the inactivating potassium currents of hair cells isolated ... · the inactivating potassium...

JOURNAL OF NEIJROPHYSIOLOGY Vol. 68, No. 5. November 1992. Prinred in U.S.A.

The Inactivating Potassium Currents of Hair Cells Isolated From the Crista Ampullaris of the Frog

C. H. NORRIS, A. J. RICCI, G. D. HOUSLEY, AND P. S. GUTH Departments of Pharmacology and Otolaryngology, Tulane University School of IWedicine, New Orleans, Louisiana 70112

SUMMARY AND CONCLUSIONS

1. A-type outward currents were studied in sensory hair cells isolated from the semicircular canals (SCC) of the leopard frog (Rana pipiens) with whole-cell voltage- and current-clamping techniques.

2. There appear to be two classes of A-type outward-conduct- ing potassium channels based on steady-state, kinetic, pharmacological parameters, and reversal potential.

3. The two classes of A-type currents differ in their steady-state inactivation properties as well as in the kinetics of inactivation. The steady-state inactivation properties are such that a significant portion of the fast channels are available from near the resting potential.

4. The inactivating channels studied do not appear to be calcium dependent.

5. The A-channels in hair cells appear to subserve functions that are analogous to IA functions in neurons, that is, modulating spike latency and Q (the oscillatory damping function). The A- currents appear to temporally limit the hair cell voltage response to a current injection.

INTRODUCTION

The transient outward K+ current, which has come to be called Z*, was first described by Connor and Stevens ( 197 1). They separated Z* from the delayed rectifier current by the use of a protocol that elicited all currents, then only the noninactivating currents. Subtraction of these two currents revealed the existence of the A current. The protocol that elicited both inactivating and noninactivating currents employed a hyperpolarizing prepulse to enable the inactivating conductances and thus allow the channels to open when the cell was depolarized. The second protocol used a depolarizing prepulse, which inactivated the A-type conductances, leaving only the noninactivating conductances.

In addition, these two potassium currents can sometimes be separated pharmacologically. In some cells, ZA is less sensitive to inhibition by tetraethylammonium (TEA) and more sensitive to block by 4-aminopyridine (4.AP) than the delayed rectifier (Rudy, 1988; Thompson, 1977). Thus the definition of A-type channels is that they are voltage- sensitive, rapidly-activating, rapidly-inactivating, outward- flowing potassium channels that are blocked by 4-AP.

Several roles have been suggested for A currents. In neurons I) they could control the spike latency, 2) they could regulate interspike interval, and 3) they could contribute to action potential repolarization (Rudy, 1988).

A great deal of information about A-channels has come from the discovery of the Shaker mutant genes of the dro-

sophila (Baumann et al. 1987; Jan et al. 1977; Kamb et al. 1987; Papazian et al. 1987). Investigations have localized the sites for voltage-dependence, ion selectivity, and the kinetics of inactivation (Catterall 1986; Greenblatt et al. 1985; Iverson and Rudy 1988; MacKinnon et al. 1988). Alternate splicing of the Shaker gene has demonstrated that multiple mRNAs can be made that produce A-channels that differ in inactivation kinetics as well as in the recovery from inactivation (Iverson and Rudy 1988; Timpe et al. 1988; Zagotta et al. 1989).

According to Hudspeth ( 1986), the bullfrog’s saccular hair cell has seven ionic currents, including an A current (Z*) . This transient K+ current in the saccular hair cells is fully inactivated at the normal resting potential (Hudspeth and Lewis 1988)) suggesting at best a minimal role in saccular hair cell function. Alternatively, Sugihara and Furu- kawa ( 1989) have suggested that the A-currents in saccule hair cells may be involved in modulating the damping function of the membrane oscillations typical of these cells. Murrow and Fuchs ( 1990) have described an A-type conductance in the bird basilar papilla that was found exclu- sively in the short hair cells and although not in a physio- logically active range, could be enabled during hyperpolarization induced by Acetylcholine, the major efferent transmitter.

