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J J Art

Publications and source records attributed to J J Art.

16 recordsLinked to original sources
PubMed2001

Contribution of the plasmalemma to Ca2+ homeostasis in hair cells.

Calcium influx through transduction channels and efflux via plasmalemmal Ca(2+)-ATPases (PMCAs) are known to contribute to calcium homeostasis and modulate sensory transduction in vertebrate hair cells. To examine the relative contributions of apical and basolateral pathways, we analyzed the calcium dynamics in solitary ciliated and deciliated guinea pig type I and type II vestibular hair cells. Whole-cell patch-clamp recordings demonstrated that these cells had resting potentials near -70 mV and could be depolarized by 10-20 mV by superfusion with high potassium. Fura-2 measurements indicated that ciliated type II cells and deciliated cells of either type had low basal [Ca(2+)](i), near approximately 90 nm, and superfusion with high potassium led to transient calcium increases that were diminished in the presence of Ca(2+) channel blockers. In contrast, measurements of type I ciliated cells, hair cells with large calyceal afferents, were associated with a higher basal [Ca(2+)](i) of approximately 170 nm. High-potassium superfusion of these cells induced a paradoxical decrease in [Ca(2+)](i) that was augmented in the presence of Ca(2+) channel blockers. Optical localization of dihydropyridine binding to the kinocilium suggests that they contain L-type calcium channels, and as a result apical calcium influx includes a contribution from voltage-dependent ion channels in addition to entry via transduction channels localized to the stereocilia. Eosin block of PMCA significantly altered both [Ca(2+)](i) baseline and transient responses only in ciliated cells suggesting that, in agreement with immunohistochemical studies, PMCA is primarily localized to the bundles.

Animals↗
PubMed1999

The functional role of alternative splicing of Ca(2+)-activated K+ channels in auditory hair cells.

Turtle auditory hair cells are frequency tuned by the activity of large-conductance calcium-activated potassium (KCa) channels, the frequency range being dictated primarily by the channel kinetics. Seven alternatively spliced isoforms of the KCa channel alpha-subunit, resulting from exon insertion at two splice sites, were isolated from turtle hair cells. These, when expressed in Xenopus oocytes, produced KCa channels with a range of apparent calcium sensitivities and channel kinetics. However, most expressed channels were less calcium sensitive than the hair cells' native KCa channels. Coexpression of alpha-subunit with a bovine beta-subunit substantially increased the channel's calcium sensitivity while markedly slowing its kinetics, but kinetic differences between isoforms were preserved. These data suggest a molecular mechanism for hair cell frequency tuning involving differential expression of different KCa channel alpha-subunits in conjunction with an expression gradient of a regulatory beta-subunit.

Alternative Splicing↗
PubMed1996

Variations in the ensemble of potassium currents underlying resonance in turtle hair cells.

1. Potassium currents were characterized in turtle cochlear hair cells by whole-cell voltage clamp during superfusion with the potassium channel antagonists, tetraethylammonium (TEA) and 4-aminopyridine (4-AP). The estimated resonant frequency, f0, was inferred from tau, the time constant of deactivation of outward current upon repolarization to -50 mV, according to the empirical relation, f0 = k1 tau-1/2 + k2. 2. Dose-response relations for TEA and 4-AP were obtained by exposing single cells to ten concentrations exponentially distributed over four orders of magnitude. Potassium current in cells tuned to low frequencies was carried by a single class of channels with an apparent affinity constant, K1, for TEA of 35.9 mM. Half-blocking concentrations of 4-AP were correlated with the time constant of deactivation and varied between 26.2 and 102 microM. In cells tuned to higher frequencies, K+ current was carried by a single class of channels with high affinity for TEA (K1 = 0.215 mM) and low affinity for 4-AP (K1 = 12.3 mM). This pharmacological profile suggests that K+ current in low frequency cells is purely voltage gated and in high frequency cells, it is gated by both Ca2+ and voltage. 3. For each current type, the voltage dependence of activation was determined from tail current amplitude at -50 mV. The purely voltage-gated current, IK(V), was found to increase e-fold in 4.0 +/- 0.3 mV (n = 3) in low frequency cells exposed to TEA (25 mM). The Ca(2+)- and voltage-gated current, IK(Ca), was more steeply voltage dependent, increasing e-fold in 1.9 mV (n = 2) in high frequency cells exposed to 4-AP (0.8 mM). 4. IK(V) was found to inactivate slowly during prolonged voltage steps (approximately 10 s). Steady-state inactivation increased with depolarization from -70 mV and was incomplete such that on average IK(v) did not fall below approximately 0.39 of its maximum value. 5. Superfusion of 4-AP (0.8 mM) reversibly depolarized a low frequency cell and eliminated steady voltage oscillations, while TEA (6 mM) had no effect. In a high frequency cell, voltage oscillations were abolished by TEA, but not by 4-AP. 6. The differential pharmacology of IK(V) and IK(Ca) was used to measure their contribution to K+ current in cells tuned to different frequencies. Both currents exhibited a frequency-dependent increase in maximum conductance. IK(V) accounted for nearly all K+ current in cells tuned to less than 60 Hz, while IK(Ca) was the dominant current in higher frequency cells. 7. Mapping resonant frequency onto epithelial position suggests an exponential relation between K+ current size and position. IK(V) appeared to be limited to the apical or low frequency portion of the basilar papilla and coincided with maximal expression of a K(+)-selective inward rectifier, IK(IR). This finding is consistent with the notion that low frequency resonance is produced by interaction of IK(V) and IK(IR) with the voltage-gated Ca2+ current, ICa, and the cell's capacitance. The ionic events underlying higher frequency resonance are dominated by the action of IK(Ca) and ICa and include a contribution from IK(IR).

