29 results on '"FETTIPLACE, R."'
Search Results
2. The role of Ca2+-activated K+ channel spliced variants in the tonotopic organization of the turtle cochlea.
- Author
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Jones, E. M. C., Gray-Keller, M., and Fettiplace, R.
- Published
- 1999
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3. Calcium permeation of the turtle hair cell mechanotransducer channel and its relation to the composition of endolymph.
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Ricci, A. J. and Fettiplace, R.
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- 1998
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4. The effects of calcium buffering and cyclic AMP on mechano-electrical transduction in turtle auditory hair cells.
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Ricci, A. J. and Fettiplace, R.
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- 1997
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5. The role of Ca2+‐activated K+channel spliced variants in the tonotopic organization of the turtle cochlea
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Jones, E. M. C., Gray‐Keller, M., and Fettiplace, R.
- Abstract
1Turtle auditory hair cells contain multiple isoforms of the pore‐forming α‐subunit of the large‐conductance Ca2+‐activated K+(KCa) channel due to alternative splicing at two sites. Six splice variants were studied by expression in Xenopusoocytes.2The isoforms possessed differences in apparent Ca2+sensitivity and kinetics. The lowest Ca2+sensitivity was observed in a novel variant resulting from a 26 amino acid deletion around one of the splice sites.3Co‐expression of a bovine β‐subunit slowed the current relaxation 10‐fold compared with channels formed from α‐subunits alone but preserved the original order of kinetic differences. The β‐subunit also increased the Ca2+sensitivity of isoforms to bring them nearer the range of sensitivity of the native KCachannels of the hair cell.4With channels formed from α‐subunits or α+β‐subunits, the half‐activation voltage in a fixed Ca2+concentration, and the time constant of the current relaxation, varied linearly with the combined size of the insertions/deletions at the splice sites.5Experiments in which the β/α concentration ratio was varied indicated that the β‐subunit exerts an all‐or‐none effect on the Ca2+sensitivity and kinetics of the channel.6Co‐expression of an avian β2‐subunit had effects on kinetics and Ca2+sensitivity of several α‐isoforms which were qualitatively similar to those produced by the bovine β‐subunit.7We conclude that differential expression of alternatively spliced α‐subunit variants and a non‐uniform distribution of a β‐subunit can produce a range of KCachannel properties needed to explain the tonotopic organization of the turtle cochlea.
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- 1999
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6. Activation and adaptation of transducer currents in turtle hair cells.
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Crawford, A C, Evans, M G, and Fettiplace, R
- Abstract
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.
- Published
- 1989
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7. The effects of low calcium on the voltage‐dependent conductances involved in tuning of turtle hair cells.
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Art, J J, Fettiplace, R, and Wu, Y C
- Abstract
1. The voltage‐dependent conductances of turtle cochlear hair cells of known resonant frequency were characterized by tight‐seal, whole‐cell recording during superfusion with solutions containing normal (2.8 mM) and reduced (0.1‐10 microM) Ca2+. 2. In 1 microM Ca2+, the current flowing through the voltage‐dependent Ca2+ channels was increased roughly fivefold and had a reversal potential near 0 mV. This observation may be explained by the Ca2+ channels becoming non‐selectively permeable to monovalent cations in low‐Ca2+ solutions. Lowering the Ca2+ further to 0.1 microM produced little increase in the current. 3. The size of the non‐selective current increased systematically with the resonant frequency of the hair cell over the range from 10 to 320 Hz. This suggests that hair cells tuned to higher frequencies contain more voltage‐dependent Ca2+ channels. 4. There was a good correlation between the amplitudes of the non‐selective current and the K+ current which underlies electrical tuning of these hair cells. The amplitude of the K+ current also increased systematically with resonant frequency. 5. In cells with resonant frequencies between 120 and 320 Hz, the K+ current was completely abolished in 1 microM Ca2+, consistent with prior evidence that this current flows through Ca2+ activated K+ channels. In a majority of cells tuned between 50 and 120 Hz, the K+ current was incompletely blocked in 1 microM Ca2+, but was eliminated in 0.1 microM Ca2+. In all hair cells the K+ current was abolished by 25 mM tetraethylammonium chloride. 6. In cells tuned to 10‐20 Hz, the K+ current was not substantially diminished even in 0.1 microM Ca2+, which argues that it may not be Ca2+ activated. 7. In cells tuned to frequencies above 100 Hz, the K+ current could still be evoked by depolarization during superfusion with 10 microM Ca2+. However, its half‐activation voltage was shifted to more depolarized levels and its maximum amplitude was systematically reduced with increasing resonant frequency. 8. These observations are consistent with the notion that in cells tuned to more than 50 Hz, there is a fixed ratio of the number of voltage‐dependent Ca2+ channels to Ca(2+)‐activated K+ channels, the numbers of each increasing in proportion to resonant frequency. The results also provide indirect evidence that the Ca(2+)‐activated K+ channels in cells tuned to higher frequencies may be less sensitive to intracellular Ca2+.