In contradistinction to the auditory hair cells, in a preliminary study of the currents of hair cells isolated from the posterior semicircular canal (SCC) of the frog, Rana pipiens Housley et al. ( 1989), suggested that an active Z* could be elicited by depolarization from normal resting potentials. Correia et al. ( 1989) and Rennie and Ashmore ( 199 1) have described an A-type current in bird and guinea pig semicircular canal, respectively. Although the steady- state properties allow these channels to be active in a more physiological range, the relative proportion of the A-channels is significantly less than that found in the frog.

A-type currents may be of fundamental importance in SCC hair cells. We, therefore, made a more rigorous study of these inactivating outward currents revealing the existence of two classes of A-type channels, differing largely in their inactivation properties.

METHODS

Zsolation of hair cells

The procedure adopted essentially follows that reported by Lewis and Hudspeth (1983), Hudspeth and Lewis (1988), and Housley et al. ( 1989) for the isolation of hair cells from frog vestibular organs. The external medium used by Lewis and Hudspeth

1642 0022-3077192 $2.00 Copyright 0 1992 The American Physiological Society

INACTIVATING CURRENTS IN VESTIBULAR HAIR CELLS 1643

TABLE 1. Media

Dissociation, mM External, mM Internal, mM

CaCIZ . 2Hz0 KC1 MgC12 l HZ0 NaCl NaH,PO, Na,HPO, KH2P0,, K2HP04 ATP EGTA D-glucose 4-AP TEA GTP

0.02 2 3 3

1 122 119

2 2 8 8

3 3 10 10

1 115

3

1 4 3

11 3

0.5

EGTA, ethylene glycol-bis(P-aminoethyl ether)-IV,N,N’,N’-tetraacetic acid; 4-AP, 4-aminopyridine; TEA, tetraethylammonium chloride; GTP, guanosine triphosphate.

( 1983) contains Hepes buffer. In our procedure, this has been replaced by phosphate buffer ( Table 1) (Norris and Guth 1985 ) . Cells were isolated as previously described ( Housley et al. 1989).

Leopard frogs (Rana pipiens) were chilled, pithed, and then

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decapitated. With the head bathed in external medium (Table 1) and sectioned sagittally, the inner ear was exposed by opening the otic capsule. The semicircular canals were dissected free from the rest of the membranous labyrinth and trimmed leaving only the ampullae. These were opened and placed in dissociation medium (Table 1) containing the proteolytic enzyme papain (0.1 mg/ml; Calbiochem No. 5 125) and L-cysteine (0.33 mg/ml) for 5 min. The tissues were then washed in dissociation medium containing bovine serum albumin (0.5 mg/ml), and the cristae were moved to a glass-bottomed bath on an inverted microscope. Mechanical twisting of the tissues then freed the individual hair cells from the rest of the crista. The bathing medium was then changed from dissociation medium to external medium (Table 1).

Studies with whole-cell recordings required the use of various solutions (Table 1). The bath (2.ml vol) containing the isolated hair cells was perfused with external medium at a rate of l-2 ml/min, and all experiments were carried out at room tempera- ture (21°C).

Cells were selected for whole-cell patch clamping according to the criteria enumerated by Housley et al. ( 1989).

Whole-cell recording

Gigohm seals ( 1- 15 GQ) were obtained with 1.5-mm OD boro- silicate glass pipettes (Frederick Haer, capillary tubing No. 30-32-

Intermediate

FIG. 1. Current vs. time responses for three different cells (top to bottom) to 2 different whole-cell voltage-clamp protocols (A and B) . A : the cell is first voltage clamped to a conditioning prepulse of - 130 mV from a holding potential of -60 mV. After the 80 ms, - 130 mV prepulse the cell is depolarized for 200 ms to a given voltage step and then returned to the -60 mV holding potential. This cycle is repeated every 5 s with a 10 mV more positive step utilized each cycle. Depolarizing voltage steps ranged from - 130 to + 120 mV. This protocol is designed to elicit the inactivating currents as well as the noninactivating channel types such as the delayed rectifier, or the calcium-dependent potassium conductances. B: current responses to a similar protocol are depicted except that the conditioning prepulse has been changed to -20 mV. This protocol has been designed to elicit the noninactivating currents (see Fig. 5 ) . C: current responses are the differences (the inactivating currents) between those in A and those in the B.

