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Author(s): Andrei S. Kozlov [sup.1] , Johannes Baumgart [sup.2] , Thomas Risler [sup.3] [sup.4] [sup.5] , Corstiaen P. C. Versteegh [sup.1] [sup.6] , A. J. Hudspeth [sup.1] Author Affiliations: (1) [...], How inner-ear stereocilia pull together Hearing is initiated by the movement of tiny bundles of cilia located at the top of hair cells inside the ear. Despite being surrounded by the viscous fluid that bathes the inner ear, these stereocilia remain sensitive to movements of atomic dimension. The mechanism that makes this possible by eliminating most of the viscous drag in the liquid between the stereocilia has now been demonstrated. Analysis of the balance of forces in bullfrog hair cell stereocilia on a subnanometre scale shows that, at the frequencies used in hearing, most stereocilia inside the hair bundle are shielded from the external liquid and move virtually as one. The detection of sound begins when energy derived from an acoustic stimulus deflects the hair bundles on top of hair cells.sup.1. As hair bundles move, the viscous friction between stereocilia and the surrounding liquid poses a fundamental physical challenge to the ear's high sensitivity and sharp frequency selectivity. Part of the solution to this problem lies in the active process that uses energy for frequency-selective sound amplification.sup.2,3. Here we demonstrate that a complementary part of the solution involves the fluid-structure interaction between the liquid within the hair bundle and the stereocilia. Using force measurement on a dynamically scaled model, finite-element analysis, analytical estimation of hydrodynamic forces, stochastic simulation and high-resolution interferometric measurement of hair bundles, we characterize the origin and magnitude of the forces between individual stereocilia during small hair-bundle deflections. We find that the close apposition of stereocilia effectively immobilizes the liquid between them, which reduces the drag and suppresses the relative squeezing but not the sliding mode of stereociliary motion. The obliquely oriented tip links couple the mechanotransduction channels to this least dissipative coherent mode, whereas the elastic horizontal top connectors that stabilize the structure further reduce the drag. As measured from the distortion products associated with channel gating at physiological stimulation amplitudes of tens of nanometres, the balance of viscous and elastic forces in a hair bundle permits a relative mode of motion between adjacent stereocilia that encompasses only a fraction of a nanometre. A combination of high-resolution experiments and detailed numerical modelling of fluid-structure interactions reveals the physical principles behind the basic structural features of hair bundles and shows quantitatively how these organelles are adapted to the needs of sensitive mechanotransduction.

The sensitivity and frequency selectivity of hearing result from tuned amplification by an active process in the mechanoreceptive hair cells, in most vertebrates, the active process stems from the active motility of hair bundles. The mammalian cochlea exhibits an additional form of mechanical activity termed electromotility: its outer hair cells (OHCs) change length upon electrical stimulation. The relative contributions of these two mechanisms to the active process in the mammalian inner ear is the subject of intense current debate. Here, we show that active hair-bundle motility and electromotility can together implement an efficient mechanism for amplification that functions like a ratchet: Sound-evoked forces, acting on the basilar membrane, are transmitted to the hair bundles, whereas electromotility decouples active hair-bundle forces from the basilar membrane. This unidirectional coupling can extend the hearing range well below the resonant frequency of the basilar membrane. It thereby provides a concept for low-frequency hearing that accounts for a variety of unexplained experimental observations from the cochlear apex, including the shape and phase behavior of apical tuning curves, their lack of significant nonlinearities, and the shape changes of threshold tuning curves of auditory-nerve fibers along the cochlea. The ratchet mechanism constitutes a general design principle for implementing mechanical amplification in engineering applications. auditory system | cochlea | hair cell doi/ 10.1073/pnas.0914345107

