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.

SummaryOuter hair cells (OHCs) provide amplification in the mammalian cochlea using somatic force generation underpinned by voltage-dependent conformational changes of the motor protein prestin. However, prestin must be gated by changes in membrane potential on a cycle-by-cycle basis and the periodic component of the receptor potential may be greatly attenuated by low-pass filtering due to the OHC time constant (τm), questioning the functional relevance of this mechanism. Here, we measured τm from OHCs with a range of characteristic frequencies (CF) and found that, at physiological endolymphatic calcium concentrations, approximately half of the mechanotransducer (MT) channels are opened at rest, depolarizing the membrane potential to near −40 mV. The depolarized resting potential activates a voltage-dependent K+ conductance, thus minimizing τm and expanding the membrane filter so there is little receptor potential attenuation at the cell's CF. These data suggest that minimal τm filtering in vivo ensures optimal activation of prestin.

SUMMARY1. Hair cells were enzymatically isolated from identified regions of the turtle basilar papilla and studied with the patch-electrode technique. The experimental aim was to relate the resonance properties seen during current injection to the membrane currents measured in the same cell under whole-cell voltage clamp.2. Solitary hair cells had resting potentials of about -50 mV, and produced a damped oscillation in membrane potential at the onset and termination of a small current step; the resonant frequency varied from 9 to 350 Hz between cells, and was correlated with the region of papilla from which a cell had been isolated. The inferred frequency map was consistent with the tonotopic arrangement described previously in the intact papilla. J. J. ART AND R. FETTIPLACE K+ current, the Ca2+ current was activated by small depolarizations from the resting potential, and over this voltage range it was about five to ten times smaller than the K+ current. Its activation was more rapid than the fastest outward currents in high-frequency cells.8. The inward current could also be carried by Ba2+, which when substituted for external Ca2+ blocked the K+ current. Measurements on cells with resonant frequencies of 13-240 Hz indicated that the peak Ba2+ current increased systematically with resonant frequency.9. Manipulations such as external addition of Cd2+ which would be expected to reduce or abolish the Ca2+ current also blocked the K+ current, consistent with a previous suggestion (Lewis & Hudspeth, 1983b) that the hair-cell K+ conductance is gated by changes in intracellular Ca2+.10. Small steady depolarizations caused a pronounced increase in current fluctuations. The spectral density of the fluctuations in a given cell could be well fitted by the sum of two Lorentzians with half-power frequencies differing by an order of magnitude.11. Both half-power frequencies changed with the resonant frequency of the hair cell, and the lower half-power frequency was consistent with that predicted from the time constant of the current relaxation. The variance-to-mean ratio for the fluctuations was about 1 pA in all cells. It is suggested the the fluctuations are dominated by the opening and closing of the K+ channels, and that the intrinsic kinetics of these channels differ in cells with different resonant frequencies.12. Single K+ channels recorded in the cell-attached mode could be opened by depolarizations of the membrane under the patch electrode. At the start and end of a depolarizing step, the probability of channel opening rose and fell with an exponential time course, the time constant varying from 2 to 20 ms in different cells.13. The results support the following conclusions: (i) the resonance behaviour and tuning of turtle cochlear hair cells are governed by the interplay of membrane Ca2+and K+ conductances; (ii) the resonant frequency is determined by the characteristics of the K+ conductance, an increase in frequency being achieved largely by faster kinetics, but also to some extent by an increase in the size of ...

Mechanosensory hair cells of the vertebrate inner ear contribute to acoustic tuning through feedback processes involving voltage-gated channels in the basolateral membrane and mechanotransduction channels in the apical hair bundle. The specific number and kinetics of calcium-activated (BK) potassium channels determine the resonant frequency of electrically tuned hair cells. Kinetic variation among BK channels may arise through alternative splicing of slo gene mRNA and combination with modulatory beta subunits. The number of transduction channels and their rate of adaptation rise with hair cell response frequency along the cochlea's tonotopic axis. Calcium-dependent feedback onto transduction channels may underlie active hair bundle mechanics. The relative contributions of electrical and mechanical feedback to active tuning of hair cells may vary as a function of sound frequency.

SUMMARY1. The mechanical behaviour of the ciliary bundles of hair cells in the turtle cochlea was examined by deflecting them with flexible glass fibres ofknown compliance during simultaneous intracellular recording of the cell's membrane potential.2. Bundle motion was monitored through the attached fibre partially occluding a light beam incident on a photodiode array. The change in photocurrent was assumed to be proportional to bundle displacement.3. For deflexions of 1-100 nm towards the kinocilium, the stiffness of the ciliary bundles was estimated as about 6 x 10-4 N/m, with the fibre attached to the top of the bundle.4. When the fibre was placed at different positions up the bundle, the stiffness decreased approximately as the inverse square of the distance from the ciliary base. This suggests that the bundles rotate about an axis close to the apical pole of the cell and have a rotational stiffness of about 2 x 10-14 N. m/rad. 5.Step displacements of the fixed end of the flexible fibre caused the hair cell's membrane potential to execute damped oscillations; the frequency of the oscillations in different cells ranged from 20 to 320 Hz. Displacements towards the kinocilium always produced membrane depolarization.6. The amplitude of the initial oscillation increased with displacements up to 100 nm and then saturated. For small displacements of a few nanometres, the hair cell's mechanoelectrical sensitivity was estimated as about 0-2 mV/nm. 7. Force steps delivered by the flexible fibre caused the bundle position to undergo damped oscillations in synchrony with the receptor potential. The mechanical oscillations could be abolished with large depolarizing currents that attenuated the receptor potential.8. When placed against a bundle, a fibre's spontaneous motion increased and became quasi-sinusoidal with an amplitude several times that expected from the compliance of the system. It is suggested that the hair bundle drives the fibre.9. We conclude that turtle cochlear hair cells contain an active force generating mechanism.