Neuron,

Vol. 7, 409-420,

September,

1991, Copyright

0 1991 by Cell Press

localization of the Hair Cell’s Transduction Channels at the Hair Bundle’s Top by lontophoretic Application of a Channel Blocker Fern& Jaramillo and A. J. Hudspeth Department of Cell Biology and Neuroscience University of Texas Southwestern Medical Center Dallas, Texas 75235-9039

Summary In order to understand how the hair cell’s mechanoelectrical transduction channels are gated during mechanical stimulation, it is essential to determine their location with respect to the hair bundle’s constituent stereocilia. We localized the transduction channels by focally blocking receptor currents with iontophoretically ejected gentamicin, an aminoglycoside antibiotic that acts as a reversible channel blocker. The drug was most effective when directed at the top of a hair bundle, whereas application at the bundle’s bottom or at the cuticular plate had little or no effect. Computer simulations of blocking agreed with experimental data only when the transduction channels were hypothesized to occur near the bundle’s top. These results confirm that the hair cell’s transduction channels are located near the stereociliary tips. Introduction The hair bundle is theorganelle responsible for mechanoelectrical transduction by hair cells, the sensory receptors of the vertebrate auditory, vestibular, and lateral-line systems. A bundle comprises 20-300 specialized microvillar derivatives, the stereocilia, that protrude from the hair cell’s apical surface. The stereociliary lengths vary systematically so that the bundle has a sloping top surface. At its tall edge, the bundle has a single axonemal cilium, the kinocilium, which is not required for transduction (Hudspeth and Jacobs, 1979). The hair bundle owes its mechanical sensitivity to mechanoelectrical transduction channels, a population of cation-selective transmembrane channels that open when the bundle is deflected in the positive direction, toward its tallest stereocilia (Hudspeth and Corey, 1977; Shotwell et al., 1981). There are about 100 transduction channels per hair cell (Holton and Hudspeth, 1986; Howard and Hudspeth, 1988), or roughly 1 per stereocilium. The short latency of transduction (Corey and Hudspeth, 1979a, 1983b; Crawford et al., 1989) suggests that these channels are directly gated by the application of force to a hair bundle, without the intervention of a second messenger. Transduction channels are thought to be opened by elastic elements, the gating springs, which are extended when the hair bundle is deflected in the positive direction (Corey and Hudspeth, 198313; Howard and Hudspeth, 1988; for reviews, see Howard et al., 1988; Roberts et al., 1988; Hudspeth, 1989). In order to understand at a molecular level how the hair cell’s transduction channels are gated, it is

necessary to learn precisely where these channels occur. The polarity of receptor-current flow indicates that the transduction channels reside on the hair cell’s apical membrane surface (Corey and Hudspeth, 1983a), which includes the plasma membrane that covers the stereocilia as well as that on the flattened apical surface of the cell. Because no means is yet available by which to label the transduction channels, however, their location must be inferred by means of signals associated with the flow of receptor currents. The initial attempt at localizing the transduction channels (Hudspeth, 1982) relied upon the fact that the current through them is modulated by hair-bundle deflection. Focal extracellular recordings from hair cells of the bullfrog’s sacculus revealed field potentials phase-locked to a sinusoidal mechanical stimulus. That the electrical signals associated with mechanoelectrical transduction were greatest at the tops of hair bundles suggested that transduction channels are located near the stereociliary tips. Because the hair cell’s transduction channels are permeable to most small cations (Corey and Hudspeth, 1979b; Ohmori, 1985), another means by which the site of transduction might be revealed is by determining the cytoplasmic location where Ca*+ traversing such channels initially appears in a cell loaded with a Ca2+-sensitive fluorescent dye. The results obtained with this approach are contradictory. Ohmori (1988) used the indicator fura- to measure Ca2+ accumulation in mechanically stimulated hair cells of the chicken. These cells showed a peak 340/380 nm fluorescence ratio near the hair bundle’s insertion into the cuticular plate, which was taken to implicate the bases of the stereocilia as the sites of mechanoelectrical transduction. In another, preliminary study on the frog’s saccular hair cells (Huang and Corey, 1990, Biophys. J., abstract), the indicator fluo-3 was instead employed to detect Ca*+ entry through the 15% or so of transduction channels open at rest (for a review, see Roberts et al., 1988). When a hair cell’s membrane potential was changed so as to augment the driving force for Ca2+ entry, the ensuing increase in intracellular Ca2+ concentration originated at the hair bundle’s top. This result favors the possibility that transduction channels occur near the stereociliary tips. In addition to their mechanosensitivity and ionic permeability, the hair cell’s transduction channels have a third characteristic by which they may be recognized: they are subject to reversible, voltage-dependent blockage byaminoglycoside antibiotics (Hudspeth and Kroese, 1983, J. Physiol., abstract; Ohmori, 1985; Kroese et al., 1989). Because iontophoretic application of drugs can be used to map the sites to which they bind with micrometer-level precision (Peper and McMahan, 1972; Kuffler and Yoshikami, 1975a), we attempted to localize the hair cell’s transduction channels by focally inactivating receptor currents with the

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aminoglycoside antibiotic, gentamicin (jaramillo and Hudspeth, 1991, Biophys. J., abstract). Several features of aminoglycoside antibiotics make them especiallysuitablefor iontophoretic localization of the site of mechanoelectrical transduction. These drugs are quite water-soluble, so that iontophoretic pipettes can be filled with concentrated solutions of the substances. By contrast, transduction-channel blockers of another family, the amiloride derivatives (Jorgensen and Ohmori, 1988), are poorlywater-soluble. Most aminoglycoside molecules bear several positive charges (for a review, see Daniels, 1978), so they may be iontophoretically expelled from pipettes with satisfactory efficiency (Kroese et al., 1989). The diffusional properties of aminoglycoside antibiotics are also particularly felicitous for iontophoretic experiments. These drug molecules remain near their site of expulsion from a pipette’s tip long enough to achieve a concentration sufficient to block transduction channels; at the same time, the substances diffuse fast enough to produce a steep spatial gradient in concentration. At the end of an iontophoretic pulse of the duration and magnitude employed in this study, for example, the concentration of an aminoglycoside 5 urn from the pipette’s tip is only l/100 that at a distance of 1 urn. lontophoresis accordingly offers spatial resolution that is fine by comparison with the hair bundle’s length of about 8 urn (Jacobs and Hudspeth, 1990). Concentration-response plots (Kroese et al., 1989) indicate that a single molecule of an aminoglycoside antibiotic, A, suffices to block a transduction channel according to the state diagram:

Closed

k g k 21

Open

A ku Y k 32

Blocked.