1644 NORRIS, RICCI, HOUSLEY, AND GUTH

1) pulled to a resistance of -3-5 Ma. A continuous single-electrode patch-clamp amplifier ( Axopatch- 1 B, Axon Instruments) was used to record from crista hair cells during voltage or current clamp (after Hamill et al. 198 1). Unless specified, records were low-pass filtered at 5 kHz with a four-pole Bessel filter. Command potentials and currents were controlled with a 12.bit digital-to-analog converter, and data were sampled digitally at 2OO-ps intervals with a 12.bit analog-digital converter (Labmaster, Tekmar Indus- tries) coupled to a microcomputer (PC’s Limited 80286, Dell Computer). Clamp speed, which is dependent on uncompensated series resistance and cell capacitance, was estimated to be 15 ps. Data were stored on hard media for off-line analysis (p-clamp version 5.0 1, Axon Instruments). The junction potential between the pipette internal solution and the bath was nulled before seal formation. The ground electrode was placed in a separate bath of

Drug solutions (4-AP, 1 O-20 mM ) were made up in external medium (Table 1). The NaCl concentration was adjusted to ac- commodate the drug and maintain osmotic pressure. 4-AP was obtained from Sigma. It was applied by bath substitution for 3 to 5 min (i.e., 1.5-3~ bath volume) before the voltage-stepping protocol designed to elicit the inactivating current was applied to the cells ( see Fig. 7 ) . Changes in inactivating currents caused for example by 4-AP exposure were expressed as percent of maximal inactivating current elicited in that cell. The currents were elicited once before drug application, at least twice during drug application, and at least once during the drug washout period.

RESULTS

Cell parameters internal solution and coupled via an agar bridge to the recording chamber, so that no significant liquid junction potential should The input capacitance for all the cells used in this study exist between electrodes. was 5.3 t 0.2 (SE) pF (n = 25 ) ; the series resistance was

Cell conductances were then calculated from current records 9.3 t 0.7 MQ, and the zero current membrane potential resulting from various voltage-clamp step protocols. Each step was -50 t 2 mV. These properties agree very well with the change in voltage was separated by 5 s. In most of the cells, the voltage error due to series resistance was partially ( 60-80s) can-

data previously published by Housley et al. ( 1989).

celed electronically. The residual uncompensated series resistance was at most l-3 MQ, which could cause an average of 3 mV of

The major conductance is inactivating

error in the voltage protocols for the largest currents recorded and This study will suggest that two classes of inactivating a smaller error for smaller currents. channels exist in semicircular canal hair cells and that these

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($1 mo+J4p’L711 FIG. 2. A and B: examples of inactivating outward currents from 2 different cells obtained by applying the subtraction

method shown in Fig. 1 to the responses to the step to + 120 mV (maximal current response). Dotted lines represent the actual data. Each current trace was fitted with a double exponential equation (solid line, see middle of figure, and text). The double exponential was fitted to the data starting r2 ms after the start of the voltage-clamp step to avoid any clamping artifact. A : the fast portion of the double exponential predominates (A I > Al); B: the slower portion of the double exponential predominates (A, > A, ). C: histogram depicting the distribution of the fast (Al ) and slow (A,) coefficients; the bin width is 5 ms. The coefficients represent the relative proportion of a given current in a particular cell. The mean q is 4.4 ms. The mean r2 is 36.4 ms.

