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Previous Article | Next Article
The Journal of Neuroscience, April 15, 1998, 18(8):2962-2973
Electrical Properties of Frog Saccular Hair Cells: Distortion by Enzymatic Dissociation
Cecilia E. Armstrong and William M. Roberts Institute of Neuroscience, University of Oregon, Eugene, Oregon 97403-1254
ABSTRACT
Top
Abstract
Introduction
Although it is widely accepted that the electrical resonance seen in many types of auditory and vestibular hair cells contributes to frequency selectivity in these sensory systems, unexplained discrepancies in the frequency (f) and sharpness (Q) of tuning have raised serious questions. For example, enzymatically dissociated hair cells from bullfrog (Rana catesbeiana) sacculus resonate at frequencies well above the range of auditory and seismic stimuli to which the sacculus is most responsive. Such disparities, in addition to others, have led to the proposal that electrical resonance alone cannot account for frequency tuning. Using grassfrog (Rana pipiens) saccular hair cells, we show that the reported discrepancies in f and Q in this organ can be explained by the deleterious effects of enzyme (papain) exposure during cell dissociation. In patch-clamp studies of hair cells in a semi-intact epithelial preparation, we observed a variety of voltage behaviors with frequencies of 35-75 Hz. This range is well below the range of resonant frequencies observed in enzymatically dissociated hair cells and more in tune with the frequency range of natural stimuli to which the sacculus is maximally responsive. The sharpness of tuning also agreed with previous studies using natural stimuli. In contrast to results from enzymatically dissociated hair cells, both a calcium-activated K+ (KCa) current and a voltage-dependent K+ (KV) current contributed to the oscillatory responses of hair cells in the semi-intact preparation. The properties of the KCa and the Ca2+ current were altered by enzymatic dissociation. KV and a small-conductance calcium-activated K+ current were apparently eliminated.
Key words: frog saccular hair cells; semi-intact epithelial preparation; enzymatically dissociated; electrical resonance; voltage oscillations; K+ currents; Ca2+ current; papain
INTRODUCTION
Auditory and vestibular processing involves the decomposition of complex stimuli, such as sound waves and substrate vibrations, into their constituent frequency components. A remarkable variety of sophisticated mechanisms, often used in concert, have evolved for this purpose. In all vertebrates, the physical properties of structures in the inner ear surrounding the mechanosensory hair cells serve as initial filters. In mammals, there is good evidence that the exquisite sensitivity and sharp frequency discrimination of the cochlea are achieved through active mechanical feedback provided by the outer hair cells (for review, see Ashmore and Kolston, 1994
). Such a feedback mechanism has not been demonstrated in nonmammalian vertebrates, although there is ample evidence that hair cells from these species can exert forces when stimulated (for review, see Hudspeth, 1997
In an electrically tuned hair cell, the membrane potential typically undergoes damped sinusoidal oscillations in response to injected current steps (Crawford and Fettiplace, 1981
Measurements of electrical resonance in enzymatically dissociated bullfrog saccular hair cells suggest that these hair cells are most sensitive to stimuli of 120 ± 24 Hz (mean ± SD) (Hudspeth and Lewis, 1988b
Using whole-cell and perforated patch recording methods in conjunction with standard pharmacological agents, we compared the membrane properties of enzymatically dissociated grassfrog saccular hair cells and hair cells in a semi-intact epithelial preparation. Our results from enzymatically dissociated hair cells were identical to previous reports from bullfrog (Lewis and Hudspeth, 1983
Some of the results presented here have appeared in abstract form (Armstrong and Roberts, 1996
MATERIALS AND METHODS
All recordings were made from grassfrog saccular hair cells, and all procedures were performed at room temperature (20-25°C).
Hair cell preparations. Enzymatically dissociated hair cells were prepared using the same procedure and enzyme (papain 5125; Calbiochem, La Jolla, CA) as in previous experiments on frog saccular hair cells (Lewis and Hudspeth, 1983
, 3 D-glucose, 5 HEPES) to which 0.25 mg/ml papain and 2.5 mM L-cysteine had been added. Cysteine is thought to activate papain by reducing disulfide bonds. After enzyme treatment the sacculus was transferred to low-calcium solution containing 0.5 mg/ml BSA for at least 1 hr to stop the enzyme reaction. The epithelium was then transferred to a recording chamber containing low-calcium solution, in which the hair cells were dissociated by gently rubbing the saccular epithelium with a fine piece of dog hair (Canis familiaris var. Labrador).