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- 1993
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8. The frequency selectivity of auditory nerve fibres and hair cells in the cochlea of the turtle
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Crawford, A. C. and Fettiplace, R.
- Abstract
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.
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- 1980
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9. An electrical tuning mechanism in turtle cochlear hair cells
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Crawford, A. C. and Fettiplace, R.
- Abstract
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 cochlea's frequency selectivity could be accounted for by the electrical properties of the hair cells.
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- 1981
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10. Non‐linearities in the responses of turtle hair cells
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Crawford, A. C. and Fettiplace, R.
- Abstract
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.
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- 1981
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11. Synaptic hyperpolarization and inhibition of turtle cochlear hair cells.
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Art, J J, Fettiplace, R, and Fuchs, P A
- Abstract
Intracellular recordings were made from turtle cochlear hair cells in order to examine the properties of the post‐synaptic potentials evoked by electrical stimulation of the efferent axons. Single shocks to the efferents generated a hair cell membrane hyperpolarization with an average amplitude generally less than 1 mV and lasting for about 100 ms. With short trains of shocks, the size of the post‐synaptic potential grew markedly to a maximum of 20‐30 mV. The interaction between pairs of shocks separated by a varying interval was studied. For an interval of 4 ms, the response to the second shock was increased on average by a factor of 3 and the conditioning effect of the first shock decayed with a time constant of about 100 ms. We suggest the augmentation in response to trains of shocks may be partly due to facilitation of efferent transmitter release. The efferent post‐synaptic potentials could be reversibly abolished by perfusion with perilymphs containing 3 microM‐curare or atropine, and infusion of acetylcholine gave a transient membrane hyperpolarization. These observations are consistent with efferent action being mediated via a cholinergic synapse onto the hair cells. The post‐synaptic potentials could be reversed in polarity by injection of hyperpolarizing currents through the recording electrode. The reversal potential was estimated as about ‐80 mV, 30 mV negative to the resting potential. Near reversal, a small brief depolarization was evident and may constitute a minor component of the synaptic response. The value of the reversal potential was unaffected by substitution of the perilymphatic chloride, but was altered in a predictable manner by changes in extracellular potassium concentration indicating that the post‐synaptic potentials arise mainly by an increase in the permeability of the hair cell membrane to potassium ions. Throughout the post‐synaptic hyperpolarization there was a reduction in the sensitivity of the hair cell to tones at its characteristic frequency. The desensitization, maximal for low sound pressures, varied in different cells from a factor of 1.6 to 28. At the peak of the largest synaptic potentials, the receptor potential remained negative to the resting potential with all but the loudest characteristic frequency tone s. We suggest that there are two factors in efferent inhibition; one a r duction in the receptor potential at the hair cell's characteristic frequency and the other a hyperpolarization of its membrane potential which should reduce the release of excitatory transmitter onto the afferent terminals.
