Michael G. Evans

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)

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.

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 hair cells isolated from the cochlea of the chick

1. Potassium currents were characterized in tall hair cells of the chicks cochlea. Outward potassium currents were found to flow through two distinct classes of channels. 2. Individual hair cells were isolated from 200 microns long segments of the apical half of the chicks cochlea. Whole‐cell voltage‐clamp and current‐clamp recordings were made from these cells. 3. Voltage responses to injected current ranged from high‐frequency (100‐250 Hz) oscillations in some cells, to slowly repetitive Ca2+ action potentials or slow oscillations (5‐20 Hz) in others. 4. Ionic currents recorded in voltage clamp also varied in different hair cells. Cells with high‐frequency voltage oscillations had rapidly activating Ca2(+)‐dependent outward K+ current, IK(Ca). Cells that generated action potentials had slow delayed rectifier outward K+ current, IK, and inward rectifier current, IIR. All hair cells had inward Ca2+ current. 5. IK(Ca) activated positive to ‐45 mV. Tail currents reversed at the K+ equilibrium potential. This current was eliminated in Ca2(+)‐free solutions, or when exposed to 10 mM‐TEA. This outward current was fully activated within 1‐3 ms at 0 mV. The whole‐cell current was noisy and ensemble variance analysis suggested a single‐channel conductance of 63 pS near 0 mV. 6. IK activated positive to ‐50 mV. Tail currents reversed at the K+ equilibrium potential. This current was not eliminated in Ca2(+)‐free solutions, and was relatively resistant to external TEA. IK activated slowly, reaching peak values in 10‐20 ms at 0 mV. This current showed little variance and the average single‐channel conductance based on macroscopic noise near 0 mV was 8 pS. 7. External tetraethylammonium (TEA) or Ca2(+)‐free saline eliminated the high‐frequency voltage oscillations seen in many basal cells. In contrast TEA had little effect on slow action potentials (or low‐frequency oscillations) seen in cells with IK. 8. IK(Ca) was prominent in hair cells originating 1.0‐2.0 mm from the cochlear apex. IK and IIR dominated the membrane conductance of tall hair cells originating within 0.5 mm of the cochlear apex. 9. The frequency of voltage oscillation in apical cells was temperature‐dependent, nearly doubling for each 10 degrees C rise in temperature. 10. IIR activated at membrane potentials negative to ‐75 mV. The average time constant of activation at ‐100 mV was 2 ms. Tail currents reversed at the K+ equilibrium potential and did not depend on the external Na+ concentration. IIR was blocked by 5 mM‐Cs+ or 100 microM‐Ba2+ in the external saline.

A Large-Conductance Calcium-Selective Mechanotransducer Channel in Mammalian Cochlear Hair Cells

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.

Acetylcholine activates two currents in guinea-pig outer hair cells.

1. Whole‐cell voltage clamp recordings were made from outer hair cells (OHCs) isolated from the basal and middle regions of the cochlea. The current produced when acetylcholine (ACh) was applied to the basal pole was investigated. 2. Acetylcholine (50‐70 microM) activated an early inward current followed by a late outward current in most cells at ‐70 mV. The activation of the early current was very rapid, with no delay. 3. The late outward current was a K+ current and was the major component of the total ACh‐sensitive current at negative voltages. 4. The ACh‐sensitive current was voltage dependent. From tail current measurements, the current was maximal at ‐55 mV and declined steeply at more positive potentials. On average, 50% of the current was active at ‐22 mV and 10% was active at 0 mV. This decline was caused by a reduction in the K+ current with depolarization. Between +20 and +40 mV, the major component of the ACh‐sensitive current was the early current. 5. The steady‐state I‐V curve for the ACh‐sensitive current was N‐shaped, with a peak at ‐36 mV. The instantaneous I‐V curve for the ACh‐sensitive current taken from measurements just after a voltage step was outwardly rectifying and did not show this peak. This difference occurred because the voltage dependence was time dependent. 6. The reversal potential for the early current was estimated to be close to+13 mV, in accordance with it being a non‐specification current. 7. The K+ current was abolished by the removal of external Ca2+. The effect occurred with no measurable delay. When external Ca2+ was lowered to 0.4 mM, the peak of the steady‐state I‐V curve for the ACh‐sensitive current shifted by up to ‐20 mV and its amplitude was reduced. These results suggested that the K+ current was dependent on Ca2+ influx. 8. The inward current usually remained when the K+ current was abolished or greatly reduced by removing external Ca2+ or by dialysing the cells with BAPTA (5‐10 mM). 9. Both early and late currents were reversibly blocked by alpha‐bungarotoxin (0.2 microM) and curare (1 microM). 10. A simple scheme is proposed to account for the response. ACh, binding to a nicotinic receptor, directly gates the early cation current. Part of this current is carried by Ca2+, which once inside the cell leads to the activation of a Ca(2+)‐dependent K+ current.

