The hair cell as a presynaptic terminal.
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Animals↗
Biomedical subjects
A J Hudspeth
Publications and source records attributed to A J Hudspeth.
Ultrastructural correlates of mechanoelectrical transduction in hair cells of the bullfrog's internal ear.
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Acoustic Maculae↗
Colocalization of ion channels involved in frequency selectivity and synaptic transmission at presynaptic active zones of hair cells.
Calcium ions serve as intracellular messengers in 2 activities of hair cells: in conjunction with Ca2(+)-activated K+ channels, they produce the electrical resonance that tunes each cell to a specific frequency of stimulation, and they trigger the release of a chemical synaptic transmitter. Our experiments indicate that both of these functions are conducted within a region that extends a few hundred nanometers around each presynaptic active zone. In focal electrical recordings from the plasma membranes of isolated anuran hair cells, we found nearly all of a cell's Ca2+ channels and Ca2(+)-activated K+ channels clumped at a fixed ratio in an average of 20 clusters on the basolateral membrane surface. Because serial-section electron microscopy indicated that each hair cell has approximately 19 afferent synaptic contacts with a similar distribution upon its basolateral surface, we conclude that the channel clusters coincide with synaptic active zones. Ensemble-variance analysis of current fluctuations indicated that each cell has a total of approximately 1800 Ca2+ channels and approximately 700 Ca2(+)-activated K+ channels; if these are uniformly divided, we estimate that each channel cluster contains approximately 90 Ca2+ and approximately 40 Ca2(+)-activated K+ channels. Freeze-fracture electron microscopy demonstrated an average of 133 large intramembrane particles in the presynaptic membrane at each active zone, an observation that suggests that the particles are the clustered channels. We used the K+ channel's sensitivity to intracellular Ca2+ to assay the concentration of free Ca2+ in the presynaptic cytoplasm, which we found to vary between 10 microM and 1 mM over the physiological range of membrane potentials. The inferred concentrations agreed with the values predicted for free diffusion of Ca2+ away from Ca2+ channels scattered randomly within a 300-nm-diameter synaptic active zone. The close association among Ca2+ channels, Ca2(+)-activated K+ channels, and synaptic active zones is necessary both for the rapid activation of K+ currents required in electrical resonance and for the transmission at afferent synapses of information about the phases of high-frequency stimuli.
Animals↗
Movement of microtubules by single kinesin molecules.
Kinesin is a motor protein that uses energy derived from ATP hydrolysis to move organelles along microtubules. Using a new technique for measuring the movement produced in vitro by individual kinesin molecules, it is shown that a single kinesin molecule can move a microtubule for several micrometers. New information about the mechanism of force generation by kinesin is presented.
Adenosine Triphosphatases↗
How the ear's works work.
The senses of hearing and equilibrium depend on sensory receptors called hair cells which can detect motions of atomic dimensions and respond more than 100,000 times a second. Biophysical studies suggest that mechanical forces control the opening and closing of transduction channels by acting through elastic components in each hair cell's mechanoreceptive hair bundle. Other ion channels, as well as the mechanical and hydrodynamic properties of hair bundles, tune individual hair cells to particular frequencies of stimulation.
Cochlea↗
Blockage of the transduction channels of hair cells in the bullfrog's sacculus by aminoglycoside antibiotics.
The action of aminoglycoside antibiotics on transduction by hair cells was investigated in isolated preparations of the bullfrog's sacculus. Bath application of aminoglycosides produced a reversible blockage of extracellularly recorded responses to displacements of the otolithic membrane. The half-blocking concentrations for various drugs were in the range 2-95 microM. The effect of dihydrostreptomycin on the receptor currents of individual hair cells was studied under two-electrode, voltage-clamp conditions. Iontophoretic application of drug to the apical cellular surface caused a reduction of the receptor current within 20 ms; the reduction was reversible within 1 s. The effect was most striking at holding potentials more negative than -60 mV and was relieved by depolarization. The effect of intracellular aminoglycosides was investigated in cells voltage-clamped with the tight-seal, whole-cell technique. Gentamicin and dihydrostreptomycin, at concentrations near 100 microM, did not block transduction under these conditions. The acute, reversible blocking effect of aminoglycosides therefore occurs from the extracellular membrane surface. The results are consistent with aminoglycosides' plugging the poorly ion-selective transduction channels of hair cells.
Animals↗
Mechanoelectrical transduction by hair cells of the bullfrog's sacculus.