INACTIVATING CURRENTS IN VESTIBULAR HAIR CELLS

conductances, defined as fast and slow, are of the A-type. The two conductances exist in varying proportions between cells, so that any given cell will be dominated by either the fast or slow channel type, but in general will contain both types. For the purpose of distinguishing between channel characteristics, cells were grouped on the basis of the relative amount of the fast and slow component. That is, cells that contain mostly fast channels are fast cells, and cells that contain mostly slow channels are slow cells. Where rele- vant, intermediate cells are shown. The proportion of fast and slow conductances were measured from the coefficients of the double exponential kinetic fit to the inactivating current obtained from subtracted data described in Figs. 1 and 2. The ratio AI /(A, + A, + A,) = (R f) reflects the proportion of fast current in the cell, whereas the ratio AJ (A, + A, + AZ) = (R,) reflects the proportion of slow current. For fast cells Rf > R, and for slow cells R, > R f. It is important to realize that the classification is a tool for distinguishing channel types and does not necessarily imply two physiological cell types. Where comparisons between fast and slow channels were performed, cells were chosen that were dominated by either the fast or slow component (>90%). Choosing the two extreme groups allows for a bio- logical separation of the channel types. Where no differences were found between the fast and slow components, the cells were pooled and a composite figure is presented.

Preliminary evidence for two independent inactivating channels

KINETICS. All the inactivating current responses, derived as in Fig. 1 A, were examined in 26 different cells. A least- squares curve fitting procedure (clampfit, P-clamp version 5.01, Axon Instruments) was applied to the rising phase (activating phase) and to the falling phase (inactivating phase) of each current response to each voltage step. The

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STEPS (mv) FIG. 3. Tail current responses to 2 voltage-clamp protocols. In the pro-

tocol, cells are held at -60 mV and then clamped for an 80-ms long prepulse at - 130 mV. After the prepulse, the cells are briefly depolarized to +30 mV for 5 ms, then stepped back to a less depolarized level for 60 ms, and finally returned to the holding potential. The cycle is repeated each 5 s with a - 10 mV increment. The protocol is repeated except that the prepulse used is -20 mV. By subtracting the responses to the 2 protocols, the tail currents for the inactivating channels can be obtained. The results are plotted to show the reversal potential for the channels. Increasing the potassium concentration in the external medium of the cells from 3 to 30 mM shifts the reversal potential to an -53 mV more positive level.

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FIG. 4. Current and chord conductance vs. membrane voltage. A: the peak of the inactivating current derived as in Fig. 1 A is plotted as a function of membrane voltage for fast cells (A, > A2 see RESULTS in text) and for slow cells (A, > A, see RESULTS in text). Cells dominated by the slow channels show a much larger macroscopic current than do the cells dominated by the fast current. B: from these plots, normalized chord conductance (G/G,,,) was calculated and is plotted. The solid circles are the calculated normalized conductance and for comparison, the solid line is a Boltzmann equation fitted to the data with a half-maximal voltage of -2 mV and a gating charge of - 1. (R = 0.985).

kinetics of activation were fitted with a single exponential whose time constant was (1 ms. No detectable difference was found between fast and slow channels, however, both had significantly faster activation than reported for other hair cells (Hudspeth and Lewis 1988; Lang and Correia 1989; Rennie and Ashmore 199 1) . Analysis of these parameters of activation are limited by the sampling and settling times as dictated by the cells and the electronics. That is, often the curve fitting program applied to the rising phase of the current response only had two or three valid points with which to work.