Access to hair cells in the semi-intact epithelial preparation was achieved by removing the overlying otolithic membrane and then plowing a furrow (Fig. 1) through the saccular epithelium with a beveled piece of dog hair. The epithelium was then transferred to a recording chamber, mounted, and the debris surrounding live hair cells was removed with a suction pipette. Consistently better preparations were obtained when the epithelium was incubated in low-calcium solution for at least 1.5 hr before the furrow was plowed. Although this preparation gave us access to hair cells throughout the saccular epithelium, no attempt was made to correlate position with variations in membrane properties or to identify the hair cells morphologically.
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Figure 1. Scanning electron micrograph illustrating the semi-intact epithelial preparation. A beveled piece of hair was used to plow a furrow through the saccular epithelium (A), revealing basolateral aspects of hair cells (B). Scale bars: A, 50 µm; B, 5 µm.
Recording procedures. Both preparations were visualized on an upright microscope (Zeiss, Thornwood, NY) equipped with a 40× water immersion objective and differential interference contrast optics. Whole-cell (Hamill et al., 1981
. Correction for the liquid junction potential (Vjp) at the pipette tip was made using the values for K+-aspartate and Cs+-aspartate internal solutions from previous work (Vjp =
13 mV for both; Roberts et al., 1990
pClamp6 (Axon Instruments, Foster City, CA) was used to generate voltage- and current-clamp commands and to record the resulting data. Data were filtered at either 5 or 10 kHz and digitized at 20 kHz. All voltage-clamp recordings were made from a holding potential of
Solutions and pharmacological agents. All solutions were adjusted to have a pH of 7.25 and an osmotic strength of ~220 mOsm. The recording chamber was continuously perfused with a perilymph-like solution (normal extracellular solution) containing (in mM): 112 Na+, 2 K+, 1.8 Ca2+, 0.7 Mg2+, 119 Cl
For experiments in which the papain solution was perfused onto the semi-intact preparation, papain (0.25 mg/ml) and L-cysteine (2.5 mM) were added to normal extracellular solution (the same concentrations used in the dissociation procedure). In control experiments, papain was heat-inactivated by boiling for ~5 min, either before or after adding the remaining components of the solution. In either case the osmolarity was adjusted to ~220 mOsm after boiling.
Data analysis. Most data analysis was performed using pClamp6. Additional analysis and plotting were performed using Excel 4.0 (Microsoft Corp., Redmond, WA), IgorPro (WaveMetrics, Lake Oswego, OR), and Mathcad (MathSoft, Cambridge, MA). Despite using low-resistance recording pipettes and active series resistance compensation, the residual uncompensated series resistance (Rseries; ~4 M
), fitted to the falling phase of the capacitive transient evoked by a small voltage step, by the membrane capacitance determined from the area under the capacitive transient. The Rseries correction made it more difficult to calculate average currents at a given potential, because the corrected membrane potentials varied between cells. Therefore, the data (see Fig. 10D,E), as well as the average outward current at
Another consequence of residual Rseries was that the recorded membrane currents were low-pass-filtered. The only noticeable effect of this filtering was an apparent slowing of the Ca2+ current activation. To correct for this when measuring Ca2+ current activation kinetics, we deconvolved the digitized current traces shown in Figure 10 using the equation:
where Im(n) is the nth point in the digitized current trace, I'm(n) is the deconvolved current trace,
t is the digital sampling interval (50 µsec), and
Quality of resonance. As in previous studies of electrical resonance in hair cells, we measured the quality of resonance (Q) using equations that describe a circuit containing a capacitor in parallel with a resistor and inductor connected in series (Crawford and Fettiplace, 1981
Larger values of Q correspond to more prolonged oscillations and a more sharply tuned cell.