(1)

For simplicity’s sake, the transduction channel is here considered to have only a single closed and a single open state; the actual state diagram may be more complex (Corey and Hudspeth, 198313; Holton and Hudspeth, 1986; Jaramillo et al., 1990). Several lines of evidence indicate that aminoglycoside antibiotics act as open-channel blockers. The blockage produced by an aminoglycoside is voltagedependent (Huspeth and Kroese, 1983, J. Physiol., abstract; Ohmori, 1985; Kroese et al., 1989), so the drug molecule evidently enters a channel’s aqueous pore far enough to experience a fraction of the transmembrane potential difference. Because gentamicin abolishes the gating compliance associated with the opening and closing of transduction channels (Howard and Hudspeth, 1988), the antibiotic appears to arrest the channels in one kinetic state;thetransient mechanical response of hair bundles to an aminoglycoside suggests that the drug blocks channels whose gates are in the open position (Denk, 1989).

Results Receptor-Current Blockage by Aminoglycoside Antibiotics A fine, glass probe attached near the top of the hair bundle provides aconvenient means of evoking a hair cell’s electrical response (Figure IA). A positive stimulus, deflection of the bundle toward its tall edge, opens some portion of the mechanically sensitive transduction channels. The resultant influx of cations through the plasma membrane constitutes the receptor current that underlies the hair cell’s receptor potential. When the hair bundle is abruptly displaced in the positive direction and held there, the receptor current commences rapidly and peaks within a few milliseconds (Figure 2). The response then declines as a result of an adaptation process that resets the hair bundle’s range of mechanical sensitivity (Eatock et al., 1987; Howard and Hudspeth, 1987; Assad et al., 1989; Crawford et al., 1989; Hacohen et al., 1989). In order to study the local sensitivity of the receptor current to blockage byaminoglycosideantibiotics, we used focal iontophoretic application of these drugs. The distal portion of an iontophoretic electrode was bent so that it was perpendicular to the bottom of the recording chamber, and thus to the long axis of the hair bundle. This arrangement, which provided a clear, cross-sectional view of the electrode’s tip, allowed us to determine accurately the electrode’s position with respect to the bundle (Figures IB and IC). Approximately 5 ms after the onset of a mechanical stimulus pulse, when the hair bundle was stationary and the receptor current had reached its peak, we ejected gentamicin from a microelectrode with a 5-ms iontophoretic pulse of 2-10 nA (Figure 2). As the drug occluded transduction channels, partial blockage of the receptor current commenced as soon as 1 ms after the onset of iontophoresis. After ejection ceased, and as the drug left the channels and diffused away, the receptor current returned toward its control level along a roughly exponential time course with a time constant of 25-30 ms. Spatial localization of Transduction Channels The spatial sensitivity of transduction to blockage by iontophoretically applied gentamicin was assayed at 63 different locations for each of the 12 hair bundles studied in detail. In every instance, the effectiveness of iontophoretic application was greatest near the top of the hair bundle, submaximal at locations intermediate between the tips and the bases of the stereocilia, and minimal, or more commonly absent, at locations near the bottom of the bundle and the cuticular plate (Figure 3). Blockage was observed in response to gentamicin application near the bases of stereocilia only when a cell’s sensitivity to the drug was unusually high as a consequence of either a large iontophoretic current or placement of the iontophoretic electrode less than 0.5 pm above the hair bundle. Three sets of

Localization 411

of Transduction

Channels

. . . . . . . .

Figure 1. Experimental Antibiotics

Arrangement

Used

to Study

the

Blockage

of Receptor

Currents

by Focal

lontophoresis

of Aminoglycoside

(A) A fine glass probe attached to the kinociliary bulb, shown at the top of the micrograph, deflected the hair bundle in the positive stimulus direction, to the right. This differential interference contrast micrograph shows a plane of focus roughly coincident with the hair bundle’s plane of mirror symmetry. (B) Focusing the microscope above the hair bundle revealed the diffracted image of the iontophoretic electrode’s tip, which was usually situated 1 Rm above the bundle’s side. The electrode was bent so that its distal portion was oriented perpendicular to the bundle’s plane of symmetry. (C) lontophoretic pulses were delivered at 63 different locations on an 8 x 8 grid. The two axes of movement of the piezoelectrical micromanipulator that positioned the iontophoretic electrode were not perpendicular and did not have identical voltage sensitivities. The grid was accordingly somewhat rhomboidal, and the horizontal and vertical movement increments were respectively about 570 nm and 1100 nm. While the electrode paused at each of the indicated points, the hair bundle was transiently displaced and an electrical response recorded.

representative responses to gentamicin iontophoresis are shown in Figure 4; application of dihydrostreptomycin produced a similar pattern of blockage. Computer Modeling of Responses for Hypothetical Channel Distributions To demarcate more precisely the region within which the transduction channels are confined, we used a diffusion equation to estimate the spatial and temporal extents of the cloud of aminoglycoside molecules released by an iontophoretic pipette. We simplified a

very complex geometrical problem by assuming that the hair bundle is not a significant barrier to diffusion; this assumption is supported by evidence detailed in the Discussion. The concentration of blocker, [A](r,t), following the onset of an iontophoretic ejection can then be estimated from the equation for free diffusion from a point source (Dionne, 1976; Purves, 1977a; Berg, 1983), [A](r,t)

= [~/,,,,,/[.‘r; (47Or)]j . IS - erf[r/(4ti)1/2]j.

Here r is a channel’s distance from the iontophoretic pipette, t is time, I,,,, is the iontophoretic current, .Y is the Faraday constant, % is the diffusion coefficient of the blocker, and < is the ratio of the blocker’s transference number to its valence. The value of B is unity at all times during an iontophoretic pulse; for times following the termination of a pulse of duration fronto, B = erfrrl[4~t-t,,,,,)11’*J. This expression assumes that iontophoresis is the only transport process and that release is directly proportional to the iontophoretic current. Because transduction channels are evidently dispersed within the hair bundle (Hudspeth and Jacobs, 1979), the concentration of gentamicin that they experience is not uniform. It is nevertheless possible to predict the extent to which a given iontophoretic ejection of blocker should reduce the receptor current. If the extracellular drug concentration at the nth transduction channel is [A],, that channel’s average contribution to the receptor current, i,,, is

---I r-+-r-“OOPA v 20 ms

l-l Figure 2. Blockage of Receptor Application of Gentamicin Near

IlOnA Currents by lontophoteric the Hair Bundle’s Top

An inward receptorcurrent (uppertraces, thin line) ensued when the bundlewas displaced in the positivedirection (middle trace). The gradual decline in this control response resulted from adaptation of the transduction process, theexponential timeconstant of which is typically 20-30 ms. When gentamicin was iontophoretically applied near the top of the hair bundle (bottom trace), the receptor current was partially blocked (upper traces, thick line).