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- 1984
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12. Efferent desensitization of auditory nerve fibre responses in the cochlea of the turtle Pseudemys scripta elegans.
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Art, J J and Fettiplace, R
- Abstract
Extracellular recordings were made from single auditory afferents in the isolated half‐head of the turtle, and changes in their acoustic sensitivity were examined following electrical stimulation of the efferent fibres to the basilar papilla. Short trains of efferent shocks caused a prolonged elevation of the pure tone thresholds of the auditory afferents and an abolition of their spontaneous activity. These changes could be demonstrated in a majority of recordings and without antidromic firing of the afferent. The amount of desensitization increased steeply with shock number and a train of ten closely spaced shocks could elevate the threshold at the most sensitive or characteristic frequency by four orders of magnitude. Desensitization also occurred with single efferent shocks at repetition frequencies exceeding 25/s. Discharge rate versus sound pressure functions were constructed for a number of afferents. The maximum slope of the functions, and the saturated firing rates were both reduced by efferent stimulation; there was also an over‐all shift of the rate‐intensity function to higher stimulus levels. Such effects would enable the afferent to signal a wider range of sound pressures. Efferent stimulation caused a broadening of the afferent frequency‐threshold curves by removal of the narrowly‐tuned region around the characteristic frequency. We suggest that the loss in tuning and concomitant improvement in temporal resolution may be a functionally important consequence of efferent action.
- Published
- 1984
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13. Efferent modulation of hair cell tuning in the cochlea of the turtle.
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Art, J J, Crawford, A C, Fettiplace, R, and Fuchs, P A
- Abstract
Intracellular recordings were made from turtle cochlear hair cells in order to study the changes in their tuning properties resulting from electrical stimulation of the efferent axons. Efferent stimulation caused a reduction in the amplitude of the receptor potential at the hair cell's most sensitive or characteristic frequency, an increased amplitude at frequencies more than an octave below the characteristic frequency, and no change at very high frequencies. These differential effects resulted in a broadening of each cell's tuning curve, which, during maximal efferent stimulation degenerated from a sharply tuned resonance to a critically damped low‐pass filter. Efferent alterations in tuning were also inferred from the oscillations in membrane potential produced by acoustic clicks or extrinsic currents. The quality factor (Q) of tuning, derived from the decay of the oscillations, was progressively reduced with synaptic hyperpolarizations up to about 5 mV in amplitude. A consequence of efferent action was that the wave forms of transient pressure changes were more faithfully encoded as changes in hair cell membrane potential. Hyperpolarization of a hair cell by steady current injection resulted in a lowering of its characteristic frequency and quality factor, and an increase in steady‐state resistance. By comparison, for a given reduction in quality factor, efferent stimulation was associated with a smaller change in characteristic frequency. This difference is expected if the resonance is also damped by the shunting action of the synaptic conductance. Perfusion with perilymphs containing 0.5‐15 mM of the potassium channel blocker, tetraethylammonium bromide (TEA) reduced the hair cell's frequency selectivity, whether assayed acoustically or with extrinsic currents. Lower TEA concentrations abolished the efferent inhibitory post‐synaptic potential with only a minor change in tuning. TEA produced other effects different from efferent stimulation including (i) a lowering of the characteristic frequency, and (ii) a highly asymmetric receptor potential. These observations suggest that the efferents do not simply block membrane conductances associated with tuning. We conclude that the efferent modification of the shape of the tuning curve may be a composite result of the synaptic conductance and the hyperpolarization of the hair cell membrane.
- Published
- 1985
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14. The mechanical properties of ciliary bundles of turtle cochlear hair cells.
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Crawford, A C and Fettiplace, R
- Abstract
The mechanical behaviour of the ciliary bundles of hair cells in the turtle cochlea was examined by deflecting them with flexible glass fibres of known compliance during simultaneous intracellular recording of the cell's membrane potential. 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. 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. 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. 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. 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. 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. 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. We conclude that turtle cochlear hair cells contain an active force generating mechanism.