![Q=[(&pgr;&tgr;<SUB><UP>d</UP></SUB>f)<SUP>2</SUP>+0.25]<SUP>1/2</SUP>.](/content/vol18/issue8/fulltext/2962/img002.gif)
RESULTS
Voltage oscillations: variations on the theme of resonance
Enzymatically dissociated grassfrog saccular hair cells that had been exposed to papain for ~30 min before mechanical dissociation exclusively exhibited stereotypical electrical resonance (Fig. 2A), as previously described in bullfrog (Lewis and Hudspeth, 1983
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Figure 2. Current-clamp recordings showing stereotypical electrical resonance from an enzymatically dissociated hair cell (A) and the variations seen in the semi-intact epithelial preparation (B-D), evoked by depolarizing current steps (top trace) of amplitude Istim. A, Damped sinusoidal voltage oscillations (Istim = 225 pA; f = 185 Hz; Q = 15.3; Vrest =
For any given amplitude current injection, enzymatically dissociated hair cells oscillated at approximately three times the frequency of hair cells in the semi-intact preparation (Fig. 3A). For example, the average oscillation frequency (f) evoked by a 100 pA depolarizing current step was 149 Hz in enzymatically dissociated hair cells (SD, 23 Hz; range, 103-190 Hz; n = 40) and 55 Hz in hair cells in the semi-intact preparation (SD, 18 Hz; range, 24-105 Hz; n = 50). For both populations, f increased with larger amplitude current steps (Fig. 3A). Average frequencies ranged from 100 to 221 Hz (total range, 58-271 Hz) in enzymatically dissociated hair cells and from 35 to 75 Hz (total range, 19-170 Hz) in the semi-intact preparation for current injections of 25-400 pA.
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Figure 3. Average current-clamp behaviors of enzymatically dissociated cells ( ; n = 40) and hair cells in the semi-intact preparation (
; n = 50). A, Frequency-current relationships. Not all cells oscillated at every current amplitude tested. B, Voltage-current relationships. Membrane potential was measured either as the steady-state voltage at the end of the current step or, when the oscillations lasted for the duration of the step, as the average potential around which the cell oscillated. Both panels show mean ± SD.
Another common descriptor of the exponentially damped sinusoidal oscillations seen in most enzymatically dissociated hair cells (Fig. 2A) is the quality of resonance (Q), a measure of the sharpness of tuning (see Materials and Methods). The more prolonged (less damped) the voltage oscillations, the larger the Q value and the more tightly tuned the hair cell is to that frequency. Like f, Q varied with the size of injected current. In most cells, the peak Q was seen during small current steps (25-100 pA) that produced voltage oscillations between
) raised the median Q to 3.5 (n = 32).
In response to small (<150 pA) depolarizing current steps, enzymatically dissociated hair cells tended to depolarize a few millivolts more than cells in the semi-intact preparation (Fig. 3B), but otherwise the two populations operated in identical voltage ranges. The average resting potential in both populations was
K+ currents in enzymatically dissociated hair cells
Previous biophysical and modeling studies have attributed the generation of resonance exhibited in enzymatically dissociated bullfrog saccular hair cells to the interplay of a voltage-dependent Ca2+ current with a single outward current, KCa (Lewis and Hudspeth, 1983
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Figure 4. Effects of 1 mM 4-AP and 6 mM TEA on current-clamp recordings from enzymatically dissociated hair cells. A, Resonance recorded in normal extracellular solution (control: Istim = 200 pA; f = 225 Hz; Vrest =
An A-type, inactivating K+ current (KA) has also been reported in enzymatically dissociated bullfrog saccular hair cells (Lewis and Hudspeth, 1983
Together, these findings support the conclusion that in enzymatically dissociated grassfrog saccular hair cells, as in bullfrog, the only K+ current integrally involved in the generation of resonance is KCa. The story is, however, quite different and more complex in hair cells that have suffered neither enzymatic treatment nor dissociation from the epithelium.
K+ currents in hair cells in the semi-intact preparation
Comparison of Figures 4 and 5 shows that the effects of 1 mM 4-AP and 6 mM TEA on hair cells in the semi-intact epithelial preparation were quite different from their effects on enzymatically dissociated hair cells. Application of 1 mM 4-AP eliminated voltage oscillations in the majority (11 of 14) of hair cells in the semi-intact preparation (Fig. 5A). Because, as mentioned above, 1 mM 4-AP is expected to block most of KA in addition to KV, these data do not address whether only one or both of these conductances were involved. Interestingly, the three cells on which 4-AP had no discernible effect also oscillated at higher frequencies (90-125 Hz in response to 100 pA current steps) than the 11 others (57-76 Hz), making them appear more like enzymatically dissociated cells. Application of 6 mM TEA to hair cells in the semi-intact preparation never eliminated the oscillations as it did in enzymatically dissociated cells (Fig. 4B). In fact, in the majority (8 of 11) of these cells, sinusoidal oscillations were replaced by spike-like behavior (Fig. 5B), similar to the spiking found in some cells in the absence of TEA. This suggests that the cells that spiked in the absence of TEA contained a dominating KV current, as in chick cochlea (Fuchs and Evans, 1990
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Figure 5. Effects of 1 mM 4-AP and 6 mM TEA on current-clamp recordings from hair cells in the semi-intact epithelial preparation. A, Voltage oscillations recorded in normal extracellular solution (control, solid line: Istim = 225 pA; f = 82 Hz; Vrest =
These results indicate that in the preponderance of hair cells in the semi-intact preparation, a voltage-dependent K+ current (KV and/or KA) was integral to the generation of voltage oscillations. Furthermore KCa, although present, was not necessary for the generation of voltage oscillations (spikes) in these cells but did appear to play role in modulating the shape of the response.