(2)

Neuron 412

A

c

Figure 3. Spatial Sensitivity of Receptor Currents to Blockage by Centamicin

d

(A) During mechanical stimulation, 5-ms iontophoretic pulses of 5 nA were delivered at 63 locations on agrid approximately 1 urn above the hair bundle. The hairbundle diagram indicates the positions of the iontophoretic electrode’s tip; in this and subsequent figures, the diagram was constructed from a tracing of the experiment’s video record. The usual pattern of iontophoresis was along each of transects a through h, sequentially from position 7 through position 8; about half of the hair bundles were oriented in the opposite direction. Because the entire scan was repeated several times in each experiment with similar results, the pattern of blockage was not caused by deterioration of the response during protracted recording. (B) The receptor currents for 63 positions of the iontophoretic pipette are arranged 50 pA and labeled in a grid corresponding to that looms in the adjacent diagram. In addition to the slow decline in each responseas a result of adaptation, some traces display a sharp, transient upstroke due to channel blockage by iontophoresed gentamicin (for an example, note the arrow at trace ~2). Blockage was extensive when the pipette was oriented toward the bundle’s top, but the drug’s effect was minimal when the electrode was directed at the bundle’s bottom or well beyond its top. Because of the compressed time base in this display, the records were digitally filtered at a frequency of 250 Hz. abcdefgh

in = icoJU + &41,&)1,

(3)

in which i,,, is the channel’s average control response in the absence of the drug and Kd is the dissociation constant of the blocker from the channel. A hair cell’s receptor current is simply the sum of the individual

B

n

contributions from the total complement of transduction channels. We first employed computer modeling of the blockage of transduction channels by aminoglycosides to discriminate between two specific distributions of the transduction channels: at the top or at the bottom of

Figure 4. Blockage of Receptor bycentamicin inThreeDifferent

.

n

n

IlOnA

Currents Hair Cells

In each panel, receptor currents (thick lines) are shown during iontophoresis at eight equally spaced positions (7 through 8, from top to bottom) along a transect down the hair bundle. lontophoretic pulses (bottom traces) were delivered ap proximately 5 ms after the onset of mechanical stimulation (second traces from bottom). The hair-bundle diagram above each panel indicates the positions of the iontophoretic electrode corresponding to the records shown. (A) Blockage was greatest with the iontophoretic electrode in position 3, which corresponded to the bundle’s top edge. The control traces in this panel (thin lines) were taken during iontophoresis at a distance from the bundle. (B) A larger iontophoretic current produced extensive blockage in another cell. Here the most effective sites of ejection, which corresponded to positions 3-5, again lay at the bundle’s top. In this and the next panel, control receptor currents in the absence of iontophoresis (thin traces) are superimposed for comparison. (0 Blockage was most complete in a third cell around electrode position 3, which coincided with the bundle’s distal region.

Localization

of Transduction

Channels

413

A

I3

Experimental

results

C Model channels

with at top

Model with channels at bottom

lontophoresis at bottom

Figure

5. Comparison

of Experimental

Data with

Computer-Simulated

Responses

for Plausible

Distributions

of Transduction

Channels

(A) Experimental records of receptor currents during iontophoretic ejection of gentamicin at a hair bundle’s top (upper traces, thick line) or bottom (lower traces, thick line). In each instance, and in the other panels, the control response in the absence of gentamicin (thin line) is superimposed. (6) If the transduction channels were located at the stereociliary tips, modeling predicts the records shown for iontophoretic pulses directed at the bundle’s top (upper traces, thick line) or bottom (lower traces, thick line). The calculated responses correspond closely to those observed experimentally. Note that, because the calculated blockage was applied to actual control records, the simulated responses in this and the following figures display electrical noise. (C) Situating the transduction channels at the bases of the stereocilia leads to the traces shown with the iontophoretic electrode pointed at the bundle’s top (upper traces, thick line) or bottom (lower traces, thick line). Here the correspondence between computed and observed responses is poor.

the hair bundle. Morphometrical data on the bullfrog’s saccular hair cells (Jacobs and Hudspeth, 1990) provided the basis for a computer program that calculated the spatial coordinates of the stereociliary tips and bases in each experimentally studied hair bundle. For each location of the iontophoretic electrode, and at 500~ps intervals following the onset of iontophoretie ejection, the concentration of aminoglycoside was calculated with Equation 2 for the base and tip of every stereocilium. On the assumption that the transduction channels are uniformly apportioned among the stereocilia (Hudspeth and Jacobs, 1979), we then employed Equation 3 to estimate the blockage of the receptor current expected for the two channel distributions. Finally, to produce a simulated response, a control record was reduced in proportion to the calculated blockage. In each of four instances tested, disposition of transduction channels near the tips of stereocilia accounted for the observed effects of aminoglycoside application much better than did a basal channel distribution (Figure 5). Iterative Computer Estimation of Channel Distributions The results above indicate that transduction channels located at the bundle’s top mimic the actual channel distribution more faithfully than do channels situated at the bundle’s bottom. In a second approach to modeling the channels’ distribution, we made no a priori assumption about their location along the bundle’s length. We instead assumed the channels to be uniformlydistributedalonganoriented Iinesegmentthat could be situated anywhere in and around the hair bundle. Because of the bundle’s plane of morphological symmetry, we approximated a presumably mirrorsymmetrical channel distribution by constraining the line segment within the plane of symmetry. We then specified the shape of each individual hair bundle,

obtained from the videotape record of the experiment, and an initial location for the line segment, usually within and near the bottom of the bundle. For each of the 63 positions in which the iontophoretic pipette was placed, we used Equation 2 to compute the average aminoglycoside concentration along the line segment at 500-ps intervals following the onset of iontophoretic ejection. The drug’s predicted effect on the receptor current was calculated from Equation 3, and the error in the prediction was quantified by summing the squared difference between the predicted and observed responses. The Simplex algorithm (Caceci and Cacheris, 1984) then systematically varied the length and position of the line segment until the predicted responses agreed optimally with the experimental data. Figures 6A and 6B display a representative comparison between the experimental data and the responses simulated by use of the diffusion equation. The line segmentsthat best represented thechannels’position were located near the top of the hair bundle (Figure 6C). The iterative fitting procedure consistently converged to a similar position, regardless of the coordinates of the line segment used as an initial guess. Similar results ensued when the 63 traces were simultaneously fitted with a single line segment. A distribution of channels near the hair bundle’s top best fit the observations from each of the three experiments examined in detail (Figures 6C and 6D). Similar results ensued when the calculations were performed with more complex hypothetical distributions of channels, for example when the line segment was not constrained to lie in the bundle’s plane of symmetry. A Temporal Bound on the Positions of Transduction Channels The boundaries within which the transduction channels are confined could also be delimited by analyzing