The initial synapses of the auditory system, which connect hair cells to afferent nerve fibers, display two unusual features. First, synaptic transmission occurs in a multiquantal fashion: the contents of multiple synaptic vesicles are discharged simultaneously. Second, synaptic transmission may be tuned to specific frequencies of stimulation. We developed a minimal theoretical model to explore the possibility that hair-cell synapses achieve both multiquantal release and frequency selectivity through a cooperative mechanism for the exocytotic release of neurotransmitter. We first characterized vesicle release as a four-step cycle at each release site, then generalized the result to an arbitrary number of steps. The cyclic process itself induces some degree of resonance, and may display a stable, underdamped fixed point of the release dynamics associated with a pair of complex eigenvalues. Cooperativity greatly enhances the frequency selectivity by moving the eigenvalues toward the imaginary axis; spontaneously oscillatory release can arise beyond a Hopf bifurcation. These phenomena occur both in the macroscopic limit, when the number of release sites involved is very large, and in the more realistic stochastic regime, when only a limited number of release sites participate at each synapse. It is thus possible to connect multiquantal release with frequency selectivity through the mechanism of cooperativity. afferent fiber | auditory system | multiquantal release | multivesicular release | vestibular system doi/10.1073/pnas.0914372107

The development of functional neural circuits requires that connections between neurons be established in a precise manner. The mechanisms by which complex nervous systems perform this daunting task remain largely unknown. In the posterior lateral line of larval zebrafish, each afferent neuron forms synaptic contacts with hair cells of a common hair-bundle polarity. We investigated whether afferent neurons distinguish hair-cell polarities by analyzing differences in the synaptic signaling between oppositely polarized hair cells. By examining two mutant zebrafish lines with defects in mechanoelectrical transduction, and by blocking transduction during the development of wild-type fish, we found that afferent neurons could form specific synapses in the absence of stimulus-evoked patterns of synaptic release. Asking next whether this specificity arises through intrinsically generated patterns of synaptic release, we found that the polarity preference persisted in two mutant lines lacking essential synaptic proteins. These results indicate that lateral-line afferent neurons do not require synaptic activity to distinguish hair-cell polarities and suggest that molecular labels of hair-cell polarity guide prepatterned afferents to form the appropriate synapses. calcium channel | hair cell | neuromast | planar cell polarity | protocadherin doi/10.1073/pnas.0912082106

Little is known about the proteins that mediate mechanoelectrical transduction, the process by which acoustic and accelerational stimuli are transformed by hair cells of the inner ear into electrical signals. In our search for molecules involved in mechanotransduction, we discovered a line of deaf and uncoordinated zebrafish with defective hair-cell function. The hair cells of mutant larvae fail to incorporate fluorophores that normally traverse the transduction channels and their ears lack microphonic potentials in response to vibratory stimuli. Hair cells in the posterior lateral lines of mutants contain numerous lysosomes and have short, disordered hair bundles. Their stereocilia lack two components of the transduction apparatus, tip links and insertional plaques. Positional cloning revealed an early frameshift mutation in tmie, the zebrafish ortholog of the mammalian gene transmembrane inner ear. The mutant line therefore affords us an opportunity to investigate the role of the corresponding protein in mechanoelectrical transduction. auditory system | hair cell | lateral line | mechanoelectrical transduction | vestibular system doi/ 10.1073/pnas.0911632106

An understanding of the molecular bases of the morphogenesis, organization, and functioning of hair cells requires that the genes expressed in these cells be identified and their functions ascertained. After purifying zebrafish hair cells and detecting mRNAs with oligonucleotide microarrays, we developed a subtractive strategy that identified 1,037 hair cell-expressed genes whose cognate proteins subserve functions including membrane transport, synaptic transmission, transcriptional control, cellular adhesion and signal transduction, and cytoskeletal organization. To assess the validity of the subtracted hair-cell data set, we verified the presence of 11 transcripts in inner-ear tissue. Functional evaluation of two genes from the subtracted data set revealed their importance in hair bundles: zebrafish larvae bearing the seahorse and ift 172 mutations display specific kinociliary defects. Moreover, a search for candidate genes that underlie heritable deafness identified a human ortholog of a zebrafish hair-cell gene whose map location is bracketed by the markers of a deafness locus. auditory system | balance | hearing | vestibular system | zebrafish

The restoration of planar cell polarity is an essential but poorly understood step toward physiological recovery during sensoryorgan regeneration. Investigating this issue in the lateral line of the zebrafish, we found that hair cells regenerate in pairs along a single axis established by the restricted localization and oriented division of their progenitors. By analyzing mutants lacking the planar-polarity determinant Vangl2, we ascertained that the uniaxial production of hair cells and the subsequent orientation of their hair bundles are controlled by distinct pathways, whose combination underlies the establishment of hair-cell orientation during development and regeneration. This mechanism may represent a general principle governing the long-term maintenance of planar cell polarity in remodeling epithelia. auditory system | balance | hearing | lateral line | vestibular system