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- 1985
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15. Light path and photon capture in turtle photoreceptors.
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Baylor, D A and Fettiplace, R
- Abstract
1. The directional selectivity of individual cones was examined by intracellular recording in the eye of the turtle. Sensitivites were determined from linear responses to dim flashes of monochromatic light incident on a cell over a range of angles to its long axis. 2. With light near the optimum wave‐length, some red‐ and green‐sensitive cones showed a high sensitivity for light entering axially and lower sensitivities for light entering obliquely. In contrast, other cells had lower peak sensitivities and less pronounced directional selectivities. The highest axial sensitivities observed in red receptors were about 320 muV photon(−1) mu2; in these cells, the sensitivity declined to half for rays 6–9 degrees off the axis as measured in the retina. Green receptors had lower axial sensitivities and broader angular profiles. 3. On the assumption that rays at all angles contribute independently to the over‐all sensitivity, the sensitivity of a cell to large cones of rays was successfully predicted from the angular selectivity determined with a narrow pencil of rays. The shape of small responses to dim stimuli delivered on and off the axis of the cell was invariant, implying that a cone signals the number of photons absorbed but not their angle of incidence. 4. Short wave‐lengths have previously been shown to be filtered out by the oil droplets present in turtle cones. At short wave‐lengths, the angular profiles showed a depression in axial sensitivity consistent with this filtering action. 5. Diameters of inner segments, oil droplets, and outer segments were measured in red‐, green‐, and blue‐sensitive cones, since these dimensions are expected to influence the cones' angular acceptances and ability to collect light. The diameters of the structure were in approximately the same proportions for each type of receptor, but the absolute values of the diameters were found to be scaled in relation to the wave‐length of maximum sensitivity. 6. Optical determinations of the efficiency with which axial rays are concentrated by red receptors gave a mean value of 55%. 7. Receptors in histological sections of the whole eye were found to be oriented with their long axes directed approximately toward the pupil. 8. The observed directional selectivities and collecting efficiencies agree well with the behaviour of a model retinal cone developed by Winston & Enoch (1971) on a geometrical optical treatment. 9. Effective collecting areas are derived for red‐, green‐ and blue‐sensitive cones; these permit conversion of observed flash sensitivities into the mean peak hyperpolarization produced by isomerization of a visual pigment molecule. The figure obtained is about 25 muV for red‐sensitive cones and 21muV for green‐sensitive cones.
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- 1975
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16. Transmission from photoreceptors to ganglion cells in turtle retina
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Baylor, D. A. and Fettiplace, R.
- Abstract
1. Synaptic transfer between photoreceptors and impulse‐generating cells was studied in isolated eyecups from turtles. Single red‐sensitive cones or rods were stimulated by current passed through an intracellular electrode, and impulses generated by the resulting synaptic action were recorded with an external micro‐electrode. This technique permits study of retinal transmission without the operation of the visual transduction mechanism. Antidromic stimulation of the optic nerve indicated that most of the impulse‐generating cells were ganglion cells.
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- 1977
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17. Kinetics of synaptic transfer from receptors to ganglion cells in turtle retina
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Baylor, D. A. and Fettiplace, R.
- Abstract
1. Synaptic transfer between the retinal input and output was studied in turtle eyecups by injecting rectangular current pulses into a single cone or rod while recording externally from a ganglion cell.
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- 1977
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18. Synaptic drive and impulse generation in ganglion cells of turtle retina.