Neuron 414

+ A

A

-

r

>‘-

I200pA

vFigure

6. Estimation

of the

Location

50 ms

of Transduction

Channels

by Iterative

Comparison

of Responses

with

Model

Data

(A) A set of eight receptor-current traces obtained during gentamicin iontophoresis (thick lines). In this and the adjacent panel, the control response in the absence of iontophoresis is shown for comparison (thin lines). (B) The calculated responses (thick lines) for the distribution of transduction channels that best mimicked the actual experimental records. (C) For the data in the previous panels, the bars superimposed upon the upper hair-bundle diagram represent the 63 line segments upon which linear distributions of transduction channels would best account for the extent and time course of blockage. The lower diagram presents the average distribution of channels calculated for responses with the iontophoretic electrode in all 63 standard positions. The coordinates of each end of every line segment were separately averaged; the average line segment extends between the resultant points. The error bars at each end of the average line segment indicate the standard deviations, in the vertical and horizontal dimensions, of the individual line segments from the average line segment. (D) These hair-bundle diagrams indicate the averages of the optimal channel distributions for 2 other hair cells, together with the corresponding standard deviations. In each instance, the optimal channel distribution lies near the bundle’s beveled top.

cated on the hair-bundle diagram. At least 50% of the cell’s mechanically activated conductance was blocked before the termination of the 5-ms iontophoretie pulse. This implies that the concentration of

the effects of the channel blockers on receptor currents at relatively short times. Consider the receptorcurrent record shown in Figure 7, obtained with the iontophoretic electrode placed at the position indi-

/

I 1 w-n

\ l-l

Figure

7. The Time

(10nA

Course

of Diffusion

Imposes

a Spatial

Bound

on the

Position

of Transduction

Channels

(A) With an iontophoretic electrode situated 1.5 urn from the bundle’s beveled top surface, the receptor current (upper traces, thick line) was reduced by more than 50% before the termination of a 5-ms iontophoretic pulse. The superimposed control record (upper traces, thin line) depicts transduction in the absence of iontophoresis. This level of channel blockage implies that the concentration of blocker experienced by the average transduction channel exceeded &, 16 PM. (B) Concentric circles represent the spherical volumes within which the average concentration of blocker was at least 16 uM at various times during and after a 5-ms iontophoretic pulse. The five inner circles, three of which are labeled, correspond to the spread of drug at I-ms intervals following the onset of iontophoresis. The outermost circle represents the maximal excursion of the 16 uM boundary, which was reached slightly more than 2 ms after ejection concluded. The central dot indicates the position of the electrode’s tip. At least half of the transduction channels must have been confined within the next-to-largest, 5-ms circle.

Localization

of Transduction

Channels

415

blocker experienced by the average channel at this time must have been at least 16 j.tM, the experimentally determined & for gentamicin in the presence of 4 mM Ca2+. The locus of points at which the concentrationof blocker reached16uMatl-Smsaftertheonset of the pulse can be estimated from Equation 2; these volumes are represented as concentric circles in Figure 7. It is clear that 50% or more of the cell’s transduction channels occur within 1 pm of the beveled top edge of the hair bundle. Ensemble-variance analysis of the receptor currents (Holton and Hudspeth, 1986) from 2 cells revealed that at least 90% of the cells’transduction channels were activated by our usual mechanical stimuli (data not shown). Aminoglycoside application immediately above the tops of these 2 hair bundles led to the nearly complete blockage of receptor currents. This finding indicates that essentially the entire complement of transduction channels occurs near the stereociliary tips.

Specificity of the Aminoglycoside Effect The iontophoretic ejection of aminoglycoside drugs, which involved current flow through the fluid in which the hair cells lay, might have produced artifactitious electrical signals resembling transductionchannel blockage. We accordingly performed control experiments with iontophoretic electrodes filled with 3 M KCI, rather than with aminoglycoside antibiotics. When a current pulse of the usual magnitude was passed from such an electrode at a position adjacent to the hair bundle, whole-cell recording showed only small, capacitative transients at the pulse’s onset and termination. In no position of the iontophoretic electrode was the receptor current interrupted as found with aminoglycoside iontophoresis. Another confirmation of the specific effect of amino glycoside iontophoresis was that the receptor current was unaffected by passage of negative current pulses from the iontophoretic electrode. Current of this poA

B

larity should retard, rather than promote, expulsion of the positively charged gentamicin molecules. It might be argued that the reduction in transmembrane current following the application of aminoglycosides resulted from an effect of the drugs on ion channels other than the transduction channels themselves. Three lines of evidence speak against this possibility. First, hair bundles of the frog’s sacculus evidently posssess no ion channels other than the transduction channels, which account for the resting conductanceof theapical membranesurface(Roberts et al., 1990). There are thus no other known channels that could be targets for aminoglycoside antibiotics. Second, the electrical response required the normal operation of mechanically sensitive channels. When applied to otherwise competent hair cells that had high input resistances and normal voltage-activated currents, but that did not respond to mechanical stimulation, gentamicin had no effect on membrane currents. Finally, a high concentration of aminoglycoside directed against a hair bundle reduced the membrane current to the same level achieved when the bundle was given a saturating negative stimulus (data not shown; Kroese et al., 1989). Another potential source of artifactitious electrical signals was hair-bundle motion (discussed in Hudspeth, 1982; Ohmori, 1988). To eliminate this threat, we ordinarily performed iontophoretic ejections while the hair bundles were stationary, but displaced from their resting positions. We found, moreover, that the aminoglycoside effect did not require mechanical stimulation per se. When a hair bundle is undisturbed, a small fraction of the transduction channels are open; these too could be blocked by drug application at the tips, but not at the bases, of the stereocilia (Figure 8A). A similar effect was observed when a stimulus probe was affixed to the hair bundle, but left stationary throughout the experiment. A final possible concern was that gentamicin ejected near the bundle’s bottom was prevented by a hypothetical diffusional barrier from reaching channels sitFigure 8. Control Experiments to Exclude Movement Artifacts and Diffusional Barriers