The development of the verterbrate inner ear depends on the precise expression of fibroblast growth factors. In a mutagenisis screen for zebrafish with abnormalities of inner-ear development and behavior, we isolate a mutant line, ru622, whose phenotypic characteristics resembled those of null mutants for the gene encoding fibroblast growth factor 8 (Fgf8): an inconsistent startle response. circular swimming, fused otoliths, and abnormal semi-circular canals. Positional cloning disclosed thE the mutant gene encodes that the transcriptional corepressor ATrophin2. Both the Fgf8 itself , were found to be overexpressed in ru622 mutants. We therefore hyphothesized that an excess of Sef eliminates Fgf8 signals and produced a Fgf8 null phenotype in ru622 mutants. In support of this idea we could rescue larvae whose atrophin2 plays a role in the feedback regulation of Fgf8 signaling. When mutation of the atrophin2 gene results in the overexpression of both Fgf8 and Sef, the excessive Sef inhibits Fgf8 signaling. The resultant imbalance of Fgf8 and Sef signals then underlies the abnormal aural development observed in ru622. auditory system | hearing | vestibular system

The sense of hearing depends on fast, finely graded neurotransmission at the ribbon synapses connecting hair cells to afferent nerve fibers. The processing that occurs at this first chemical synapse in the auditory pathway determines the quality and extent of the information conveyed to the central nervous system. Knowledge of the synapse's input--output function is therefore essential for understanding how auditory stimuli are encoded. To investigate the transfer function at the hair cell's synapse, we developed a preparation of the bullfrog's amphibian papilla. In the portion of this receptor organ representing stimuli of 400-800 Hz, each afferent nerve fiber forms several synaptic terminals onto one to three hair cells. By performing simultaneous voltage-clamp recordings from presynaptic hair cells and postsynaptic afferent fibers, we established that the rate of evoked vesicle release, as determined from the average postsynaptic current, depends linearly on the amplitude of the presynaptic [Ca.sup.2+] current. This result implies that, for receptor potentials in the physiological range, the hair cell's synapse transmits information with high fidelity. auditory system | exocytosis | glutamate | ribbon synapse | synaptic vesicle

When a hair cell of the bullfrog's sacculus is maintained in vitro under native ionic conditions, its mechanosensitive hair bundle may oscillate spontaneously. This movement has been hypothesized to result from the interaction of the bundle's negative stiffness, which creates a region of mechanical instability, with a myosin-based adaptation mechanism that continually repositions the bundle there. To test this proposition, we used a flexible stimulus fiber in an analog feedback loop to measure the displacement-force relation of an oscillating hair bundle. A digital signal processor was used to monitor spontaneous oscillations in real time and trigger measurements at particular phases of the movement cycle. By comparing the displacement-force curves obtained at the two extremes of a hair bundle's motion, we demonstrated a shift in the negative-stiffness region whose direction, orientation, magnitude, and kinetics agreed with the predictions of the gating-spring theory. The results are in accordance with the idea that adaptation underlies spontaneous hair-bundle oscillation, and therefore powers the active process that also amplifies and tunes the hair cell's mechanical responsiveness. auditory system | hair cell, hearing | myosin | vestibular system

The lateral line and its associated sensory nerves develop from cephalic epithelial thickenings called neurogenic placodes. In the zebrafish, the transcription factor neurogenin 1 is essential for the generation of the sensory ganglion from the placode, but is dispensable for the migration of the primordium and the initial development of neuromasts. We find that inactivation of the gene encoding neurogenin 1 leads to the development of over twice the normal number of neuromasts along the posterior lateral line of zebrafish larvae. Mutation of the gene encoding another transcription factor, sox10, has a similar effect. After a normal number of proneuromasts is initially deposited by the migrating primordia, interneuromast cells divide and differentiate to form the extra neuromasts. Our results indicate that the development of these intercalary neuromasts occurs principally because of the absence of neural crest-derived peripheral gila, which evidently inhibit the assembly of interneuromast cells into neuromasts. acousticolateralis system | hair cell | neurogenin

