Jong-Hoon Nam

Localization of inner hair cell mechanotransducer channels using high speed calcium imaging

Hair cells detect vibrations of their stereociliary bundle by activation of mechanically sensitive transducer channels. Although evidence suggests the transducer channels are near the stereociliary tops and are opened by force imparted by tip links connecting contiguous stereocilia, the exact channel site remains controversial. We used fast confocal imaging of fluorescence changes reflecting calcium entry during bundle stimulation to localize the channels. Calcium signals were visible in single stereocilia of rat cochlear inner hair cells and were up to tenfold larger and faster in the second and third stereociliary rows than in the tallest first row. The number of functional stereocilia was proportional to transducer current amplitude, indicating that there were about two channels per stereocilium. Comparable results were obtained in outer hair cells. The observations, supported by theoretical simulations, suggest there are no functional mechanically sensitive transducer channels in first row stereocilia and imply the channels are present only at the bottom of the tip links.

The Actions of Calcium on Hair Bundle Mechanics in Mammalian Cochlear Hair Cells

Sound stimuli excite cochlear hair cells by vibration of each hair bundle, which opens mechanotransducer (MT) channels. We have measured hair-bundle mechanics in isolated rat cochleas by stimulation with flexible glass fibers and simultaneous recording of the MT current. Both inner and outer hair-cell bundles exhibited force-displacement relationships with a nonlinearity that reflects a time-dependent reduction in stiffness. The nonlinearity was abolished, and hair-bundle stiffness increased, by maneuvers that diminished calcium influx through the MT channels: lowering extracellular calcium, blocking the MT current with dihydrostreptomycin, or depolarizing to positive potentials. To simulate the effects of Ca(2+), we constructed a finite-element model of the outer hair cell bundle that incorporates the gating-spring hypothesis for MT channel activation. Four calcium ions were assumed to bind to the MT channel, making it harder to open, and, in addition, Ca(2+) was posited to cause either a channel release or a decrease in the gating-spring stiffness. Both mechanisms produced Ca(2+) effects on adaptation and bundle mechanics comparable to those measured experimentally. We suggest that fast adaptation and force generation by the hair bundle may stem from the action of Ca(2+) on the channel complex and do not necessarily require the direct involvement of a myosin motor. The significance of these results for cochlear transduction and amplification are discussed.

Calcium balance and mechanotransduction in rat cochlear hair cells.

Auditory transduction occurs by opening of Ca(2+)-permeable mechanotransducer (MT) channels in hair cell stereociliary bundles. Ca(2+) clearance from bundles was followed in rat outer hair cells (OHCs) using fast imaging of fluorescent indicators. Bundle deflection caused a rapid rise in Ca(2+) that decayed after the stimulus, with a time constant of about 50 ms. The time constant was increased by blocking Ca(2+) uptake into the subcuticular plate mitochondria or by inhibiting the hair bundle plasma membrane Ca(2+) ATPase (PMCA) pump. Such manipulations raised intracellular Ca(2+) and desensitized the MT channels. Measurement of the electrogenic PMCA pump current, which saturated at 18 pA with increasing Ca(2+) loads, indicated a maximum Ca(2+) extrusion rate of 3.7 fmol x s(-1). The amplitude of the Ca(2+) transient decreased in proportion to the Ca(2+) concentration bathing the bundle and in artificial endolymph (160 mM K(+), 20 microM Ca(2+)), Ca(2+) carried 0.2% of the MT current. Nevertheless, MT currents in endolymph displayed fast adaptation with a submillisecond time constant. In endolymph, roughly 40% of the MT current was activated at rest when using 1 mM intracellular BAPTA compared with 12% with 1 mM EGTA, which enabled estimation of the in vivo Ca(2+) load as 3 pA at rest. The results were reproduced by a model of hair bundle Ca(2+) diffusion, showing that the measured PMCA pump density could handle Ca(2+) loads incurred from resting and maximal MT currents in endolymph. The model also indicated the endogenous mobile buffer was equivalent to 1 mM BAPTA.

Computational models of hair cell bundle mechanics: III. 3-D utricular bundles

Six utricular hair bundles from a red-eared turtle are modeled using 3-D finite element analysis. The mechanical model includes shear deformable stereocilia, realignment of all forces during force load increments, and tip and lateral link inter-stereocilia connections. Results show that there are two distinct bundle types that can be separated by mechanical bundle stiffness. The more compliant group has fewer total stereocilia and short stereocilia relative to kinocilium height; these cells are located in the medial and lateral extrastriola. The stiff group are located in the striola. They have more stereocilia and long stereocilia relative to kinocilia heights. Tip link tensions show parallel behavior in peripheral columns of the bundle and serial behavior in central columns when the tip link modulus is near or above that of collagen (1x10(9) N/m(2)). This analysis shows that lumped parameter models of single stereocilia columns can show some aspects of bundle mechanics; however, a distributed, 3-D model is needed to explore overall bundle behavior.