(A) The hair bundle’s spatial sensitivity to blockage of receptor current is independent of mechanical stimulation. When the bundle was free of the stimulus probe and remained stationary throughout the experiment, iontophoretic ejection at the bundle’s top nevertheless blocked the resting current through transduction channels (upper trace). No effect was observed when the electrode lay at the bundle’s bottom (lower trace). Restoration of mechanical stimulation confirmed that this cell remained fully responsive after exposure to gentamicin. (B) The receptor current of a mechanically stimulated hair cell was partially blocked by iontophoresis of gentamicin at the bundle’s top (top traces, thick line). Blockage was much less extensive when an identical iontophoretic pulse was delivered at the bundle’s bottom (middle traces, thick line). No additional blockage ensued when the iontophoretic electrode’s tip was advanced into the hair bundle’s bottom, so that it lay extracellularly among the basal tapers of the stereocilia (bottom traces, thick line). The control record in the absence of iontophoresis is superimposed on each response (thin lines).

Neuron 416

uated at the stereociliary insertions. To exclude this possibility, we ejected gentamicin in one instance while the iontophoretic electrode was insinuated deeply into the basal region of a stimulated hair bundle, among the basal tapers of the stereocilia. This procedure did not enhance the slight blockage that occurred upon iontophoresis just outside and at the bottom of the bundle (Figure 8B).

Discussion Transduction Channels Are Blocked Aminoglycoside Application at the Tops of Hair Bundles

by

In every hair cell that we examined, aminoglycoside antibiotics effectively blocked receptor currents when focally applied near the tops of hair bundles. In contrast, application of the drugs near the bottoms of bundles consistently proved far less effective. These results imply that the transduction channels occur near the stereociliary tips. Our data also indicate that few if any transduction channels exist at the stereociliary bases. It is extremely unlikely that the observed lack of channel blockage when the iontophoretic pipette was positioned at a bundle’s bottom was due to inadequate diffusional access of the drugs to their site of action. There is no electron microscopic evidence for diffusional barriers around hair bundles, including those of the bullfrog’s sacculus (Jacobs and Hudspeth, 1990); macromolecular markers readily label the surfacesof all stereocilia, even those in a bundle’s interior (Santi and Anderson, 1987). Moreover, the swift capacitative charging of a hair cell’s apical membrane surface (Roberts et al., 1990) indicates that the bundle possesses no significant barrier to diffusion by small ions. Channel blockage commences as rapidly as expected if gentamicin diffuses untrammeled from an electrode to its target channels. Finally, we observed little change in the extent of channel blockage when we ejected gentamicin from an iontophoretic electrode whose tip was placed among the basal tapers of the stereocilia. The experiment documented in Figure 7 bears critically on the possibility of diffusional barriers around or within the hair bundle. The demonstrated rapidity of blockage indicated that more than half of the transduction channels lay so close to the bundle’s top that gentamicin occluded them before the drug’s concentration at the bundle’s base even approached the Kd for interaction with the channels. Because any barrier to free diffusion could only retard the spread of drug, the existence of such a barrier would if anything strengthen theargumentforthechannels’occurrence at the bundle’s top.

The Time Course of Channel The extent experimental transduction

and time conditions, channels

Blockage

course could reside

of

blockage, be best near the

under explained tips of

all if ste-

reocilia. A hypothetical location of transduction channels near the stereociliary bases, by contrast, always provided an unsatisfactory correspondence between experiments and models. Open-channel blockage by many other drugs is known to be very fast. For the nicotinic acetylcholine receptor at the neuromuscular junction, the blocking rate constant for carbachol is approximately IO* s-‘.M-l (Ogden and Colquhoun, 1985), a value close to the limit imposed by the drug’s rate of diffusion (Dionne, 1976). If in the state diagram of Equation 1 we assume a comparable blocking rate constant, k23, for the effect of aminoglycoside molecules on the transduction channel, then the Kd value of 16 PM implies an unblocking rate constant, kj2, near 1600 ~0. These values suggest that the relaxation time constant for blockage, (k,[A] + kJ1, is small on the time scale characteristic of drug diffusion. Experimental evidence supports the supposition that gentamicin’s action on receptor currents is quite fast. In transepithelial voltage-clamp recordings (Corey and Hudspeth, 1983a), we found that 50 PM gentamitin blocks receptor currents within 100-150 ps after the onset of a stimulus, as soon as the channels open (data not shown). This result suggests that the rate of blockage in our iontophoretic experiments is limited by the availability of the relatively slowly diffusing drug. It is therefore reasonable to assume that the blockage of transduction channels is in equilibrium with the local drug concentration, and to use Equation 3, not only to estimate the fraction of channels blocked at a given concentration of aminoglycoside, but also to predict the time course of the blockage that results from iontophoretic application of these drugs. Even if the rate constants for drug binding and unbinding were smaller than we believe, the fundamental conclusions of our study would not be affected. Slow blocking and unblocking would have an effect similar to that of a diffusion barrier, strengthening the association of the transduction channels with the bundle’s top in experiments such as that of Figure 7. It is noteworthy that the predicted receptor currents in Figure 5 were calculated without free parameters whose values could be varied to optimize the correspondence between the computed and observed blockage of responses. The agreement between the simulated responses and the experimental data therefore provides especially strong evidence that the transduction channels occur at the hair bundle’s top. We estimated the concentration of a drug at potential sites for the transduction channels by a diffusion relation (Equation 2) derived on the assumption that the aminoglycoside flux is directly proportional to the iontophoretic current. At the onset of an iontophoretie pulse, however, there is a surge of capacitative current across the wall of the iontophoretic electrode that carries no drug. For iontophoretic electrodes whose time constants were typically about 1 ms, we ascertained that the delay in release associated with

Localization

of Transduction

Channels

417

capacitative currents was minor. We calculated the drug concentration by numericallyconvolvingthe impulse response for the drug concentration (Dionne, 1976; Purves, 1977a; Berg, 1983),

c’[Al(r,t) = lV,o,,,(t)dt/e'r(4~t)3'2]1

(4)