In a three-generation screen of chemically mutagenized zebrafish, we identified a group of mutations that affect the development and function of hair cells, the mechanically sensitive cells of the inner ear and lateral-line organ. One mutant line, ru920, was discovered in a behavioral screen for defects in the acoustically evoked escape response. Despite apparently normal numbers of hair cells, mutants lack an inner-ear microphonic potential and exhibit reduced labeling of hair cells by a fluorophore that traverses transduction channels. This hair-cell-specific phenotype suggested a defect in the mechanoelectrical transduction apparatus. Positional cloning revealed that the recessive mutation introduces a premature stop codon in the ORF of myosin6b (myo6b), one of the two zebrafish orthologs of the human gene myosin W. The ru920 line therefore provides an animal model with which to study the role of class VI myosin proteins in mechanotransduction.

Proteins of the ezrin-radixin-moesin family are ubiquitous constituents of the submembrane cortex, especially in epithelial cells. Earlier biochemical results suggested that a protein of this family occurs in the hair bundle, the cluster of actin-filled stereocilia that serves as the mechanoreceptive organelle of each hair cell in the inner ear. We prepared antipeptide antisera directed against chicken radixin and ezrin and demonstrated their specificity and absence of crossreactivity. When used in immunocytochemical studies of isolated hair cells, anti-radixin produced an intense band of labeling at the bases of hair bundles from the chicken, frog, mouse, and zebrafish. Electron microscopic immunocytochemistry disclosed that radixin labeling commenced in the stereociliary taper, peaked in the lower stereociliary shaft, and declined progressively toward the hair bundle's top. Labeling with anti-ezrin produced no signal in hair bundles. Radixin is thus a prominent constituent of stereocilia, where it may participate in anchoring the 'pointed' ends of actin filaments to the membrane.

To identify genes important for hair-cell function, we conducted a mutagenic screen in zebrafish. Larvae from one mutant line, ru848, were unresponsive to acoustic stimuli and unable to balance. The mutation results in a 90% reduction in hair-cell number and partial retinal degeneration by 5 days postfertilization. We localized the recessive ru848 mutation by positional cloning to the zebrafish homolog of the human Choroideremia gene, which encodes Rab escort protein 1. This protein is essential for the normal prenylation of Rabs. Mutations in the human gene induce choroideremia, a disease marked by slow-onset degeneration of rod photoreceptors and retinal pigment epithelial cells. The degenerative phenotype resulting from a null mutation in the zebrafish gene indicates that hair cells and retinal cells require Rab escort protein 1 for survival.

The association of [Ca.sup.2+]-activated [K.sup.+] channels with voltage-gated [Ca.sup.2+] channels at the presynaptic active zones of hair cells, photoreceptors, and neurons contributes to rapid repolarization of the membrane after excitation. [Ca.sup.2+] channels have been shown to bind to a large set of synaptic proteins, but the proteins interacting with [Ca.sup.2+]-activated [K.sup.+] channels remain unknown. Here, we report that the large-conductance [Ca.sup.2+]-activated [K.sup.+] channel of the chicken's cochlear hair cell interacts with [beta]-catenin. Yeast two-hybrid assays identified the $10 region of the [K.sup.+] channel's [alpha]-subunit and the ninth armadillo repeat and carboxyl terminus of [beta]-catenin as necessary for the interaction. An antiserum directed against the [alpha]-subunit specifically coprecipitated [beta]-catenin from brain synaptic proteins. [beta]-Catenin is known to associate with the synaptic protein Lin7/Velis/MALS, whose interaction partner Lin2/CASK also binds voltage-gated [Ca.sup.2+] channels. [beta]-Catenin may therefore provide a physical link between the two types of channels at the presynaptic active zone.