4-Aminopyridine↗
PubMed1996

Positive feedback by a potassium-selective inward rectifier enhances tuning in vertebrate hair cells.

Electrical resonance in vertebrate hair cells shapes receptor potentials and tunes each cell to a narrow band of frequencies. We have investigated the contribution of a potassium-selective inward rectifier (IR) to electrical resonance, isolating outward current carried by IR from other ionic currents active in the physiological voltage range (-75 to -30 mV) using a combination of potassium and calcium channel antagonists. IR expression is tightly regulated in the turtle's auditory epithelium, as revealed by the observation that its size declines systematically with resonant frequency. A critical feature of IR is the rapid inhibition produced by depolarization, which results in a negative slope in the steady-state current-voltage relation in the vicinity of the resting potential (-50 mV). The increasing block of outward current produced by depolarization is functionally equivalent to activating an inward current, suggesting that IR provides positive feedback and, in hair cells, serves an electrical function ordinarily reserved for voltage-dependent sodium and calcium currents. Additional support for this idea comes from the observation that superfusion with cesium selectively reduces IR and eliminates resonance in cells tuned to low frequencies and degrades resonant quality in cells tuned to more than 50 Hz.

Animals↗
PubMed1995

The calcium-activated potassium channels of turtle hair cells.

A major factor determining the electrical resonant frequency of turtle cochlear hair cells is the time course of the Ca-activated K current (Art, J. J., and R. Fettiplace. 1987. Journal of Physiology. 385:207-242). We have examined the notion that this time course is dictated by the K channel kinetics by recording single Ca-activated K channels in inside-out patches from isolated cells. A hair cell's resonant frequency was estimated from its known correlation with the dimensions of the hair bundle. All cells possess BK channels with a similar unit conductance of approximately 320 pS but with different mean open times of 0.25-12 ms. The time constant of relaxation of the average single-channel current at -50 mV in 4 microM Ca varied between cells from 0.4 to 13 ms and was correlated with the hair bundle height. The magnitude and voltage dependence of the time constant agree with the expected behavior of the macroscopic K(Ca) current, whose speed may thus be limited by the channel kinetics. All BK channels had similar sensitivities to Ca which produced half-maximal activation for a concentration of approximately 2 microM at +50 mV and 12 microM at -50 mV. We estimate from the voltage dependence of the whole-cell K(Ca) current that the BK channels may be fully activated at -35 mV by a rise in intracellular Ca to 50 microM. BK channels were occasionally observed to switch between slow and fast gating modes which raises the possibility that the range of kinetics of BK channels observed in different hair cells reflects a common channel protein whose kinetics are regulated by an unidentified intracellular factor. Membrane patches also contained 30 pS SK channels which were approximately 5 times more Ca-sensitive than BK channels at -50 mV. The SK channels may underlie the inhibitory synaptic potential produced in hair cells by efferent stimulation.

Animals↗
PubMed1993

The effects of low calcium on the voltage-dependent conductances involved in tuning of turtle hair cells.

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+.

Animals↗
PubMed1987

Variation of membrane properties in hair cells isolated from the turtle cochlea.

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)

Action Potentials↗
PubMed1986

Electrical resonance and membrane currents in turtle cochlear hair cells.

The electrical and mechanical properties of single hair cells from the turtle's cochlea were examined to investigate the basis of their electrical resonance. Receptor potentials were measured with intracellular micropipettes in the isolated basilar papilla. At the onset and termination of a step displacement of the ciliary bundle the receptor potential showed a damped oscillation reflecting the frequency selectivity of the cell. Resonance frequencies increased systematically from apex to base of the cochlea. Similar oscillations could be elicited by a current step injected through the recording electrode. Solitary hair cells enzymatically isolated from the papilla were investigated with the tight-seal whole-cell recording method. Cells retained their properties in response to current steps and had resonance frequencies between 10 and 350 Hz. In voltage clamp such cells displayed a large outward K+ current and an inward Ca2+ current both activated by depolarization from the resting potential. The relaxation time constant of the K+ current was inversely correlated with the resonance frequency of the cell, varying from 150 ms in the lowest frequency cells to less than 1 ms in the highest ones. It is argued that variation in the kinetics of this current is the major factor responsible for the range of resonance frequencies. In preparations of the isolated papilla a flexible glass fibre, attached to the tip of a ciliary bundle, was used to deliver constant force steps to the bundle and to monitor its displacement. Receptor potentials were simultaneously recorded. At the beginning and end of a force step towards the kinocilium, the bundle vibrated at a frequency which coincided with the electrical resonance frequency of the cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Efferent modulation of hair cell tuning in the cochlea of the turtle.

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.

Action Potentials↗
PubMed1984

Efferent desensitization of auditory nerve fibre responses in the cochlea of the turtle Pseudemys scripta elegans.

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.

Acoustic Stimulation↗
PubMed1984

Synaptic hyperpolarization and inhibition of turtle cochlear hair cells.

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.

Acetylcholine↗
PubMed1982

Efferent regulation of hair cells in the turtle cochlea.

Intracellular recordings were made from hair cells in the isolated cochlea of the turtle to characterize the inhibition achieved by the cochlea's efferent innervation. A short train of shocks delivered to the efferent axons produced in the hair cells slow hyperpolarizing synaptic potentials which could be reversed by shifting the membrane potential more negative than about -80 mV. Throughout the efferent hyperpolarization, there was a reduction of up to 25-fold in the amplitude of the receptor potential for tones presented at the hair cell's characteristic frequency. Efferent stimulation also was shown to degrade the cell's tuning properties. It is argued that the combined effects of the hyperpolarization and the loss in hair cell sensitivity could account for a threshold elevation of at least 70 dB in the auditory nerve fibres.

Action Potentials↗