Voltage-clamp recordings
We now turn our attention to more directly assessing which ionic currents are present in each hair cell preparation by examining, under voltage-clamp conditions, the time-dependence, voltage- dependence, and pharmacological sensitivity of the currents.
Outward currents in enzymatically dissociated hair cells
Currents recorded from enzymatically dissociated hair cells bathed in normal extracellular solution were remarkably stereotyped. In response to depolarizing voltage steps from a holding potential of
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Figure 6. Effects of TEA, Ibtx, and 4-AP on outward currents in enzymatically dissociated hair cells. Essentially all of the outward current recorded in normal extracellular solution (control) was eliminated by 6 mM TEA (A) or 2 µM Ibtx (B). Application of 1 mM 4-AP (C) had no effect. The panels to the right show the I-V relationship for each cell before (
Our pharmacological dissection of this large, rapidly activating outward current under voltage-clamp conditions is consistent with our current-clamp data that, in turn, agree with previous findings from bullfrog (Lewis and Hudspeth 1983
Outward currents in hair cells in the semi-intact epithelial preparation
As in enzymatically dissociated hair cells, depolarizing voltage steps applied to hair cells in the semi-intact preparation evoked a small inward current that was rapidly overtaken by a large outward current. However, the shape of the outward current was quite different from that seen in enzymatically dissociated hair cells (compare Figs. 6, 7, left panels). At all voltages the outward current rose quickly, leveled off or decayed slightly, and then continued rising. During large-amplitude voltage steps the current reached a second peak and then decayed, whereas with smaller steps the current was often still rising at the end of the 50 msec step (Fig. 7A-D, left panels). Although nearly all cells in this preparation possessed these major attributes, there was much more variability in the recordings from these cells (Fig. 7, compare left panels) than in enzymatically dissociated hair cells. The average size of the outward current at
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Figure 7. Effects of TEA, Ibtx, and 4-AP on outward currents from hair cells in the semi-intact epithelial preparation. Application of 6 mM TEA (A) or 1 µM Ibtx (B) blocked a fast component of the outward current, leaving a more slowly activating component. 1 mM 4-AP (C) blocked a slow component of the outward current, leaving a rapidly activating outward current that partially inactivated. Nearly all outward current was eliminated by applying both 1 mM 4-AP and 6 mM TEA (D). The panels to the right show the I-V relationship for each cell before (
Rather than eliminating nearly all outward current, as in enzymatically treated hair cells, 6 mM TEA blocked only about half of the outward current in the semi-intact preparation (Fig. 7A; n = 8), leaving behind a slowly activating component. Ibtx had a similar effect (Fig. 7B; n = 5). Application of 1 mM 4-AP also blocked about half of the outward current but left behind a current that activated rapidly and then partially inactivated (Fig. 7C; n = 8). Co-application of 4-AP and TEA eliminated essentially all of the outward current (Fig. 7D; n = 11).