Electrically evoked otoacoustic emission is a manifestation of reverse transduction by the inner ear. We present evidence for a single-cell correlate of this phenomenon, hair-bundle movement driven by transepithelial electrical stimulation of the frog's sacculus. Responses could be observed at stimulus frequencies up to 1 kHz, an order of magnitude higher than the organ's natural range of sensitivity to acceleration or sound. Measurements at high-stimulus frequencies and pharmacological treatments allow us to distinguish two mechanisms that mediate the electrical responses: myosin-based adaptation and [Ca.sup.2+]-dependent reclosure of transduction channels. These mechanisms also participate in the active process that amplifies and tunes the mechanical responses of this receptor organ. Transient application of the channel blocker gentamicin demonstrated the crucial role of mechanoelectrical transduction channels in the rapid responses to electrical stimulation. A model for electrically driven bundle motion that incorporates the negative stiffness of the hair bundle as well as its two mechanisms of motility captures the essential features of the measured responses.

The [beta] subunits of voltage-gated [Ca.sup.2+] channels are known to be regulators of the channels' gating properties. Here we report a striking additional function of a [beta] subunit. Screening of chicken cochlear and brain cDNA libraries identified [[beta].sub.4c], a short splice variant of the [[beta].sub.4] subunit. Although [[beta].sub.4c] occurs together with the longer isoforms [[beta].sub.4a] or [[beta].sub.4b] in the brain, eye, heart, and lung, the cochlea expresses exclusively [[beta].sub.4c]. The association of [[beta].sub.4c] with the [Ca.sup.2+]-channel [[alpha].sub.1] subunit has slight but significant effects on the kinetics of channel activation and inactivation. Yeast two-hybrid and biochemical assays revealed that [[beta].sub.4c] interacts directly with the chromo shadow domain of chromobox protein 2/heterochromatin protein 1[gamma] (CHCB2/HP1[gamma]), a nuclear protein involved in gene silencing and transcriptional regulation. Coexpression of this protein specifically recruits [[beta].sub.4c] to the nuclei of mammalian cells. Furthermore, [[beta].sub.4c] but not [[beta].sub.4a] dramatically attenuates the gene-silencing activity of chromobox protein 2/heterochromatin protein 1[gamma]. The [[beta].sub.4c] subunit is therefore a multifunctional protein that not only constitutes a portion of the [Ca.sup.2+] channel but also regulates gene transcription.

Hearing relies on active filtering to achieve exquisite sensitivity and sharp frequency selectivity. In a quiet environment, the ears of many vertebrates become unstable and emit one to several tones. These spontaneous otoacoustic emissions, the most striking manifestation of the inner ear's active process, must result from self-sustained mechanical oscillations of aural constituents. The mechanoreceptive hair bundles of hair cells in the bullfrog's sacculus have the ability to amplify mechanical stimuli and oscillate spontaneously. By comparing a hair bundle's spontaneous oscillations with its response to small mechanical stimuli, we demonstrate a breakdown in a general principle of equilibrium thermodynamics, the fluctuation-dissipation theorem. We thus confirm that a hair bundle's spontaneous movements are produced by energy-consuming elements within the hair cell. To characterize the dynamical behavior of the active process, we introduce an effective temperature that, for each frequency component, quantifies a hair bundle's deviation from thermal equilibrium. The effective temperature diverges near the bundle's frequency of spontaneous oscillation. This behavior, which is not generic for active oscillators, can be accommodated by a simple model that characterizes quantitatively the fluctuations of the spontaneous movements as well as the hair bundle's linear response function.

The auditory system's ability to interpret sounds over a wide range of amplitudes rests on the nonlinear responsiveness of the ear. Whether measured by basilar-membrane vibration, nerve-fiber activity, or perceived loudness, the ear is most sensitive to small signals and grows progressively less responsive as stimulation becomes stronger. Seeking a correlate of this behavior at the level of mechanoelectrical transduction, we examined the responses of hair bundles to direct mechanical stimulation. As reported by the motion of an attached glass fiber, an active hair bundle from the bullfrog's sacculus oscillates spontaneously. Sinusoidal movement of the fiber's base by as little as [+ or -] 1 nm, corresponding to the application at the bundle's top of a force of [+ or -] 0.3 pN, causes detectable phase-locking of the bundle's oscillations to the stimulus. Although entrainment increases as the stimulus grows, the amplitude of the hair-bundle movement does not rise until phase-locking is nearly complete. A bundle is most sensitive to stimulation at its frequency of spontaneous oscillation. Far from that frequency, the sensitivity of an active hair bundle resembles that of a passive bundle. Over most of its range, an active hair bundle's response grows as the one-third power of the stimulus amplitude; the bundle's sensitivity declines accordingly in proportion to the negative two-thirds power of the excitation. This scaling behavior, also found in the response of the mammalian basilar membrane to sound, signals the operation of an amplificatory process at the brink of an oscillatory instability, a Hopf bifurcation.