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Baylor, D A and Fettiplace, R
- Abstract
1. Light reponses and electrical constants of ganglion cells in the retina of the turtle were examined by intracellular recording in eyecup preparations. 2. In 'on', 'off', and 'on/off' cells, the impulses produced by illumination of the centre of the receptive field arose from slow synaptic depolarizations. The ganglion cells also exhibited inhibitory synaptic potentials. 3. The synaptic depolarization evoked by a step change in light intensity rose more slowly than the response of the cones in which the excitation originated, and the depolarization then declined in spite of a well maintained cone response. This behaviour is consistent with the notion advanced previously that, during transmission to ganglion cells, receptor signals are relayed through the equivalent of a bandpass filter. 4. The e.p.s.p.s evoked by light grew when the membrane was hyperpolarized by injected current and decreased when the membrane was depolarized. The i.p.s.p.s reversed at a level slightly negative to the resting potential in darkness. 5. In neither 'on' nor 'off' ganglion cells did the synaptic potentials evoked by step changes in illumination show the hyperpolarizing phases expected of a linear filter. The absence of hyperpolarizations is consistent with a rectification which permits transmission of depolarizations but not hyperpolarizations from bipolar to ganglion cells. 6. In darkness the membrane potential of some ganglion cells showed random depolarizations which brought the potential near the threshold for impulse generation. 7. With very small spots in the receptive field centre the 'on' responses of ganglion cells to flashes and steps of light grew approximately linearly with stimulus intensity. The step reponse was not, however, related to the flash response by superposition. Larger spots in the field centre gave responses which grew non‐linearly with the intensity of even dim stimuli. 8 Depolarizing current passed through the recording electrode elicited a repetitive discharge of impulses. The frequency of firing increased linearly with current strength above a rheobase value of about 10(‐10) A. Accommodation occurred during steady currents, the main decline taking place with a time constant of about 15 msec. 9. Strength‐latency measurements and bridge records of ganglion cell charging by constant currents gave time constants of 10‐‐20 msec and input resistances of 100‐‐150 M omega.
- Published
- 1979
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19. Variation of membrane properties in hair cells isolated from the turtle cochlea.
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Art, J J and Fettiplace, R
- Abstract
1. 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. 3. Depolarizations from the resting potential under voltage clamp activated a large net outward current with a steep voltage dependence, and the steady‐state current‐voltage relationship was strongly rectified about the resting potential. Input resistances tended to be smaller in cells with higher resonant frequencies, although there was no concurrent variation in membrane area as inferred from the cell capacitance. 4. The kinetics of the outward current evoked by a small depolarizing step depended upon the resonant frequency, fo, of the hair cell, and were slower in low‐frequency cells. On repolarization to the resting potential the current decayed exponentially with a time constant that changed from 150 ms in the lowest‐frequency cell to less than 1 ms in the highest‐frequency one. The time constant was approximately proportional to 1/f0(2). 5. Following repolarization to different membrane potentials, the tail current was found to reverse around ‐80 mV, indicating that the outward current was due mainly to K+. 6. The outward current was abolished by extracellular application of 25 mM‐tetraethylammonium chloride (TEA), or on exchange of Cs+ for K+ in the intracellular medium filling the recording electrode, each experiment supporting the contention that K+ is the major current carrier. Such treatments also removed the oscillations in membrane potential evoked by imposed current steps. 7. Addition of TEA or intracellular perfusion with Cs+ also revealed a fast inward current with an ionic sensitivity consistent with its being carried by Ca2+. Like the K+ current, the Ca2+ current was activated by small depolarization 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.(ABSTRACT TRUNCATED AT 400 WORDS)
- Published
- 1987
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20. The actions of calcium on the mechano‐electrical transducer current of turtle hair cells.
- Author
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Crawford, A C, Evans, M G, and Fettiplace, R
- Abstract
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 current's 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)
- Published
- 1991
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21. Monitoring calcium in turtle hair cells with a calcium‐activated potassium channel.