The inactivating phase of the maximal current response (i.e., the response to the step to + 120 mV) was fitted best with a double exponential [y = A, + A, exp( -t/T,) + A2 exp( -t/T2)] where R = 0.998 or better (Fig. 2). In this equation, the mean fast-time constant (T] ) was 4.44 ms (n = 25), and the mean slow-time constant (Q) was 36.4 ms ( n = 25) (Fig. 2C). No significant voltage dependence


of the time constants was observed. Analysis of the propor- was increased from 3 to 30 mM, the reversal potential be- tions between the coefficients of the two exponentials for came 53 mV less negative. The Nernst equation predicts a each double exponential fit indicated that the proportions change of 58 mV for a potassium selective channel. No of fast- and slow-inactivating channels in these cells varied. difference in this reversal potential was found for fast and Figure 2C is a histogram showing the range in the coeffi- slow channels. Initial comparisons between fast and slow cients, suggesting a range in the proportion of channel types channels were not different so the data has been pooled and found between cells. These results suggest that there are two presented in Fig. 3. inactivating channels, which vary in number but not charac- Additional evidence that potassium is the current carrier ter among cells. for these channels was provided by the observation that

Potassium selectivity replacement of the potassium in the internal solution in the patching pipette with cesium eliminates the outward

Evidence that potassium is the major current carrier in current response to the voltage-clamping protocol (n = 4, the inactivating channels was provided by tail current analy- data not shown). A similar finding was reported by Fuchs sis. Prepulse protocols, similar to those described in Fig. 1, et al. ( 1990) and in Table 1 of Rudy’s review ( 1988). were used to isolate the tail currents produced by the inactivating conductances (Fig. 3; see legend for protocol detail). Activation

The responses to the two protocols were subtracted to Depicted in Fig. 4A is the peak of the inactivating current reveal the tail currents for the A-type inactivating channels. isolated as in Fig. 1 and plotted as a function of the mem- Then the tail current responses were plotted as a function of brane voltage with fast and slow groups of cells. The main the clamped tail voltage steps to reveal a reversal potential difference between fast and slow cells in this figure is that of -77 mV for the inactivating channels (Fig. 3). When the slow cells have almost six times more current than fast cells. potassium concentration in the medium bathing the cells Chord conductance allows for the evaluation of the volt-

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PREPULSE (mV) FIG. 5. Inactivation curves for 3 groups of cells. A : protocol used showing that cells are voltage-clamped and held at -60

mV and then stepped to a prepulse for 200 ms. After the prepulse, they are depolarized to 0 mV for 50 ms (test level), then stepped back to the prepulse level for 200 ms (tail current level), and finally returned to the holding level. C: peak current at the test level is plotted as a function of the prepulse level voltage. Examples of the current responses are shown in B where kinetically, fast cells (the /ej-most responses) have sharply inactivating currents, slow cells (the right-most responses) inactivate with much slower time constants, and intermediate cells (the center set of responses) have inactivating rates between the 2 extremes. A separate single Boltzmann equation is fitted to the fast and slow cells’ responses plotted in C, and a double Boltzmann equation is fitted to the intermediate cells’ responses (R = 0.99). The double Boltzmann equation is composed of elements identical to the scaled sum of the 2 single Boltzmann equations, suggesting that the hair cells contain both fast- and slow-inactivating channels in different proportions.