Force Transmission in the Organ of Corti Micromachine

Auditory discrimination is limited by the performance of the cochlea whose acute sensitivity and frequency tuning are underpinned by electromechanical feedback from the outer hair cells. Two processes may underlie this feedback: voltage-driven contractility of the outer hair cell body and active motion of the hair bundle. Either process must exert its mechanical effect via deformation of the organ of Corti, a complex assembly of sensory and supporting cells riding on the basilar membrane. Using finite element analysis, we present a three-dimensional model to illustrate deformation of the organ of Corti by the two active processes. The model used available measurements of the properties of structural components in low-frequency and high-frequency regions of the rodent cochlea. The simulations agreed well with measurements of the cochlear partition stiffness, the longitudinal space constant for point deflection, and the deformation of the organ of Corti for current injection, as well as displaying a 20-fold increase in passive resonant frequency from apex to base. The radial stiffness of the tectorial membrane attachment was found to be a crucial element in the mechanical feedback. Despite a substantial difference in the maximum force generated by hair bundle and somatic motility, the two mechanisms induced comparable amplitudes of motion of the basilar membrane but differed in the polarity of their feedback on hair bundle position. Compared to the hair bundle motor, the somatic motor was more effective in deforming the organ of Corti than in displacing the basilar membrane.

Theoretical Conditions for High-Frequency Hair Bundle Oscillations in Auditory Hair Cells

Substantial evidence exists for spontaneous oscillations of hair cell stereociliary bundles in the lower vertebrate inner ear. Since the oscillations are larger than expected from Brownian motion, they must result from an active process in the stereociliary bundle suggested to underlie amplification of the sensory input as well as spontaneous otoacoustic emissions. However, their low frequency (<100 Hz) makes them unsuitable for amplification in birds and mammals that hear up to 5 kHz or higher. To examine the possibility of high-frequency oscillations, we used a finite-element model of the outer hair cell bundle incorporating previously measured mechanical parameters. Bundle motion was assumed to activate mechanotransducer channels according to the gating spring hypothesis, and the channels were regulated adaptively by Ca(2+) binding. The model generated oscillations of freestanding bundles at 4 kHz whose sharpness of tuning depended on the mechanotransducer channel number and location, and the Ca(2+) concentration. Entrainment of the oscillations by external stimuli was used to demonstrate nonlinear amplification. The oscillation frequency depended on channel parameters and was increased to 23 kHz principally by accelerating Ca(2+) binding kinetics. Spontaneous oscillations persisted, becoming very narrow-band, when the hair bundle was loaded with a tectorial membrane mass.

Optimal Electrical Properties of Outer Hair Cells Ensure Cochlear Amplification

The organ of Corti (OC) is the auditory epithelium of the mammalian cochlea comprising sensory hair cells and supporting cells riding on the basilar membrane. The outer hair cells (OHCs) are cellular actuators that amplify small sound-induced vibrations for transmission to the inner hair cells. We developed a finite element model of the OC that incorporates the complex OC geometry and force generation by OHCs originating from active hair bundle motion due to gating of the transducer channels and somatic contractility due to the membrane protein prestin. The model also incorporates realistic OHC electrical properties. It explains the complex vibration modes of the OC and reproduces recent measurements of the phase difference between the top and the bottom surface vibrations of the OC. Simulations of an individual OHC show that the OHC somatic motility lags the hair bundle displacement by ∼90 degrees. Prestin-driven contractions of the OHCs cause the top and bottom surfaces of the OC to move in opposite directions. Combined with the OC mechanics, this results in ∼90 degrees phase difference between the OC top and bottom surface vibration. An appropriate electrical time constant for the OHC membrane is necessary to achieve the phase relationship between OC vibrations and OHC actuations. When the OHC electrical frequency characteristics are too high or too low, the OHCs do not exert force with the correct phase to the OC mechanics so that they cannot amplify. We conclude that the components of OHC forward and reverse transduction are crucial for setting the phase relations needed for amplification.