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Tucker, T R and Fettiplace, R
- Abstract
1. An apamin‐sensitive Ca(2+)‐activated K+ channel was characterized in turtle hair cells and utilized to monitor submembranous intracellular Ca2+ and to evaluate the concentration of the mobile endogenous calcium buffer. 2. Isolated hair cells were voltage clamped with whole‐cell patch electrodes filled with a Cs(+)‐based intracellular solution to block the large‐conductance Ca(2+)‐activated K+ (BK) channel. Ca2+ currents evoked by depolarization were followed by inward tail currents lasting several hundred milliseconds. Both the Ca2+ current and slow tail current were abolished by nifedipine. 3. The tail current was carried by K+ and Cs+ (relative permeabilities PCa/PK = 0.22), and was fully blocked by 0.1 microM apamin and half blocked by 5 mM external TEA. These properties suggest the tail current flows through a Ca(2+)‐activated K+ channel distinct from the BK channels. 4. Intracellular Ca2+ was imaged with a confocal microscope in hair cells filled with the indicator Calcium Green‐5N introduced via the patch pipette. Increases in Ca2+ evoked by depolarization were localized to hotspots on the basolateral surface of the cell. The time course of the tail current closely matched the fast component of the fluorescenece monitored at a hotspot. 5. Ca(2+)‐ATPase pump inhibitors thapsigargin, 2,4‐di‐(t‐butyl)hydroquinone (BHQ) and vanadate, which are known to influence calcium regulation in turtle hair cells, prolonged the time course of the tail current, supporting the idea that the channel monitors cytoplasmic Ca2+. 6. The mobile endogenous buffer was estimated by combining perforated‐patch and whole‐cell recordings on a single cell. After recording tail currents with an amphotericin‐perforated patch, the patch was ruptured to obtain the whole‐cell mode, thus allowing washout of soluble cytoplasmic proteins and exchange with pipette buffers. By varying the concentration of Ca2+ buffer in the pipette, the mobile endogenous buffer was found to be equivalent to about 1 mM BAPTA.
- Published
- 1996
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22. The contribution of TMC1 to adaptation of mechanoelectrical transduction channels in cochlear outer hair cells.
- Author
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Goldring AC, Beurg M, and Fettiplace R
- Subjects
- Animals, Calcium metabolism, Cells, Cultured, Hair Cells, Auditory physiology, Ion Channel Gating, Ion Channels metabolism, Membrane Proteins genetics, Mice, Mice, Inbred C57BL, Mutation, Adaptation, Physiological, Hair Cells, Auditory metabolism, Mechanotransduction, Cellular, Membrane Proteins metabolism
- Abstract
Key Points: Hair cell mechanoelectrical transducer channels are opened by deflections of the hair bundle about a resting position set by incompletely understood adaptation mechanisms. We used three characteristics to define adaptation in hair cell mutants of transmembrane channel-like proteins, TMC1 and TMC2, which are considered to be channel constituents. The results obtained demonstrate that the three characteristics are not equivalent, and raise doubts about simple models in which intracellular Ca
2+ regulates adaptation. Adaptation is faster and more effective in TMC1-containing than in TMC2-containing transducer channels. This result ties adaptation to the channel complex, and suggests that TMC1 is a better isoform for use in cochlear hair cells. We describe a TMC1 point mutation, D569N, that reduces the resting open probability and Ca2+ permeability of the transducer channels, comprising properties that may contribute to the deafness phenotype., Abstract: Recordings of mechanoelectrical transducer (MET) currents in cochlear hair cells were made in mice with mutations of transmembrane channel-like (TMC) protein to examine the effects on fast transducer adaptation. Adaptation was faster and more complete in Tmc2-/- than in Tmc1-/- , although this disparity was not explained by differences in Ca2+ permeability or Ca2+ influx between the two isoforms, with TMC2 having the larger permeability. We made a mouse mutation, Tmc1 p.D569N, homologous to a human DFNA36 deafness mutation, which also had MET channels with lower Ca2+ -permeability but showed better fast adaptation than wild-type Tmc1+/+ channels. Consistent with the more effective adaptation in Tmc1 p.D569N, the resting probability of MET channel opening was smaller. The three TMC variants studied have comparable single-channel conductances, although the lack of correlation between channel Ca2+ permeability and adaptation opposes the hypothesis that adaptation is controlled simply by Ca2+ influx through the channels. During the first postnatal week of mouse development, the MET currents amplitude grew, and transducer adaptation became faster and more effective. We attribute changes in adaptation partly to a developmental switch from TMC2- to TMC1- containing channels and partly to an increase in channel expression. More complete and faster adaptation, coupled with larger MET currents, may account for the sole use of TMC1 in the adult cochlear hair cells., (© 2019 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)- Published
- 2019
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23. PIEZO2 as the anomalous mechanotransducer channel in auditory hair cells.