A vertebrate osmoreceptor candidate has been identified in the vanilloid receptor-related osmotically activated channel (VR-OAC). Researchers cloned cDNAs encoding the VR-OAC from rats, mice, humans and chickens. A candidate-gene approach was based on genes encoding members of the TRP superfamily of ion channels. In the central nervous system, the channel is expressed in neurons of the circumventricular organs. These are neurosensory cells that respond to systemic osmotic pressure. The channel is in other neurosensory cells as well.

Amplification of auditory stimuli by hair cells augments the sensitivity of the vertebrate inner ear. Cell-body contractions of outer hair cells are thought to mediate amplification in the mammalian cochlea. In vertebrates that lack these cells, and perhaps in mammals as well, active movements of hair bundles may underlie amplification. We have evaluated a mathematical model in which amplification stems from the activity of mechanoelectrical-transduction channels. The intracellular binding of [Ca.sup.2+] to channels is posited to promote their closure, which increases the tension in gating springs and exerts a negative force on the hair bundle. By enhancing bundle motion, this force partially compensates for viscous damping by cochlear fluids. Linear stability analysis of a six-state kinetic model reveals Hopf bifurcations for parameter values in the physiological range. These bifurcations signal conditions under which the system's behavior changes from a damped oscillatory response to spontaneous limit-cycle oscillation. By varying the number of stereocilia in a bundle and the rate constant for [Ca.sup.2+] binding, we calculate bifurcation frequencies spanning the observed range of auditory sensitivity for a representative receptor organ, the chicken's cochlea. Simulations using prebifurcation parameter values demonstrate frequency-selective amplification with a striking compressive nonlinearity. Because transduction channels occur universally in hair cells, this active-channel model describes a mechanism of auditory amplification potentially applicable across species and hair-cell types.

The L-type voltage-gated [Ca.sup.2+] channels that control tonic release of neurotransmitter from hair cells exhibit unusual electrophysiological properties: a low activation threshold, rapid activation and deactivation, and a lack of [Ca.sup.2+]-dependent inactivation. We have inquired whether these characteristics result from cell-specific splicing of the mRNA for the L-type [[Alpha].sub.1D] subunit that predominates in hair cells of the chicken's cochlea. The [[Alpha].sub.1D] subunit in hair cells contains three uncommon exons: one encoding a 26-aa insert in the cytoplasmic loop between repeats I and II, an alternative exon for transmembrane segment IIIS2, and a heretofore undescribed exon specifying a 10-aa insert in the cytoplasmic loop between segments IVS2 and IVS3. We propose that the alternative splicing of the [[Alpha].sub.1D] mRNA contributes to the unusual behavior of the hair cell's voltage-gated [Ca.sup.2+] channels.

The voltage-gated [Ca.sup.2+] channels that effect tonic release of neurotransmitter from hair cells have unusual pharmacological properties: unlike most presynaptic [Ca.sup.2+] channels, they are sensitive to dihydropyridines and therefore are L-type. To characterize these [Ca.sup.2+] channels, we investigated the expression of L-type [[Alpha].sub.1] subunits in hair cells of the chicken's cochlea. In PCRs with five different pairs of degenerate primers, we always obtained [[Alpha].sub.1D] products, but only once an [[Alpha].sub.1C] product and never an [[Alpha].sub.1S] product. A full-length [[Alpha].sub.1D] mRNA sequence was assembled from overlapping PCR products: the predicted amino acid sequence of the [[Alpha].sub.1D] subunit was about 90% identical to those of the mammalian [[Alpha].sub.1D] subunits. In situ hybridization confirmed that the [[Alpha].sub.1D] mRNA is present in hair cells. By using a quantitative PCR assay, we determined that the [[Alpha].sub.1D] mRNA is 100-500 times more abundant than the [[Alpha].sub.1C] mRNA. We conclude that most, if not all, voltage-gated [Ca.sup.2+] channels in hair cells contain an [[Alpha].sub.1D] subunit. Furthermore, we propose that the [[Alpha].sub.1D] subunit plays a hitherto undocumented role at tonic synapses.

