SUMMARY1. Transducer currents were recorded in turtle cochlear hair cells during mechanical stimulation of the hair bundle. The currents were measured under wholecell voltage clamp in isolated cells that were firmly stuck to the floor of the recording chamber.2. Stimuli were calibrated by projecting the image of the hair bundle onto a rapidly scanned 128 photodiode array. This technique showed that, while the cell body was immobilized, the tip of the bundle would follow faithfully the motion of an attached glass probe up to frequencies of more than 1 kHz.3. The relationship between inward transducer current and bundle displacement was sigmoidal. Maximum currents of 200-400 pA were observed for deflections of the tip of the bundle of 0 5 ,tm, equivalent to rotating the bundle by about 5 deg.4. In response to a step deflection of the bundle, the current developed with a time constant (about 0 4 ms for small stimuli) that decreased with the size of displacement. This suggests that the onset of the current was limited by the gating kinetics of the transduction channel. The onset time course was slowed about fourfold for a 20 'C drop in temperature.5. For small maintained displacements, the current relaxed to about a quarter of the peak level with a time constant of 3-5 ms. This adaptation was associated with a shift of the current-displacement relationship in the direction of the stimulus. The rate and extent of adaptation were decreased by lowering external Ca2+.6. Adaptation was strongly voltage sensitive, and was abolished at holding potentials positive to the reversal potential of the transducer current of about 0 mV. It was also diminished by loading cells with 10 mm of the Ca2+ chelator BAPTA. These observations suggest that adaptation may be partly controlled by influx of Ca2+ through the transducer channels.7. Removal of adaptation produced asymmetric responses, with fast onsets but slow decays following return of the bundle to its resting position; the offset time course depended on both the magnitude and duration of the prior displacement.8. In some experiments, hair bundles were deflected with a flexible glass fibre whose motion was monitored using a dual photodiode arrangement. Positive holding potentials abolished adaptation of the transducer currents, but had no influence on the time course of motion of the fibre. We have no evidence therefore that adaptation is caused by a mechanical reorganization within the bundle.NIS 77W0

SUMMARY1. Mechano-electrical transducer currents evoked by deflections of the hair bundle were recorded in turtle isolated hair cells under whole-cell voltage clamp. The outcome of perfusing with solutions of reduced Ca2+ concentration was investigated.2. The transducer current was roughly doubled by lowering the concentration of divalent cations from normal (2-2 mM-Mg2+, 2-8 mM-Ca2+) to 0 Mg2+, 0 5 mM-Ca2+. No significant effects on the current's kinetics or reversal potential, or on the currentdisplacement relationship, were noted.3. If the Ca2+ concentration was lowered to 50 ,UM (with no Mg2+), there was about a threefold increase in the maximum current but other changes, including loss of adaptation and a decreased slope and negative shift in the current-displacement relationship, were also observed. As a result, more than half the peak transducer current became activated at the resting position of the hair bundle compared to about a tenth in the control solution.4. The extra changes manifest during perfusion with 50 /LM-Ca2+ had also been seen when the cell was held at positive potentials near the Ca2+ equilibrium potential. This supports the view that some consequences of reduced external Ca2+ stem from a decline in its intracellular concentration.5. With 20,tM-Ca2+, a standing inward current developed and the cell became unresponsive to mechanical stimuli, which may be explained by the transducer channels being fully activated at the resting position of the bundle.6. The results are interpreted in terms of a dual action of Ca2+: an external block of the transducer channel which reduces the maximum current, and an intracellular effect on the position and slope of the current-displacement relationship; the latter effect can be modelled by internal Ca2+ stabilizing one of the closed states of the channel.7. During perfusion with 1 gM-Ca2 , the holding current transiently increased but then returned to near its control level. There was a concomitant irreversible loss of sensitivity to hair bundle displacements which we suggest is due to rupture of the mechanical linkages to the transducer channel.

Sound stimuli are detected in the cochlea by opening of hair cell mechanotransducer (MT) channels, one of the few ion channels not yet conclusively identified at a molecular level. To define their performance in situ, we measured MT channel properties in inner hair cells (IHCs) and outer hair cells (OHCs) at two locations in the rat cochlea tuned to different characteristic frequencies (CFs). The conductance (in 0.02 mM calcium) of MT channels from IHCs was estimated as 260 pS at both low-frequency and mid-frequency positions, whereas that from OHCs increased with CFs from 145 to 210 pS. The combination of MT channel conductance and tip link number, assayed from scanning electron micrographs, accounts for variation in whole-cell current amplitude for OHCs and its invariance for IHCs. Channels from apical IHCs and OHCs having a twofold difference in unitary conductance were both highly calcium selective but were distinguishable by a small but significant difference in calcium permeability and in their response to lowering ionic strength. The results imply that the MT channel has properties possessed by few known candidates, and its diversity suggests expression of multiple isoforms.

Outer hair cells are centrally involved in the amplification and frequency tuning of the mammalian cochlea, but evidence about their transducing properties in animals with fully developed hearing is lacking. Here we describe measurements of mechanoelectrical transducer currents in outer hair cells of rats between postnatal days 5 and 18, before and after the onset of hearing. Deflection of hair bundles using a new rapid piezoelectric stimulator evoked transducer currents with ultra-fast activation and adaptation kinetics. Fast adaptation resembled the same process in turtle hair cells, where it is regulated by changes in stereociliary calcium. It is argued that sub-millisecond transducer adaptation can operate in outer hair cells under the ionic, driving force and temperature conditions that prevail in the intact mammalian cochlea.

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