrane. With maturation (P21, P70), NHERF-1 immunolabeling was reduced in the above structures, whereas labeling increased in the apical membrane of the interdental cells of the spiral limbus and the inner and outer sulcus cells, Hensen's cells, the inner and outer pillar cells, Deiters cells, the inner border cells, spiral ligament fibrocytes, and spiral ganglion neurons (particularly type II). NHERF-1 expression in strial basal and intermediate cells was persistent. NHERF-2 immunolabeling was similar to that for NHERF-1 during postnatal development, with the exception of expression in the synaptic regions beneath the outer hair cells. NHERF-1 and NHERF-2 co-localized with glial fibrillary acidic protein and vimentin in glia. The cochlear localization of NHERF scaffolds suggests that they play important roles in the developmental regulation of ion transport, homeostasis, and auditory neurotransmission.

Immunocytochemical and pharmacological characterization of metabotropic glutamate receptors of the vestibular end organs in the frog.

Using immunocytochemistry and multiunit recording of afferent activity of the whole vestibular nerve, we investigated the role of metabotropic glutamate receptors (mGluR) in the afferent neurotransmission in the frog semicircular canals (SCC). Group I (mGluR1alpha) and group II (mGluR2/3) mGluR immunoreactivities were distributed to the vestibular ganglion neurons, and this can be attributed to a postsynaptic locus of metabotropic regulation of rapid excitatory transmission. The effects of group I/II mGluR agonist (1S,3R)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD) and antagonist (R,S)-alpha-methyl-4-carboxyphenylglycine (MCPG) on resting and chemically induced afferent activity were studied. ACPD (10-100 microM) enhanced the resting discharge frequency. MCPG (5-100 microM) led to a concentration-dependent decrease of both resting activity and ACPD-induced responses. If the discharge frequency had previously been restored by L-glutamate (L-Glu) in high-Mg2+ solution, ACPD elicited a transient increase in the firing rate in the afferent nerve suggesting that ACPD acts on postsynaptic receptors. The L-Glu agonists, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and N-methyl-D-aspartate (NMDA), were tested during application of ACPD. AMPA- and NMDA-induced responses were higher in the presence than absence of ACPD, implicating mGluR in the modulation of ionotropic glutamate receptors. These results indicate that activation of mGluR potentiates AMPA and NMDA responses through a postsynaptic interaction. We conclude that ACPD may exert modulating postsynaptic effects on vestibular afferents and that this process is activity-dependent.

Defining Atoh1 function and regulation in avian supporting cells during auditory hair cell regeneration

Thesis (Ph.D.)--University of Washington, 2016-06