. exp(-r*/4gt),

with the time course of the iontophoretic current, I,,,,,(t), which was measured from the voltage drop across the iontophoretic electrode. The main consequence of considering the electrode’s time constant was the introduction of an approximately in the predicted onset of blockage. In the presence of a backing current, released iontophoreticallycan bedepleted

I-ms

delay

a drug to be at the elec-

trode’s tip (Purves, 1977b). Restoration of the average drug concentration following the onset of iontophoretie ejection could account for the discrepancy of l-3 ms in the onset of blockage that we observed between some experimental traces and the corresponding models (Figure 5). If we were to compensate for the delays in the release of blockers, our data would suggest an even closer proximity of the transduction channels to the top of the hair bundle. Relation of Findings to Earlier Results The present results indicate that mechanoelectrical transduction occurs at or near the tips of stereocilia. This conclusion accordswith those from earlier investigations by two other, independent techniques: analysis of current sinks by extracellular recording (Hudspeth, 1982) and measurement of Ca2+ entry with the fluorescent dye flue-3 (Huang and Corey, 1990, Biophys. J., abstract). The resultsof all three studies seemingly conflict with those of another investigation, in which detection of Ca2+ with the dye furawas taken to reveal a transduction site at the hair bundle’s bottom (Ohmori, 1988). In an effort to determine the basis of the discrepency between our results and those of Ohmori (1988), we attempted to repeat and improve upon the earlier experiments with fura(unpublished data). Ohmori loaded the chicken’s hair cells with the fluorescent dye as its membrane-permeant acetoxymethyl ester. This approach can yield misleading results when the dye enters mitochondria and Ca2+-sequestering elements of the endoplasmic reticulum and reports an elevated concentration, not in the cytoplasm near where Ca2+ enters the cell, but in organelles where it accumulates (Williams et al., 1985). We instead employed 100 PM furain its charged form in an internal solution without other Ca2+ buffers. Ohmori did not record electrical responses from the cells whose fluorescence he measured; the Ca2+ signals therefore may not have been associated with mechanoelectrical transduction. We made tight-seal, whole-cell, voltage-clamp recordings from numerous hair cells of the bullfrog’s sacculus and analyzed the results from 6 cells whose receptor currents at -70 mV exceeded

-100 pA in standard saline solution and from 4 cells with responses larger than -50 pA in a solution containing 50 mM Ca2+ as the dominant cation. In the original study, fluorescence was measured after stimulation for up to 410 ms, a time long enough for Ca2+ to diffuse extensively from its site of entry. We instead made measurements during the first 50 ms of hairbundle deflections and obtained a satisfactory optical signal by accumulating the results of 100 successive stimulations with a charge-coupled-device camera of high sensitivity. To minimize possible movement artifacts in the fluorescence ratio, we interleaved successive responses taken with illumination at 340 nm and 380 nm. Despite these efforts, we were unable to elicit a significant change in the 3401380 nm fluorescence ratio at any consistent site in a stimulated hair cell. A healthy cell, whose input resistance exceeded 200 MQ typically displayed a fluorescence ratio consistent with an intracellular Ca2+ concentration of 30100 nM. This value did not change detectably during hair-bundle stimulation. As the cell’s input resistance deteriorated during protracted recording, the apparent Ca2+ concentration usually rose in the apical cytoplasmic region, including the cuticular plate. Until a cell began to disintegrate, however, the Ca2+ concentration reported by the dye ordinarily remained much lower in the hair bundle than elsewhere in the cell. The concentration of free Ca2+ in stereocilia appears to be highly regulated; in addition to being buffered by Ca2’-binding proteins such as calmodulin, calbindin,and perhapsfimbrin (Shepherd et al., 1989; Gillespie and Hudspeth, 1991), the ion may be efficiently extruded by an exchanger or pump. We believe that the concentration of free Ca2+ increases at the bottom of a stimulated hair bundle under some conditions (Ohmori, 1988), not as a result of Ca2+ influx through transduction channels there, but instead because of Ca2+ accumulation in a deteriorating cell. By implicating the hair bundle’s top as the site of transduction, our data indicate that the gating springs that open transduction channels (Corey and Hudspeth, 1983b) are to be found near the stereociliary tips. The results are consistent, for example, with the hypothesis that each gating spring is a tip link, a filamentous strand that connects the tip of a stereocilium to its tallest neighbor (Pickles et al., 1984). The major experimental challenge in confirming this model for mechanoelectrical transduction remains a demonstration that the tip links are, in fact, stressed when the hair bundle is deflected. Experimental Hair

Procedures

Cell Preparation

Solitary hair cells were enzymatically isolated from the sacculi of adult bullfrogs (Rana catesbeiana) digested in situ with papain (Calbiochem Corp., San Diego, CA; Assad et al., 1989). The cells were maintained in oxygenated saline solution containing 110 mM Na’, 2 mM K+, 4 mM Ca*+, 118 mM Cl-, 5 mM HEPES, and 3 mM o-glucose; the pH was adjusted to 7.25. The solution in the experimental bath was renewed between successive recordings.

Neuron 418

Cellular dissociation and experimentation were conducted at room temperature (21OC). Dissociated cells were placed in a 500~ul experimental chamberandallowed tosettleonto,and toadherefirmlyto, theconcanavalin A-coated coverslip bottom. Each of the cells used in experiments was so situated that the hair bundle’s plane of mirror symmetry paralleled the chamber’s bottom surface (Figure 1A). A rotating stagewas used to orient each cell so that mechanical stimuli were directed along the bundle’s axis of morphological symmetry, along which mechanical sensitivity is greatest (Shotwell et al., 1981). The cells were observed with a mechanically stabilized microscope (UEM, Carl Zeiss, Oberkochen, Germany) equipped with a 40 x, water-immersion objective lens (numerical aperture 0.75) and differential interference contrast optics. A chalnicon video camera (5440, Cohu Inc., San Diego, CA) and VHS casette tape recorder (NV-8950, Panasonic Industrial Co., Secaucus, NJ) were used throughout each experiment to document the stimulus’s amplitude, the hair bundle’s dimensions, and the sites of iontophoretic ejections. The optical and videotape systems were calibrated with a IO-urn stage reticle (Carl Zeiss). The freeze-frame feature of the video recorder was employed after each experiment to display the iontophoretic electrode’s positions, which were transferred from the monitor to a plastic overlay and measured with a precision of *I60 nm.