Hair cells of the internal ear respond to excitatory mechanical stimulation of their hair bundles by the rapid opening of poorly cation-selective transduction channels. These channels, which are located near the bundles' tips, may be gated by forces applied through elastic linkages between adjacent stereocilia.
Action Potentials↗
Mechanical properties of sensory hair bundles are reflected in their Brownian motion measured with a laser differential interferometer.
By optically probing with a focused, low-power laser beam, we measured the spontaneous deflection fluctuations of the sensory hair bundles on frog saccular hair cells with a sensitivity of about 1 pm/square root of Hz. The preparation was illuminated by two orthogonally polarized laser beams separated by only about 0.2 microns at their foci in the structure under investigation. Slight movement of the object from one beam toward the other caused a change of the phase difference between the transmitted beams and an intensity modulation at the detector where the beams interfered. Maintenance of the health of the cells and function of the transduction mechanism were occasionally confirmed by measuring the intracellular resting potential and the sensitivity of transduction. The root-mean-square (rms) displacement of approximately 3.5 nm at a hair bundle's tip suggests a stiffness of about 350 microN/m, in agreement with measurements made with a probe attached to a bundle's tip. The spectra resemble those of overdamped harmonic oscillators with roll-off frequencies between 200 and 800 Hz. Because the roll-off frequencies depended strongly on the viscosity of the bathing medium, we conclude that hair-bundle motion is mainly damped by the surrounding fluid.
Animals↗
Compliance of the hair bundle associated with gating of mechanoelectrical transduction channels in the bullfrog's saccular hair cell.
Mechanical stimuli are thought to open the transduction channels of a hair cell by tensing elastic components, the gating springs, that pull directly on the channels. To test this model, we measured the stiffness of hair bundles during mechanical stimulation. A bundle's compliance increased by about 40% at the position where half of the channels opened. This we attribute to conformational changes of transduction channels as they open and close. The magnitude and displacement dependence of the gating compliance provide quantitative information about the molecular basis of mechanoelectrical transduction: the force required to open each channel, the number of transduction channels per hair cell, the stiffness of a gating spring, and the swing of a channel's gate as it opens.
Animals↗
Kinetic analysis of voltage- and ion-dependent conductances in saccular hair cells of the bull-frog, Rana catesbeiana.
1. By the use of whole-cell and excised-patch tight-seal recording techniques, we studied ionic conductances in voltage-clamped solitary hair cells isolated from the bull-frog's sacculus. As a basis for assessing their contributions to hair cell electrical resonance, we developed kinetic models describing voltage-dependent Ca2+ and Ca2+-dependent K+ conductances. 2. A transient K+ current (IA) was activated by steps to potentials positive to -50 mV from holding potentials more negative than -70 mV. In the steady state, the current was fully inactivated at the normal resting potential. Possibly due to the dissipation of a Donnan potential between the pipette's interior and the cell, the voltage dependence of IA inactivation slowly shifted in the negative direction during whole-cell recording. 3. The voltage-gated Ca2+ current (ICa) was isolated by blocking IA with 4-aminopyridine (4-AP) and Ca2+-activated K+ current with tetraethylammonium (TEA). The ICa was activated at potentials more positive than -60 to -50 mV and was maximal at about -10 mV. Its magnitude was highly variable among cells, with an average value of -240 pA at -30 mV. Its activation could be fitted well by a third-order (m3) gating scheme. 4. A Ca2+-activated K+ current (IK(Ca)) was isolated as the component of membrane current blocked by TEA. This current was activated at potentials more positive than -60 to -50 mV and had an average value of 1.5 nA at -30 mV. The Ca2+-activated K+ conductance (gK(Ca)) showed a high apparent voltage dependence, increasing e-fold every 3 mV at potentials between -50 and -40 mV. 5. The Ca2+-activated K+ current displayed rapid activation and deactivation kinetics. The current reached half-maximal activation in 2-4 ms at voltages between -50 and -30 mV, and the tail current decayed exponentially with a time constant of 1.0 ms at -70 mV. The activation rate and magnitude of IK(Ca) were reduced by lowering the extracellular Ca2+ concentration. 6. The open probability of Ca2+-activated K+ channels was estimated by ensemble-fluctuation analysis of whole-cell currents evoked by voltage steps to -30 mV. The average open probability was estimated to be 0.8 at this potential. 7. K+-selective channels with a high conductance (140-200 pS) were examined in excised, inside-out membrane patches. The activity of these channels depended on intracellular Ca2+ and membrane potential. These properties suggest that the channels underlie the whole-cell Ca2+-activated K+ current.(ABSTRACT TRUNCATED AT 400 WORDS)
4-Aminopyridine↗
A model for electrical resonance and frequency tuning in saccular hair cells of the bull-frog, Rana catesbeiana.