Depolarization of cochlear outer hair cells evokes active hair bundle motion by two mechanisms.

There is current debate about the origin of mechanical amplification whereby outer hair cells generate force to augment the sensitivity and frequency selectivity of the mammalian cochlea. To distinguish contributions to force production from the mechanotransducer (MET) channels and somatic motility, we have measured hair bundle motion during depolarization of individual outer hair cells in isolated rat cochleas. Depolarization evoked rapid positive bundle deflections that were reduced by perfusion with the MET channel blocker dihydrostreptomycin, with no effect on the nonlinear capacitance that is a manifestation of prestin-driven somatic motility. However, the movements were also diminished by Na salicylate and depended on the intracellular anion, properties implying involvement of the prestin motor. Furthermore, depolarization of one outer hair cell caused motion of neighboring hair bundles, indicating overall motion of the reticular lamina. Depolarization of solitary outer hair cells caused cell-length changes whose voltage-activation range depended on the intracellular anion but were insensitive to dihydrostreptomycin. These results imply that both the MET channels and the somatic motor participate in hair bundle motion evoked by depolarization. It is conceivable that the two processes can interact, a signal from the MET channels being capable of modulating the activity of the prestin motor.

Voltage oscillations and ionic conductances in hair cells isolated from the alligator cochlea

SummaryTall hair cells were isolated by enzymatic and mechanical dissociation from selected regions of the apical half of the alligator (A. mississippiensis) cochlea. Single cells were subjected to voltage-clamp and current-clamp using the tight-seal whole-cell recording technique. Most hair cells isolated from the apex of the cochlea produced slowly regenerative depolarizations or Na action potentials during current injection, whereas hair cells isolated from more basal regions usually produced voltage oscillations (ringing) in response to depolarizing current injection, an indication of electrical resonance. Resonant frequencies ranged from 50 to 157 Hz in different cells. The higher-frequency cells tended to have larger and more rapidly activating outward currents than did the lower-frequency cells. An inward Ca current and an outward Ca-activated K current were present in all hair cells. In addition, an inwardly rectifying current and a small, transient outward current were often seen. Thus, we conclude that an electrical tuning mechanism is present in alligator hair cells. The role of the ionic conductances in shaping hair cell responses to current injection, and the possible contributions of these electrical responses to cochlear function are discussed.

Tetrodotoxin-sensitive, voltage-dependent sodium currents in hair cells from the alligator cochlea

We have used whole-cell patch clamp techniques to record from tall hair cells isolated from the apical half of the alligator cochlea. Some of these cells gave action potentials in response to depolarizing current injections. When the same cells were voltage clamped, large transient inward currents followed by smaller outward currents were seen in response to depolarizing steps. We studied the transient inward current after the outward current had been blocked by external tetraethylammonium (20 mM) or by replacing internal potassium with cesium. It was found to be a sodium current because it was abolished by either replacing external sodium with choline or by external application of tetrodotoxin (100 nM). The sodium current showed voltage-dependent activation and inactivation. Most of the spiking hair cells came from the apex of the cochlea, where they would be subject to low-frequency mechanical stimulation in vivo.

Language Awareness in a Bidialectal Setting: The Oral Performance and Language Attitudes of Urban and Rural Students in Cyprus

This study addresses bidialectism in the context of language education by empirically assessing how explicit knowledge about language influences bidialectal students’ linguistic performance and language attitudes. A language-learning programme based on Language Awareness was applied in the bidialectal community of Cyprus with the primary aim of improving oral performance in urban and rural speakers’ second variety, the standard. Improvement was defined as a reduction of Cypriot dialectal interference in students’ Standard Modern Greek speech. A second aim was to document and subsequently identify changes in students’ language attitudes towards their two linguistic varieties. Quantitative analyses of the results reveal that the Language Awareness programme produced a marked improvement in students’ oral production of the standard variety and in their language attitudes.