be used to determine its resistance and to pass controlled currents, which were measured with a resistor in series with the current-injecting circuit. About 5 ms after the onset of the receptorcurrentinduced bydeflectionofthehairbundle,aminoglycoside was iontophoretically ejected by passage of a positive current of 2-10 nA for 5 ms. Between iontophoretic pulses, a continuous backing current of -200 pA (Kroese et al., 1989) was passed to minimize any deleterious effects of aminoglycosides on the hair cells. Even in the longest recording, 50 min in duration, thecumulative bath concentration of gentamicin following numerous iontophoretic pulses was less than 1 nM. During a typical recording, S-30 min in duration, the receptor current of a hair cell subjected to aminoglycoside iontophoresis did not deteriorate conspicuously morequickly than did that of a control cell. The iontophoretic electrode was positioned with a twodimensional piezoelectrical stimulator (Corey and Hudspeth, 1980) controlled by a computer. This electrode ordinarily delivered drug pulses to the hair bundle at 63 locations on an 8 x 8 grid that lay parallel to the chamber’s bottom and about 1 urn above the hair bundle’s side. On some occasions, we performed high resolution measurements at 255electrode positions.To preclude any possible movement artifacts, each iontophoretic pulse was delivered 5 ms after the deflected hair bundle had reached its final position.

Mechanical Stimulation The hair bundlewas stimulated with aglass probe, whose500-nm tip either adhered to the kinociliary bulb or pressed against the beveled back edge of the bundle (Hudspeth, 1982). The probe was moved in the horizontal plane with a one-dimensional, piezoelectrical stimulator (Corey and Hudspeth, 1980); the standard stimuli were 700~nm deflections of the hair bundle’s top, delivered for 50 ms at approximately 2-s intervals. To avoid mechanical resonance, the input signal to the stimulator was low pass-filtered at a half-power frequency of 500 Hz through an (I-pole Bessel filter (852, Wavetek, San Diego, CA). To minimize mechanical disturbances, the apparatus was assembled on a damped, air-suspended table (CS-34, Newport Bio-Instruments, Fountain Valley, CA). Experiments were conducted in an electrically shielded, acoustically isolated room, which was isolated from building vibrations by being constructed atop a 6.6 x 4.9 x 2.1 m pad containing approximately 160 metric tons of concrete and situated on bedrock.

Diffusion Coefficients of Aminoglycoside Antibiotics The diffusion coefficients (9) for aminoglycosides mated from the Stokes and the Einstein-Sutherland (Cantor and Schimmel, 1980),

lontophoresis of Aminoglycoside Drugs Aminoglycoside antibiotics were obtained as sulfates (Sigma Chemical Co., St. Louis, MO). The gentamicin salt, which in its pure form contains 61.4% gentamicin base (M, = 464), was specified by the manufacturer to include 10.6% water. Dihydrostreptomycin sesquisulfate, which when pure consists of 79.9% base (M, = 583.6), contained 2.4% water of hydration. For the iontophoretic application of the drugs to hair bundles, conventional, capillary-filled glass microelectrodes were filled with a 500 mM aqueous solution of gentamicin or dihydrostreptomycin sulfate. In most experiments, the solution was acidified to a pH of 3.5 with 1 N sulfuricacid toincrease thepositivechargeon thedrugs and enhance their iontophoretic mobility. Similar results were obtained, however, when the pipette solution was instead neutralized with KOH. The electrodes had resistances of 60-100 MD when filled with 3 M KCI and about 450 MD when filled with aminoglycosides. Each iontophoretic electrode was bent through an angle of approximately 70” near its tip (Hudspeth and Corey, 1978) so as to impinge vertically upon a hair bundle. Because the electrode’s tip could readily be seen in this orientation, the calibrated fine focus of the microscope could be used to situate the electrode’s tip about 1 urn above the hair bundle’s side. Vertical incidence of the electrode additionally ensured that any possible electro-osmotic jetting of its contents (Hill-Smith and Purves, 1978) would be directed at a right angle to the axis along which we studied drug diffusion, as well as to the hair bundle’s axis of mechanical sensitivity (Shotwell et al., 1981). The iontophoretic electrode was connected to an amplifier (AxoclampZA, Axon Instruments Inc., Foster City, CA) that could

5’ = kT/6nqRF,

were estirelations

(5)

in which k is the Boltzmann constant, T is the absolute temperature, and n is water’s coefficient of viscosity. For each aminoglycoside molecule, the radius of the equivalent sphere, R, was estimated as half the geometric mean of the three orthogonal molecular dimensions measured from space-filling models (CPK, Ealing Corp., South Natick, MA) of the protonated molecular forms. The Perrin shape factor, F, was calculated from the measured molecular asymmetries as 1.05 forgentamicin and 1.03 for dihydrostreptomycin. Application of Equation 5 yielded estimated diffusion coefficients of 4.0 x IO-“’ m*.s-’ for gentamicin and 3.8 x lO-‘O m*.s-’ for dihydrostreptomycin. We confirmed the accuracy of our estimation procedure by considering raffinose, a trisaccharide of size (M, = 504.4) and form (shape factor 1.02) comparable to those of the aminoglycosides. The diffusion coefficient estimated for raffinose by Equation 5,4.15 x lO-‘O m*.sQ, agreed within 7% with the experimental value of 3.85 x lO-‘O m*.sG (adjusted to 21°C; Longsworth, 1953). Blocking Affinity of Centamicin In order to estimate the effect of an aminoglycoside antibiotic on the receptor current, it was necessary to determine the affinity of the drug for the transduction channel. The I&, or drug concentration that blocked half the channels, could be readily determined by extracellular recordings from numerous hair cells. The blockage of transduction channels appears to be a unimolecular process (Kroese et al., 1989). Moreover, because the transduction channels are few in number (Holton and Hudspeth, 1986; Howard and Hudspeth, 1988), they do not occur in excess of drug molecules. The IC, may therefore be safely equated with the dissociation constant, &, of the drug from the channel. Because Ca2+ antagonizes the interaction of aminoglycoside antibiotics with the transduction channel (Kroese and van den Bercken, 1982), we ascertained the Kd of gentamicin for saccular hair cells in the saline solution containing 4 mM Ca*+. Using a two-compartment experimental chamber, we made transepithelial recordings of the summated receptor current from about 1000 hair cells in a macular preparation from which the otolithic membrane had been partially removed (Corey and Hudspeth, 1979a, 1983a). The amplitude of the peak response to 30-ms deflections of the otolithic membrane was determined in standard