1. Electrical resonance in solitary hair cells was examined under several experimental conditions using the tight-seal recording technique in the whole-cell current-clamp mode. 2. Resonance was characterized by the frequency and quality factor of oscillations in membrane potential evoked by depolarizing current pulses. Oscillation frequency increased with depolarization, from about 90 Hz at the resting potential to a limiting value of about 250 Hz. The quality factor of the oscillations was a bell-shaped function of membrane potential that reached a maximum of up to 12.6 at a potential slightly positive to the resting potential. 3. Pharmacological experiments were performed to assess which of three ionic currents participate in electrical resonance. Reduction of the voltage-gated Ca2+ current (ICa) and the Ca2+-activated K+ current (IK(Ca)) by lowering the extracellular Ca2+ concentration, or reduction of IK(Ca) with tetraethylammonium ion (TEA) degraded the resonance. In contrast, blockade of the transient K+ current (IA) with 4-aminopyridine (4-AP) had no significant effect. 4. To test the sufficiency of the Ca2+ and the Ca2+-activated K+ currents to account for resonance, we developed a model using mathematical descriptions of the two currents derived in the preceding paper (Hudspeth & Lewis, 1988), with additional terms for leakage conductance and membrane capacitance. The model correctly predicts the oscillatory responses to applied current pulses, including the non-linear dependences of oscillation frequency and quality factor on membrane potential. 5. Simulations of current-clamp experiments in the presence of a reduced extracellular Ca2+ concentration or of TEA were generated respectively by decreasing the model's values for the maximal Ca2+ or Ca2+-activated K+ conductances. The model's predictions of membrane-potential oscillations under these conditions agree qualitatively with experimental results, providing further support for the model as a description of the resonance mechanism. 6. To identify the factors most important in determining the hair cell's resonance properties, we systematically altered the values of selected parameters in the model. Frequency was most profoundly influenced by increasing the magnitude and activation rate of the Ca2+-activated K+ conductance, whereas the quality factor was most sensitive to increases in the level of the Ca2+ conductance. 7. By including a term describing activation of the hair cell's mechanically sensitive transduction conductance, we used the model to predict a tuning curve for responses to mechanical inputs of various frequencies.(ABSTRACT TRUNCATED AT 400 WORDS)
4-Aminopyridine↗
Mechanoelectrical transduction by hair cells.
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Animals↗
Hair cells: transduction, tuning, and transmission in the inner ear.
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Adaptation, Physiological↗
Mechanical relaxation of the hair bundle mediates adaptation in mechanoelectrical transduction by the bullfrog's saccular hair cell.
Mechanoelectrical transduction by hair cells of the frog's internal ear displays adaptation: the electrical response to a maintained deflection of the hair bundle declines over a period of tens of milliseconds. We investigated the role of mechanics in adaptation by measuring changes in hair-bundle stiffness following the application of force stimuli. Following step stimulation with a glass fiber, the hair bundle of a saccular hair cell initially had a stiffness of approximately equal to 1 mN X m-1. The stiffness then declined to a steady-state level near 0.6 mN X m-1 with a time course comparable to that of adaptation in the receptor current. The hair bundle may be modeled as the parallel combination of a spring, which represents the rotational stiffness of the stereocilia, and a series spring and dashpot, which respectively, represent the elastic element responsible for channel gating and the apparatus for adaptation.
Acclimatization↗
Adaptation of mechanoelectrical transduction in hair cells of the bullfrog's sacculus.
Adaptation in a vestibular organ, the bullfrog's sacculus, was studied in vivo and in vitro. In the in vivo experiments, the discharge of primary saccular neurons and the extracellular response of saccular hair cells were recorded during steps of linear acceleration. The saccular neurons responded at the onset of the acceleration steps, then adapted fully within 10-50 msec. The extracellular (microphonic) response of the hair cells adapted with a similar time course, indicating that the primary sources of the neural adaptation are peripheral to the afferent synapse--in the hair cell, its mechanical inputs, or both. Evidence for hair cell adaptation was provided by 2 in vitro preparations: after excising the sacculus and removing the accessory structures, we recorded either the extracellular hair cell response to displacement of the otolithic membrane or the intracellular hair cell response to hair bundle displacement. In both cases the response to a step stimulus adapted. The adaptation involved a shift in the displacement-response curve along the displacement axis, so that the cell's operating point was reset toward the static position of its hair bundle. This displacement shift occurred in response to both depolarizing and hyperpolarizing stimuli. Its time course varied among cells, from tens to hundreds of milliseconds, and also varied with the concentration of Ca2+ bathing the apical surfaces of the hair cells. Voltage-clamp experiments suggested that the displacement shift does not depend simply on ion entry through the hair cell's transduction channels and can occur at a fixed membrane potential. The possible role of the displacement-shift process in the function of the frog's sacculus as a very sensitive vibration detector is discussed.