age dependence of activation of a channel independent mann equation was fitted to the data for fast cells ( Rf > from the changing driving force and is given as ga = ( ZA / 90% ) and ‘to that for slow cells (R, > 90% ) . A double Boltz- ( V- IQ. Here ga reflects conductance at a given voltage; ZA mann equation was fitted to the data for intermediate cells. is the peak current plotted in Fig. 4A and obtained from the The elements for each half of this double Boltzmann equa- subtracted data described in Fig. 1; Vis the voltage step, and tion were not significantly different from the analogous ele- & is the reversal potential calculated from Fig. 3 so that the ments of the separate single Boltzmann equations. That is, difference ( V - VR) is equivalent to the driving force. The for very fast cells the voltage of half-maximal conductance, plot in Fig. 4 B is chord conductance (G) normalized to V0 = -59t l.OmVandtheslope(ze/KT),S= -7.8t0.8 maximal conductance ( G,,,) . No significant difference ( normalized current/volt). For very slow cells, V0 = -95 t was seen in the conductance plots between fast and slow so 0.5 mV and S = -13.4 t 0.4. For the two halves of the cells were grouped and analyzed together. The conductance double Boltzmann equation, the fast V0 = -63 t 0.7 mV plot was shown to fit a single Boltzmann function (-), with S = -5 t 0.8 and the slow V0 = - 10 1 t 5.0 mV with where G/G,,, = [ 1 + ecvo-v,Ze/KT]-l. Here V0 is the voltage S = - 18 + 3.0. The results then suggest that each cell can be of half-maximal conductance ( -2 mV), z is the effective represented by the scaled sum of two independent Boltz- gating charge ( 1 ), and e/ KT is a constant. The single Boltz- mann equations. Thus, these results provide further evi- mann fit (R = 0.985) suggests that the fast and slow chan- dence that there are two types of inactivating channels. As nels have similar activation gates, that the channels operate already seen with the kinetics, the difference between cells is independently, and that the channels exist either in an open the proportion of fast and slow channels present. The very or closed state (multiple closed states may exist) ( Adams et fast cells have mostly the fast inactivating type channel al. 1982; Belluzzi et al. 1985; Hille 1984; Hodgkin and (96% in Fig. 5), whereas the very slow cells have mostly the Huxley 1952). Here too the cells were pooled after a com- slower inactivating type channel (99% in Fig. 5), and the parison of fast and slow groups did not reveal a significant intermediate cells have a mixture of the two types (49% difference in the activation curves. slow and 5 1% fast in Figure 5 ). The difference in the steady-

Voltage dependence of inactivation state inactivation properties between fast and slow cells (Fig. 5) is different from that described by Hudspeth and

Inactivation was also studied in three groups of cells as Lewis( 1988),whoreportedashifiof-lOto-15mVinthe shown in Figure 5. With the use of TABLE CURVE V3.01 voltage dependence of activation and inactivation of Z* software from Jandel Scientific, a separate single Boltz- over time. They attributed this shift in inactivation to a

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FIG. 6. Removal of inactivation from inactivating channels depends on both duration and amplitude of the cell’s potential before activation. Cells are clamped to 0 mV for long periods ( 1 to 3 s) to inactivate all the A-type type channels, and then they are clamped to a potential between -30 and -90 mV for conditioning periods of 10, 100,400, 1,000, or 4,000 ms before a voltage step to +30 mV for 120 ms (the test pulse). The cells were held close to their 0 current potential ( -60 mV) for 2 s between trials. The responses to slow cells are depicted in A and those to fast cells in B.

NORRIS, RICCI, HOUSLEY, AND GUTH 1648

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Donnan potential and as seen by Hudspeth and Lewis ( 1988). Thus the differences described here are not the same as those seen in Hudspeth and Lewis ( 1988) and appear to be of physiological significance, not due to recording artifact.

Time dependence of inactivation

1 10 100 1000 lE4

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FIG. 7. Normalized peak inactivating current as a function of prepulse duration for a -90 mV prepulse. Data were obtained with the protocol of Figure 6 with the prepulse potential fixed at -90 mV and with an additional 3 durations (40, 10,000, and 20,000).

The removal of inactivation of the A-type current is both voltage and time dependent. This was studied with the paradigm described in the legend of Fig. 6. As inactivation of the Z* channels was removed by successively longer periods at membrane prepulse potentials more negative than -50 mV, larger transient currents were produced during the step to the +30 mV test pulse ( Fig. 6). This paradigm covering seven conditioning membrane potentials (which were ran- domized) and five durations for each conditioning pulse was presented successfully to 10 cells (5 fast and 5 slow types) l

dissipation of a Donnan potential across the tip of the electrode. Our data, which shows a >50 mV difference between fast and slow components cannot be explained as a dissipation of a Donnan potential. On the basis of a Donnan equi- librium, an internal potassium concentration of >200 mM would be predicted to account for such a large shift (an unlikely possibility). Also no differences were seen in the activation properties, as would be predicted by a shift in a