- Author
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Beurg M and Fettiplace R
- Subjects
- Action Potentials, Animals, Calcium Signaling, Hair Cells, Auditory physiology, Hair Cells, Auditory ultrastructure, Ion Channels genetics, Mechanotransduction, Cellular, Mice, Hair Cells, Auditory metabolism, Ion Channels metabolism
- Abstract
Throughout postnatal maturation of the mouse inner ear, cochlear hair cells display at least two types of mechanically gated ion channel: normal mechanotransducer (MT) channels at the tips of the stereocilia, activated by tension in interciliary tip links, and anomalous mechanosensitive (MS) channels on the top surface of the cells. The anomalous MS channels are responsible for the reverse-polarity current that appears in mutants in which normal transduction is lost. They are also seen in wild-type hair cells around birth, appearing 2 days earlier than normal MT channels, and being down-regulated with the emergence of the normal channels. We review the evidence that the normal and anomalous channels are distinct channel types, which includes differences in localization, susceptibility to pharmacological agents, single-channel conductance and Ca
2+ permeability. The dichotomy is reinforced by the observation that the anomalous current is absent in cochlear cells of Piezo2-null mice, even though the normal MT current persists. The anomalous current is suppressed by high intracellular Ca2+ , suggesting that influx of the divalent ion via more Ca2+ -permeable normal MT channels inhibits the anomalous channels, thus explaining the temporal relationship between the two. Piezo2-null mice have largely normal hearing, exhibiting up to 20 dB elevation in threshold in the acoustic brainstem response, so raising questions about the significance of PIEZO2 in the cochlea. Since the anomalous current declines with postnatal age, PIEZO2 may contribute to hair cell development, but it does not underlie the normal MT current. Its role in the development of hearing is not understood., (© 2017 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)- Published
- 2017
- Full Text
- View/download PDF
24. Active hair bundle movements in auditory hair cells.
- Author
-
Fettiplace R
- Subjects
- Animals, Cochlea innervation, Cochlea physiology, Electrophysiology, Hair Cells, Auditory, Outer cytology, Rats, Turtles, Hair Cells, Auditory, Outer physiology, Hearing physiology, Mechanoreceptors physiology, Mechanotransduction, Cellular physiology
- Abstract
The frequency selectivity of mammalian hearing depends on not only the passive mechanics of the basilar membrane but also an active amplification of the mechanical stimulus by the cochlear hair cells. The common view is that amplification stems from the somatic motility of the outer hair cells (OHCs), changes in their length impelled by voltage-dependent transitions in the membrane protein prestin. Whether this voltage-controlled mechanism, whose frequency range may be limited by the membrane time constant, has the band width to cover the entire auditory range of mammals is uncertain. However, there is ample evidence for an alternative mode of force generation by hair cells of non-mammals, such as frogs and turtles, which probably lack prestin. The latter process involves active motion of the hair bundle underpinned by conformational changes in the mechanotransducer (MT) channels and activation of one or more isoforms of myosin. This review summarizes evidence for active hair bundle motion and its connection to MT channel adaptation. Key factors for the hair bundle motor to play a role in the mammalian cochlea include the size and speed of force production.
