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

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.

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.

Ototoxicity, noise overstimulation, or aging, can all produce hearing loss with similar properties, in which outer hair cells (OHCs), principally those at the high-frequency base of the cochlea, are preferentially affected. We suggest that the differential vulnerability may partly arise from differences in Ca2+ balance among cochlear locations. Homeostasis is determined by three factors: Ca2+ influx mainly via mechanotransducer (MET) channels; buffering by calcium-binding proteins and organelles like mitochondria; and extrusion by the plasma membrane CaATPase pump. We review quantification of these parameters and use our experimentally-determined values to model changes in cytoplasmic and mitochondrial Ca2+ during Ca2+ influx through the MET channels. We suggest that, in OHCs, there are two distinct micro-compartments for Ca2+ handling, one in the hair bundle and the other in the cell soma. One conclusion of the modeling is that there is a tonotopic gradient in the ability of OHCs to handle the Ca2+ load, which correlates with their vulnerability to environmental challenges. High-frequency basal OHCs are the most susceptible because they have much larger MET currents and have smaller dimensions than low-frequency apical OHCs.

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.