To visualize the current blocked by each drug, we subtracted the currents recorded during drug application from control currents recorded before drug application (Fig. 8). As suggested by Figure 7, A and B, TEA and Ibtx blocked similar components of the outward current (Fig. 8A,B). In both cases the current sensitive to these agents activated rapidly and then partially inactivated. The differences between Figure 8A and Figure 8B are indicative of the variability between cells, rather than differential effects of the drugs. These subtractions suggest that the TEA- and Ibtx-sensitive current (Fig. 8A,B) was the same component of the outward current that was insensitive to 4-AP (Fig. 7C). Conversely, the current blocked by 1 mM 4-AP (Fig. 8C) was similar to the TEA- and Ibtx-insensitive component (Fig. 7A,B). Together, the TEA- and Ibtx-sensitive component and the and 4-AP-sensitive component constituted nearly all of the outward current (Fig. 7D). Therefore, as previously demonstrated in turtle cochlear hair cells (Goodman and Art, 1996
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Figure 8. TEA-, 4-AP-, and Ibtx-sensitive currents (dark traces) in hair cells in the semi-intact epithelial preparation obtained by subtracting the currents after drug application (smaller-amplitude light traces) from the currents in control conditions (larger-amplitude light traces). A, Current blocked by 6 mM TEA activated rapidly and partially inactivated. B, One micromolar Ibtx blocked a similar component of the current. C, One millimolar 4-AP blocked a slowly activating current. Because of series resistance errors in the step potential it was necessary to estimate the current at
Based on the high specificity of Ibtx for KCa channels, we identified the TEA- and Ibtx-sensitive component as a partially inactivating KCa current. The 4-AP-sensitive component could be KV, which has been found in other hair cells, or possibly KA, although this current is expected to be inactivated at the holding potential of
Transformation of voltage oscillations and outward currents by papain
So far we have shown that the characteristics of the voltage oscillations and outward currents in enzymatically dissociated hair cells were strikingly different from what we found in the semi-intact epithelium. We now demonstrate that these differences were due to enzyme treatment, rather than dissociation of the hair cells from the saccular epithelium.
Application of the papain solution directly onto the semi-intact preparation, at the same concentration used in the standard hair cell dissociation procedure, transformed the hair cells in situ (Fig. 9). In addition to comparing populations of hair cells in the semi-intact preparation before and after enzyme exposure, we made continuous current-clamp recordings from two cells during papain application. In these cells, enzyme exposure raised the frequency of the voltage oscillations more than three times and metamorphosed the oscillation into a waveform that resembled the stereotypical resonance seen in dissociated hair cells (Fig. 9A). Subsequent application of 6 mM TEA to the cell in Figure 9A reversibly eliminated the voltage oscillations (data not shown). Such an effect of TEA was always observed in enzymatically dissociated hair cells (Fig. 4B) and never in untreated hair cells in the semi-intact preparation (Fig. 5B). Likewise, application of the papain solution to hair cells in the semi-intact preparation transformed the outward currents into those typical of enzymatically dissociated cells (Fig. 9B; n = 4). The low quality of the resonance found after papain treatment (Fig. 9A), as well as the reduction in the amplitude of the outward current after treatment (Fig. 9B), effects opposite to those seen in population studies, are likely to be due to rundown that typically occurs during prolonged recording.
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Figure 9. Papain transforms voltage oscillations and outward currents in the semi-intact epithelium in situ. Traces from continuous perforated patch recordings taken before (control) and after perfusion of papain solution onto the epithelium. A, After 15 min in papain, the frequency of the voltage oscillations recorded in current-clamp increased from 36 Hz to 164 Hz. (Istim = 200 pA; Vrest =
The enzyme solution used in these experiments was made by adding papain (a partially purified preparation of papaya latex; see Materials and Methods), plus an activating agent, L-cysteine, to the normal extracellular solution. As a first step in characterizing the active agent in this solution, we tested the effects of omitting or heat-inactivating the papain. No transformation of voltage oscillations or outward currents was seen when normal extracellular solution containing 2.5 mM L-cysteine without papain was perfused onto hair cells in the semi-intact preparation. Furthermore, hair cells in a semi-intact preparation that had been incubated in heat-inactivated papain (see Materials and Methods) for 30 min retained low-frequency oscillations and currents typical of untreated hair cells. Thus, we see that some heat-sensitive component of papain caused the transformation of the voltage oscillations and outward currents in situ and that dissociation was not required. These results suggest that the proteolytic activity of the enzyme was responsible for the differences in voltage- and current-clamp responses found in these two populations of cells.