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. [Ca.sup.2+] is thought to be required in mechanoelectrical transduction, for exposure of hair bundles to [Ca.sup.2+] chelators eliminates responsiveness by disrupting tip links, filamentous interstereociliary connections that probably are the gating springs. [Ca.sup.2+] 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 [Ca.sup.2+] concentration on stiffness before and after the disruption of gating springs. The stiffness of intact hair bundles depended nonmonotonically on the extracellular [Ca.sup.2+] concentration; the maximal stiffness of [approximately equal to] 1200 [Mu]N[center dot][m.sup.-1] occurred when bundles were bathed in solutions containing 250 [[micro]molar] [Ca.sup.2+], approximately the concentration found in frog endolymph. For cells exposed to solutions with sufficient chelator capacity to reduce the [Ca.sup.2+] concentration below [approximately equal to] 100 nM, hair-bundle stiffness fell to [approximately equal to] 200 [Mu]N[center dot][m.sup.-1] and no longer exhibited [Ca.sup.2+]-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 [Ca.sup.2+] concentration.

Neurotransmitter is released when [Ca.sup.2+] triggers the fusion of synaptic vesicles with the plasmalemma. To study factors that regulate [Ca.sup.2+] concentration at the presynaptic active zones of hair cells, we used laser-scanning confocal microscopy with the fluorescent [Ca.sup.2+] indicator fluo-3. The experimental results were compared with the predictions of a model of presynaptic [Ca.sup.2+] concentration in which [Ca.sup.2+] enters a cell through a point source, diffuses from the entry site, and binds to fixed or mobile [Ca.sup.2+] 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 [Ca.sup.2+] buffer. The results confirm the localized entry of [Ca.sup.2+] underlying neurotransmitter release and suggest that [Ca.sup.2+] is cleared from an active zone almost exclusively by mobile buffer.

Calmodulin is a cytoplasmic Casuper2+ receptor found in the hair bundle at a median concentration equivalent to 70 microM. The adaptation motor potential of calmodulin was evaluated through a calmodulin-alkaline phosphotase conjugate that sought myosin isozymes in isolated bundles. The receptor's antagonists increased photolabeling of hair-bundle myosin I by nucleotides, which proved calmodulin capacity to regulate the adaptation motor.

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 [Ca.sup.2+] indicator fura-2 has instead suggested that [Ca.sup.2+] traverses channels at the bundle's base. To examine the site of [Ca.sup.2+] entry through transduction channels, we used laser-scanning confocal microscopy, with a spatial resolution of

cDNA library searching using a cDNA from polydenylated mRNA separated from frog brain, obtained through reverse transcription and PCR using myosin Ibeta amino acid sequence-based oligonucleotide primers, yields clones that code for myosin Ibeta isozyme that is of approximately 119 kDa length. An mRNA that codes for the same isozyme in bullfrog sacculus tissue is detected on PCR amplification. Monoclonal antibodies that interact with myosin I in hair bundles identify the myosin I isoform's tail regional domain when the protein is expressed as a bacterial protein.

Ca2+-activated k+ channels and Ca2+-channels help regulate the electrical resonance and frequency tuning of hair cells. Fluorescence confocal icroscopy of frog hair cells labeled with Ca2+ indicator fluo-3, shows that the Ca2+- and Ca2+- activated K+ channels localize at presynaptic active zones in hair cells. Current estimations at the active zones indicate that the channels are voltage-activated and mediate synaptic transmission.

Hair cells are the sensory receptors of the internal ear. They facilitate the process of mechanoelectrical transduction and encode the magnitude and time course of the stimulus as electrical responses, which are eventually transmitted to the brain as action potentials.

The determination of calmodulin concentrations and the proteins that interact with it in hair bundles are discussed. Experiments involve the isolation and detergent extraction of bullfrog saccular hair bundles, their analysis by quantitative immunoblotting and the conjugation of calmodulin to alkaline phosphatase. The role of calmodulin and its receptors in hair bundles were established.