- Published
- 2006
- Full Text
- View/download PDF
25. The role of Ca2+-activated K+ channel spliced variants in the tonotopic organization of the turtle cochlea.
- Author
-
Jones EM, Gray-Keller M, and Fettiplace R
- Subjects
- Acoustic Stimulation, Amino Acid Sequence, Animals, Cattle, Chick Embryo, Electrophysiology, Hair Cells, Auditory, Inner metabolism, Hair Cells, Auditory, Inner physiology, Isomerism, Kinetics, Large-Conductance Calcium-Activated Potassium Channel alpha Subunits, Large-Conductance Calcium-Activated Potassium Channel beta Subunits, Large-Conductance Calcium-Activated Potassium Channels, Molecular Sequence Data, Oocytes metabolism, Potassium Channels genetics, Quail, RNA Splicing, RNA, Messenger biosynthesis, Sequence Deletion genetics, Sequence Deletion physiology, Xenopus, Cochlea physiology, Potassium Channels physiology, Potassium Channels, Calcium-Activated, Turtles physiology
- Abstract
1. Turtle auditory hair cells contain multiple isoforms of the pore-forming alpha-subunit of the large-conductance Ca2+-activated K+ (KCa) channel due to alternative splicing at two sites. Six splice variants were studied by expression in Xenopus oocytes. 2. The isoforms possessed differences in apparent Ca2+ sensitivity and kinetics. The lowest Ca2+ sensitivity was observed in a novel variant resulting from a 26 amino acid deletion around one of the splice sites. 3. Co-expression of a bovine beta-subunit slowed the current relaxation 10-fold compared with channels formed from alpha-subunits alone but preserved the original order of kinetic differences. The beta-subunit also increased the Ca2+ sensitivity of isoforms to bring them nearer the range of sensitivity of the native KCa channels of the hair cell. 4. With channels formed from alpha-subunits or alpha + beta-subunits, the half-activation voltage in a fixed Ca2+ concentration, and the time constant of the current relaxation, varied linearly with the combined size of the insertions/deletions at the splice sites. 5. Experiments in which the beta/alpha concentration ratio was varied indicated that the beta-subunit exerts an all-or-none effect on the Ca2+ sensitivity and kinetics of the channel. 6. Co-expression of an avian beta2-subunit had effects on kinetics and Ca2+ sensitivity of several alpha-isoforms which were qualitatively similar to those produced by the bovine beta-subunit. 7. We conclude that differential expression of alternatively spliced alpha-subunit variants and a non-uniform distribution of a beta-subunit can produce a range of KCa channel properties needed to explain the tonotopic organization of the turtle cochlea.
- Published
- 1999
- Full Text
- View/download PDF
26. Reversal of hair cell responses by current [proceedings].
- Author
-
Crawford AC and Fettiplace R
- Subjects
- Animals, Membrane Potentials, Hair Cells, Auditory physiology, Turtles physiology
- Published
- 1979
27. Ringing responses in cochlear hair cells of the turtle [proceedings].
- Author
-
Crawford AC and Fettiplace R
- Subjects
- Animals, Membrane Potentials, Hair Cells, Auditory physiology, Mechanoreceptors physiology, Turtles physiology
- Published
- 1978
28. A method for altering the intracellular calcium concentration.
- Author
-
Crawford AC and Fettiplace R
- Subjects
- Animals, Anura, Axons drug effects, Electrophysiology, Ethanol pharmacology, In Vitro Techniques, Methods, Mollusca, Neuromuscular Junction drug effects, Calcium metabolism, Cell Membrane Permeability drug effects, Nystatin pharmacology
- Published
- 1971
29. The action of lysine vasopressin on artificial lipid bilayers.
- Author
-
Fettiplace R, Haydon DA, and Knowles CD
- Subjects
- Electric Conductivity, Lysine, Osmosis, Permeability, Water metabolism, Lipids, Membranes, Artificial, Vasopressins pharmacology
- Published
- 1972
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