Underestimated Sensitivity of Mammalian Cochlear Hair Cells Due to Splay between Stereociliary Columns

Current-displacement (I-X) and the force-displacement (F-X) relationships characterize hair-cell mechano-transduction in the inner ear. A common technique for measuring these relationships is to deliver mechanical stimulations to individual hair bundles with microprobes and measure whole cell transduction currents through patch pipette electrodes at the basolateral membrane. The sensitivity of hair-cell mechano-transduction is determined by two fundamental biophysical properties of the mechano-transduction channel, the stiffness of the putative gating spring and the gating swing, which are derived from the I-X and F-X relationships. Although the hair-cell stereocilia in vivo deflect <100 nm even at high sound pressure levels, often it takes >500 nm of stereocilia displacement to saturate hair-cell mechano-transduction in experiments with individual hair cells in vitro. Despite such discrepancy between in vivo and in vitro data, key biophysical properties of hair-cell mechano-transduction to define the transduction sensitivity have been estimated from in vitro experiments. Using three-dimensional finite-element methods, we modeled an inner hair-cell and an outer hair-cell stereocilia bundle and simulated the effect of probe stimulation. Unlike the natural situation where the tectorial membrane stimulates hair-cell stereocilia evenly, probes deflect stereocilia unevenly. Because of uneven stimulation, 1) the operating range (the 10–90% width of the I-X relationship) increases by a factor of 2–8 depending on probe shapes, 2) the I-X relationship changes from a symmetric to an asymmetric function, and 3) the bundle stiffness is underestimated. Our results indicate that the generally accepted assumption of parallel stimulation leads to an overestimation of the gating swing and underestimation of the gating spring stiffness by an order of magnitude.

Power Dissipation in the Subtectorial Space of the Mammalian Cochlea Is Modulated by Inner Hair Cell Stereocilia

The stereocilia bundle is the mechano-transduction apparatus of the inner ear. In the mammalian cochlea, the stereocilia bundles are situated in the subtectorial space (STS)--a micrometer-thick space between two flat surfaces vibrating relative to each other. Because microstructures vibrating in fluid are subject to high-viscous friction, previous studies considered the STS as the primary place of energy dissipation in the cochlea. Although there have been extensive studies on how metabolic energy is used to compensate the dissipation, much less attention has been paid to the mechanism of energy dissipation. Using a computational model, we investigated the power dissipation in the STS. The model simulates fluid flow around the inner hair cell (IHC) stereocilia bundle. The power dissipation in the STS because of the presence IHC stereocilia increased as the stimulating frequency decreased. Along the axis of the stimulating frequency, there were two asymptotic values of power dissipation. At high frequencies, the power dissipation was determined by the shear friction between the two flat surfaces of the STS. At low frequencies, the power dissipation was dominated by the viscous friction around the IHC stereocilia bundle--the IHC stereocilia increased the STS power dissipation by 50- to 100-fold. There exists a characteristic frequency for STS power dissipation, CFSTS, defined as the frequency where power dissipation drops to one-half of the low frequency value. The IHC stereocilia stiffness and the gap size between the IHC stereocilia and the tectorial membrane determine the characteristic frequency. In addition to the generally assumed shear flow, nonshear STS flow patterns were simulated. Different flow patterns have little effect on the CFSTS. When the mechano-transduction of the IHC was tuned near the vibrating frequency, the active motility of the IHC stereocilia bundle reduced the power dissipation in the STS.

Microstructures in the Organ of Corti Help Outer Hair Cells Form Traveling Waves along the Cochlear Coil

According to the generally accepted theory of mammalian cochlear mechanics, the fluid in the cochlear scalae interacts with the elastic cochlear partition to generate transversely oscillating displacement waves that propagate along the cochlear coil. Using a computational model of cochlear segments, a different type of propagating wave is reported, an elastic propagating wave that is independent of the fluid-structure interaction. The characteristics of the propagating wave observed in the model, such as the wavelength, speed, and phase lag, are similar to those observed in the living cochlea. Three conditions are required for the existence of the elastic propagating wave in the cochlear partition without fluid-interaction: 1), the stiffness gradient of the cochlear partition; 2), the elastic longitudinal coupling; and 3), the Y-shaped structure in the organ of Corti formed by the outer hair cell, the Deiters cell, and the Deiters cell phalangeal process. The elastic propagating waves in the cochlear partition disappeared without the push-pull action provided by the outer hair cell and Deiters cell phalangeal process. The results suggest that the mechanical feedback of outer hair cells, facilitated by the organ of Corti microstructure, can control the tuning and amplification by modulating the cochlear traveling wave.