A. C. Crawford

An electrical tuning mechanism in turtle cochlear hair cells

1. Intracellular recordings were made from single cochlear hair cells in the isolated half‐head of the turtle. The electrical responses of the cells were recorded under two conditions: (a) when the ear was stimulated with low‐intensity tones of different frequencies and (b) when current steps were injected through the intracellular electrode. The aim of the experiments was to evaluate the extent to which the cochleas frequency selectivity could be accounted for by the electrical properties of the hair cells.

THE ACTIONS OF CALCIUM ON THE MECHANO-ELECTRICAL TRANSDUCER CURRENT OF TURTLE HAIR CELLS

1. 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 currents kinetics or reversal potential, or on the current‐displacement relationship, were noted. 3. If the Ca2+ concentration was lowered to 50 microM (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 microM‐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 microM‐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 microM‐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. 8. Following treatment with 1 microM‐Ca2+, single‐channel currents with an amplitude of ‐9 pA at ‐85 mV were sometimes visible in the whole‐cell recording. The probability of such channels being open could be modulated by small deflections of the hair bundle which indicates that they may be the mechano‐electrical transducer channels or conductance about 100 pS. 9. Open‐ and closed‐time distributions for the channel were fitted by single exponentials, the mean open time at rest being approximately 1 ms. The mean open time was increased and the mean closed time decreased for movements of the hair bundle towards the kinocilium.(ABSTRACT TRUNCATED AT 400 WORDS)

Force generation by mammalian hair bundles supports a role in cochlear amplification

It is generally accepted that the acute sensitivity and frequency discrimination of mammalian hearing requires active mechanical amplification of the sound stimulus within the cochlea. The prevailing hypothesis is that this amplification stems from somatic electromotility of the outer hair cells attributable to the motor protein prestin. Thus outer hair cells contract and elongate in synchrony with the sound-evoked receptor potential. But problems arise with this mechanism at high frequencies, where the periodic component of the receptor potential will be attenuated by the membrane time constant. On the basis of work in non-mammalian vertebrates, force generation by the hair bundles has been proposed as an alternative means of boosting the mechanical stimulus. Here we show that hair bundles of mammalian outer hair cells can also produce force on a submillisecond timescale linked to adaptation of the mechanotransducer channels. Because the bundle motor may ultimately be limited by the deactivation rate of the channels, it could theoretically operate at high frequencies. Our results show the existence of another force generator in outer hair cells that may participate in cochlear amplification.

Activation and adaptation of transducer currents in turtle hair cells

1. Transducer currents were recorded in turtle cochlear hair cells during mechanical stimulation of the hair bundle. The currents were measured under whole‐cell 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 microns, 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 degrees 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.

The frequency selectivity of auditory nerve fibres and hair cells in the cochlea of the turtle

1. The electrical responses of single auditory nerve fibres or cochlear hair cells were recorded in the isolated half‐head of the turtle Pseudemys scripta elegans. Responses to sound stimuli presented to the tympanum could be recorded for at least 4 hr after isolation.

Fast adaptation of mechanoelectrical transducer channels in mammalian cochlear hair cells.

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.

Potassium currents in inner hair cells isolated from the guinea‐pig cochlea.

1. Inner hair cells were mechanically isolated from the apical, low‐frequency region of the guinea‐pig cochlea and maintained by superfusion with tissue‐culture medium. Membrane currents were studied under voltage clamp, using the whole‐cell recording mode of the patch‐clamp technique. 2. The cells were studied mostly at 35‐38 degrees C to obtain realistic kinetics of the currents, relevant to the functioning of these cells in vivo. 3. Isolated inner hair cells had resting potentials of about ‐65 mV. Depolarizing voltage steps from a holding potential of about ‐80 mV resulted in large time‐ and voltage‐dependent outward currents. Hyperpolarizing voltage steps from the same holding potential only showed a small leakage conductance of 0.5‐2.5 nS. 4. On repolarization to different membrane potentials, the tail currents reversed around ‐75 mV. This indicates that the outward currents were mainly carried by potassium ions. 5. Pharmacological dissection of the currents provided evidence for two different potassium conductances. The largest conductance had extremely fast kinetics. Its principal time constant of activation was about 0.15‐0.35 ms, the faster values being obtained for larger depolarizations. This fast potassium conductance was blocked by 25 mM‐tetraethylammonium chloride in the bath. 6. A smaller, slow potassium conductance, with principal time constants of activation of 2‐10 ms (speeding up with depolarization), was blocked by 10‐15 mM‐4‐aminopyridine in the patch pipette. 7. Both potassium conductances were activated over the membrane potential range of about ‐60 to ‐20 mV. This is approximately the same as the range of the receptor potential measured in vivo. Therefore these conductances should influence the properties of the receptor potential in inner hair cells. 8. Current injection experiments showed two main effects of the potassium conductances: (a) a non‐linearity in the voltage‐current relationships; (b) a strongly damped oscillation of the membrane potential in response to a large step of outward current. This oscillatory behaviour is caused by the fast potassium conductance.

Tonotopic variation in the conductance of the hair cell mechanotransducer channel.

Hair cells in the vertebrate cochlea are arranged tonotopically with their characteristic frequency (CF), the sound frequency to which they are most sensitive, changing systematically with position. Single mechanotransducer channels of hair cells were characterized at different locations in the turtle cochlea. In 2.8 mM external Ca2+, the channels chord conductance was 118 pS (range 80-163 pS), which nearly doubled (range 149-300 pS) on reducing Ca2+ to 50 microM. In both Ca2+ concentrations, the conductance was positively correlated with hair cell CF. Variation in channel conductance can largely explain the increases in size of the macroscopic transducer current and speed of adaptation with CF. It suggests diversity of transducer channel structure or environment along the cochlea that may be an important element of its tonotopic organization.

The Transduction Channel Filter in Auditory Hair Cells

In the first step in auditory transduction, sound-induced vibrations of the stereociliary bundles on the sensory hair cells are converted into electrical signals by opening of mechanotransducer channels. Faithful transduction and hence auditory performance will be limited by the kinetic properties of these channels. We have measured the time course of mechanotransducer currents in turtle and rat auditory hair cells during rapid deflections of the hair bundle. Current activation in the turtle had a time constant that decreased 10-fold with stimulus amplitude to a limiting value of ∼50 μs. Lowering the external Ca2+ concentration slowed both activation and adaptation time constants. Similar effects were seen in hair cells tuned to low and high frequencies, but the overall kinetics was slower in low-frequency cells. In rat outer hair cells, the time courses of both activation and adaptation were at least 10-fold faster. Although activation kinetics was too fast to characterize accurately, the adaptation time constants in the rat, like the turtle, were Ca2+ dependent and faster in hair cells tuned to higher frequencies. The results imply that mechanotransducer channels operate similarly in turtle and rat but are faster in the mammal to accommodate its higher frequency range of hearing. We suggest that the kinetics of channel activation and adaptation imposes a bandpass filter on transduction, with a center frequency matched to the frequencies detected by the hair cell, which may improve the signal-to-noise ratio near threshold.

Non-linearities in the responses of turtle hair cells

1. Intracellular recordings were made from single cochlear hair cells in the isolated half‐head of the turtle. Receptor potentials were recorded while the ear was stimulated with high‐intensity tones in order to examine the cochlear non‐linearities which shape the hair cell responses.