Ca2+ current isolation
To assess whether the enzymatic dissociation procedure affected the Ca2+ current, we blocked the outward currents by substituting the K+ in the internal solution with Cs+. Figure 10, A and B, shows examples of such recordings. A noticeable sag in inward current during depolarizations above
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Figure 10. Isolation of Ca2+ currents. All recordings, except for C, were made with Cs+ internal solution. A, A sag in the inward current was evident in hair cells in the semi-intact preparation but not in enzymatically dissociated hair cells (B). C, NMG internal solution blocked the sag. The corresponding voltage steps (corrected for series resistance errors) are indicated by each trace (A-C). D, Peak Ca2+ current I-V relationships from enzymatically dissociated hair cells (
We found no differences in the peak Ca2+ current recorded with Cs+ internal solution in the two preparations (363 ± 46 pA in the semi-intact preparation and 375 ± 40 pA in enzymatically dissociated hair cells; mean ± SEM; n = 10 and 12, respectively), but we did find differences in the current-voltage relationships and the activation kinetics (Fig. 10D,E). The voltage-dependence of the Ca2+ current in enzymatically dissociated hair cells (Fig. 10D, open symbols) was similar to what has previously been reported in enzymatically dissociated bullfrog saccular hair cells (Hudspeth and Lewis, 1988a
To compare the activation kinetics of the Ca2+ current in the two preparations we fit the recorded currents with a third order kinetic scheme:
where
Ca is the maximal Ca2+ current and
DISCUSSION
Tuning in isolated hair cells and intact preparations
Several lines of evidence suggest that tuning can be greatly altered when hair cells are dissociated from the sensory epithelium. Intracellular recordings in the intact turtle cochlea demonstrate that there is good agreement between a hair cells' electrical resonant frequency and characteristic auditory frequency (Crawford and Fettiplace, 1981
These differences in f and Q, in addition to discrepancies in the shapes of tuning curves and the phases of responses, and the small temperature-dependence of tuning, have led several researchers to conclude that hair cells are not exclusively tuned electrically (Lewis, 1988
As this study demonstrates, some of the evidence against electrical tuning of the frog sacculus, namely the discrepancies in f and Q noted above, is artifactual and can be directly attributed to the use of papain during cell dissociation. Hair cells in the semi-intact preparation, cells not exposed to papain, exhibited electrical resonance of frequencies between 35 and 75 Hz and Q values of ~3. These values are more consistent with those reported from more intact preparations (f < 100 Hz; Q ~ 2). Thus, the electrical tuning found in the majority of hair cells in the frog sacculus is sufficient to account for the frequency discrimination of this organ, and the need for a mechanical feedback mechanism may not be essential. Further experiments, however, are needed to account for the other discrepancies.
Resonant frequencies and K+ conductances
The Hudspeth and Lewis (1988b)
Rather than exhibiting exclusively stereotypical electrical resonance, hair cells in the semi-intact preparation, cells not subject to enzyme exposure, showed a variety of voltage behaviors in addition to damped sinusoidal oscillations. Our finding that at least two K+ currents (KV and a KCa) rather than a single K+ current are involved in the generation of voltage responses in these cells could explain this diversity. One might imagine that such variety could easily be achieved by varying the relative contribution of each current.
Studies of hair cells from the auditory and vestibular organs of several species have shown that a cell's resonant frequency is determined primarily, though not exclusively, by the properties of its outwardly rectifying K+ currents. For example, the tonotopical organization of the turtle cochlea appears to be the result of a smooth gradient in the kinetics of the hair cells' K+ currents (Art and Goodman, 1996
Given that papain alters frog saccular hair cells so dramatically, it is surprising that after enzymatic dissociation these cells exhibit such beautiful oscillatory behavior. Perhaps even more surprising is that enzymatically dissociated hair cells fit nicely in the K+ current versus frequency scheme found in turtle. These cells, which on average resonated at frequencies of 100-221 Hz, were found to contain only a KCa current. Thus, frog saccular hair cells can be transformed from mid-frequency cells, containing both KV and KCa, to high-frequency cells, containing only KCa simply by exposing them to papain. How this transformation occurs we cannot explain.
Conductances in frog saccular hair cells
Here we demonstrate that, like a number of other hair cell preparations (Fuchs and Evans, 1990
As in bullfrog (R. catesbeiana), KA (Lewis and Hudspeth, 1983
Based on its ionic permeability, sensitivity to dihydropyridines, and lack of inactivation (Fuchs et al., 1990
Enzyme effects
Perhaps our finding that proteolytic enzymes dramatically alter the ionic conductances of frog saccular hair cells is not so surprising. For years it has been known that, when applied intracellularly, proteolytic enzymes can eliminate inactivation of Na+ channels (Armstrong et al., 1973
FOOTNOTES Received Jan. 20, 1998; accepted Feb. 9, 1998.
This work was supported by National Institutes of Health Grant NS27142, predoctoral fellowship Training Grant GM07257, and a Grant from the Medical Research Foundation of Oregon. We thank Drs. B. D. Anson, B. Edmonds, D. Lenzi, and P. M. O'Day for careful dissection of this manuscript, J. W. Runyeon for scanning electron microscopy, and M. W. Armstrong for Canis familiaris hair.
Correspondence should be addressed to Dr. William M. Roberts, Institute of Neuroscience, 1254, University of Oregon, Eugene, OR 97403-1254.
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