Localization 419

of Transduction

Channels

saline solution to which gentamicin sulfte was added at seven concentrations in the range I-250 PM; control records were obtained between successive drug exposures. The concentrationresponse curve closely fit a rearranged Langmuir-isotherm relation; the average half-blocking concentration and presumptive Kd from two determinations was 19 PM. This value is in excellent agreement with earlier determinations at other Ca2+ concentrations (Kroese et al., 1989). Although the resting membrane potential of hair cells in the transepithelial recording preparation is not precisely known, microelectrode measurements suggest a value near -60 mV in a comparable ionic environment (Hudspeth and Corey, 1977; Shotwell et al., 1981). Becausechannel blockage isvoltage-dependent (Hudspeth and Kroese, 1983, J. Physiol., abstract; Ohmori, 1985; Kroese et al., 1989), Kd should be adjusted for our whole-cell recordings at a holding potential of -70 mV. On the assumption that genntamicin binds at the same site as dihydrostreptomycin, this correction (Kroeseet al,. 1989) reduces &to 16 PM, the value employed throughout our data analysis. Efficiency of lontophoretic Release of Centamicin To estimate the concentration of gentamicin in the vicinity of a hair bundle, it was necessary to relate the number of gentamicin molecules expelled from an iontophoretic electrode to the amount of current passed. Large cations are in general not efficiently ejected from fine pipettes. A clear example is that of acetylcholine, which under experimental conditions comparable to ours carries only a minute fraction (0.0048) of the charge passed during an iontophoretic pulse (Kuffler and Yoshikami, 1975b). This value is much smaller than one would expect on the basis of the charge, concentration, and relative mobility of acetylcholine in the iontophoretic electrodes. In our calculations we used a value of 0.013 for c, the ratio of gentamicin’s transference number to its valence. This value is the minimum compatible with the observed time course and extent of blockage. Our conclusion that mechanoelectrical transduction channels are located near the top of the hair bundle was reached on the basis of the relative sensitivity of transduction channels to blockage by aminoglycosides, and is therefore independent of our estimate of c. Let us contemplate the consequences of different estimates of con the interpretation of the experiment described in Figure 7. Lower values of 5 would lead to smaller radi for the spheres within which the concentration of gentamicin exceeded &, implying an even closer association between the transduction channels and the bundle’s tip. A value foriof 0.122, an upper limit calculated on the basisof the relative mobility of gentamicin, would increase the radius of the sphere at theend of the iontophoretic pulse to4.4 pm.This result would still situate the transduction channels in the upper half of the hair bundle. As further support for our estimate of r, we measured the efficiency of iontophoresis of a gentamicin congener, isepamicin (Sch 21420, a 2-hydroxy-3-aminopropanoic acid amide of gentamicin 8; base M, = 556), 14C-labeled to a specific activity of 147 CBq mol-’ with a radiochemical purity of 97%. The tip of a pipette, filled with 1 ~1 of a 500 mM solution of the drug’s sulfate salt, was immersed in 500 ~1 of standard saline solution. After passing IO-nA, 5-ms pulses of positivecurrent from theelectrode at SO&ms intervals for 2.0-3.8 hr, we ascertained the 14C activity in the solution by scintillation counting. In two determinations, the mean ratio of the drug’s transference number to its valence was 0.03. Although isepamicin differs slightly from gentamicin. it is sufficiently similar in size and charge that this control supports our estimate of c. WholeCell Recording and Data Acquisition Receptor currents were recorded using the perforated-patch variant (Horn and Marty, 1988; Korn and Horn, 1989) of the whole-cell, voltage-clamp recording technique (Hamill et al., 1981). Recording pipettes were pulled from glass capillaries 1.7 mm in outer diameter (Boralex, Rochester Scientific Co., Inc., Rochester, NY), then bent near their tips through an angle ot approximately 60° and heat-polished to internal tip diameters of 1-3 Bm. The pipettes were filled with a filtered internal solution

containing either 130 mM Cs’, 105 mM aspartate, and 10 mM Clor 130 mM Cs’, 65 mM aspartate, and 50 mM Cl-. The pH of each internal solution was buffered at 7.25 with 5 mM HEPES. The free Ca2+ concentration was maintained at 10 nM and the free Mg2+ concentration at 2 mM with 1 mM ECTA. The osmolality of the internal solutions was roughly 220 mmol.kg-‘; when filled with these solutions, pipettes typically had resistances of 5 Ma. Immediately before recordings, nystatin (Sigma Chemical Co.), dissolved in dimethyl sulfoxide at a concentration of 10 g.l-‘, was added to the internal solution to achieve a final concentration of SO-200 mg I-‘. A chlorided silver wire connected the pipette with the headstage of a voltage-clamp amplifier (EPCJ, List-Electronic, Darmstadt, Germany). The amplifiers used for whole-cell recording and iontophoresis were referenced in common to a silver-silver chloride wire, which was connected to the bath by an agarose bridge secured to the microscope’s objective lens. Receptor currents, low pass-filtered at a half-power frequency of 1.5 kHz with an 8-pole Bessel filter (Wavetek), were sampled at SOO-us intervals. The experiments were controlled by a computer (PDP11/73, Digital Equipment Corp., Maynard, MA) running programs written in BASIC-23 (Indec Systems, Sunnyvale, CA). The tip potentials for the first and second internal solutions, for which we compensated in nulling the current flowing into the bath from each pipette, were respectively -3 mV and +6 mV. After formation of a tight seal between the pipette and the cellular surface, the pipette’s voltage was clamped at a holding potential of -70 mV and part of the pipette’s capacitance was electronicallynulled. Electrical access tothecell’s interior usually developed within a minute after the tight seal was established. The series resistance of the electrode during a recording was about 10 MQ of which approximately 50% was compensated. Acknowledgments The authors thank Mr. R. A. Jacobs for perfecting the experimental apparatus, providing a computer program for estimation of stereociliary positions, and preparing the figures. Messrs. T. W. Hainze, 0. A. Mon, and K. L. Vahle effectively designed and supervised construction of the isolated recording facility. Dr. G. H. Miller of Schering-Plough Corp. kindly provided a sample of ‘QZ-isepamicin; Dr. W. M. Roberts made useful suggestions about the analysis of data. Drs. J. L. Allen, J. A. Assad, P. C. Gillespie, and P. Middleton and Messrs. Jacobs, W. P. Stanford, and R. C. Walker offered valuable comments on the manuscript. This research was supported by grant DC00317from the National Institutes of Health and by agrant from the Perot Family Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

April

16, 1991; revised

May

25, 1991.

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