Animals↗
The ionic channels of a vertebrate hair cell.
The ionic channels of hair cells play important roles in mechanoelectrical transduction, frequency tuning, and synaptic transmission. In the hair cells of the bullfrog's sacculus, transduction is mediated by mechanically gated, poorly selective channels located in the hair bundle. Statistical analysis of the variance in the transduction current measured under voltage-clamp conditions indicates that a cell has only a few hundred transduction channels, each with a conductance of approximately 17 pS at room temperature. The same cells employ a resonance in their membrane potential to enhance their frequency selectivity in responses to mechanical stimuli. Voltage-clamp studies indicate that the resonance results principally from the interplay of two ionic conductances, a voltage-sensitive Ca2+ conductance and a Ca2+-sensitive K+ conductance.
Animals↗
The transduction channel of hair cells from the bull-frog characterized by noise analysis.
Receptor currents in response to mechanical stimuli were recorded from hair cells in the excised epithelium of the bull-frog sacculus by the whole-cell, gigohm-seal voltage-clamp technique. The stimulus-dependent transduction current was separated from the cell's stimulus-independent K+ and Ca2+ currents; the K+ currents were blocked with an internal solution containing Cs+ while the Ca2+ current was reduced by holding the membrane potential below -70 mV. The temperature of the preparation was maintained at about 10 degrees C to slow the kinetics of the cells' transduction channels. Calibrated displacements of hair bundles of individual hair cells were made with a probe coupled by suction to the kinociliary bulb and moved with a piezoelectricbimorph stimulator. The root mean square noise of probe motion was less than 2 nm. The mean, I, and the variance, sigma 2, of the receptor current were measured from the response to saturating (+/- 0.5 micron) displacements of the hair bundle. I was corrected for current offsets and sigma 2 for the transduction-independent background variance. The relation between sigma 2 and I is consistent with the predictions of a two-conductance-state model of the transduction channel, a model having only one non-zero conductance state. The relation between sigma 2 and I was fitted by the equation sigma 2 = Ii-I2/N, where N is the number of transduction channels in the cell and i is the current through a single open channel. The conductance of the transduction channel is approximately ohmic with a reversal potential near 0 mV. The estimated conductance of a single transduction channel, gamma, is 12.7 +/- 2.7 pS (mean +/- S.D.; n = 18) at 10 degrees C. gamma is independent of the maximum transduction conductance of the cell, Gmax. The number of transduction channels, N, is proportional to Gmax. N ranges from 7 to 280 in cells with Gmax ranging from 0.08 to 2.48 nS. The largest values of N correspond to a few, perhaps four, active transduction channels per stereocilium. Control experiments show that transduction by the hair cell of two artifactual sources of hair-bundle stimulation, noisy or discontinuous motion of the probe, do not contribute substantially to the measured variance, sigma 2. Displacement-response curves are generally sigmoidal and symmetrical; they reasonably fit the predictions of a two-kinetic-state model, comprising one open state and one closed state. The estimated displacement-sensitive free energy, Z, is 5.7 +/- 1.1 kcal/mol micron (mean +/- S.D., n = 18).(ABSTRACT TRUNCATED AT 400 WORDS)
Action Potentials↗
The cellular basis of hearing: the biophysics of hair cells.
A crucial event in the hearing process is the transduction of mechanical stimuli into electrical signals by hair cells, the sensory receptors of the internal ear. Stimulation results in the rapid opening of ionic channels in the mechanically sensitive organelles of these cells, their hair bundles. These transduction channels, which are nonselectively permeable, are directly excited by hair-bundle displacement. Hair cells are selectively responsive to particular frequencies of stimulation, both due to the mechanical properties of their hair bundles and because of an ensemble of ionic channels that constitute an electrical resonator.
Acoustic Stimulation↗