Activation of the Z* was noticeable in a few cells after holding the membrane potentials at -40 mV for several seconds. However, on average, significant removal of inactivation of Z* occurred after 1 s at -50 mV. As the membrane was held at more negative potentials, the time required to achieve a similar level of removal of inactivation (enabling of the Z* channel) decreased. In several cells, it was possible to show that maximal removal of inactivation of Z* occurred at potentials of -80 mV and more negative

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FIG. 8. Responses of fast and slow cells to 4-aminopyridine (4-AP). A: control current responses are shown with the voltage-clamp protocol inset in the center (similar to that used in Fig. 1 A). B: current responses to the same protocol are shown after 10 mM 4-AP was added to the medium bathing the cells. C: responses in B subtracted from the responses in A and thus represents the currents that are blocked by 4-AP. The currents blocked by 4-AP have characteristics similar to those of both the fast- and slow-channel types separated by voltage-clamp protocol subtractions, suggesting that both the fast and slow channels are of the A-type.


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FIG. 9. Normalized activation conductance was calculated for the currents blocked by 4-AP as derived by the subtraction method of Figure 7 ( l ). Fast and slow cells were pooled because there was no difference between them. For comparison, the normalized activation conductance as shown in Figure 4 (prepulse voltage subtraction method) is also depicted (o ). Although the plots are similar, significant heterogeneity exists between isolation procedures. This is most likely due to the nonspecific actions of 4-AP.

after - 10 s. However, most cells could not withstand such treatment. Figure 6 also shows that fast and slow cells are different in their responses to various prepulses. Figure 7 is a plot showing how the inactivating currents vary with prepulse duration at one particular prepulse voltage. Again, differences between fast and slow cells can be seen. The data suggests that the slow channels are more temporally dependent.

Antagonism of ,4-type channels by 4-AP

The inactivating outward potassium current seen in hair cells is not sensit&e to 10 mM TEA (Housley et al. 1989)

but is sensitive to block by 4-AP (Hudspeth and Lewis 1988 ) . We therefore decided to test 4-AP in our prepara- tion. The results are shown in Fig. 8. The results in Fig. 8C show that 4-AP blocks both fast- and slow-type channels. With the currents as derived in Fig. 8C and a reversal potential of -77 mV (Fig. 3)) conductance curves were calculated for each cell studied with 4-AP. These are normalized, averaged, and plotted in Fig. 9 (0). For comparison, the analogous, normalized conductance from Fig. 4 is also plotted on this figure (0). Thus, although the currents used were derived differently [one method with a differential prepulse protocol and the other with a pharmacological (4- AP) protocol], the activation conductances appear similar. Kinetic analysis of the 4-AP-sensitive currents give results similar to those obtained from the voltage clamp subtracted data suggesting that the inactivating currents are of the A- type (Fig. 2). 4-AP results were not completely satisfactory. The nonspecific actions of the drug made further characterization impossible. Apparently the drug increased a noninactivating component. The differences between the voltage- current plots may be a reflection of the nonspecific action of the drug.

It should also be noted that there are some inactivating currents that are not blocked by 4-AP (Fig. 8B) and in Fig. 1 there are some inactivating currents that are not elimi- nated with a -20 mV prepulse. We are not sure if any of these latter inactivating currents are of the A-type. How- ever, the 4-AP data does suggest that the channels we describe as being fast and slow are of the A-type.

Culcium dependence

Protocols identical to those in Fig. 1 were used to test the dependency of I* on external calcium. One example of the

-60 mV

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A-A B-B C-C

FIG. 10, Apparent lack of dependency of the inactivating channels on calcium. Current traces in the top TOW are control responses to the same voltage-clamp prepulse protocols as are used in Figure 1. This is a fast cell. The responses in the middle row are to the same protocols in the same cell after the external medium bathing the cells was replaced with one in which calcium was replaced by magnesium. The traces in the bottom YOW are those in the middle TOW subtracted from those in the upper TOW. Slow cells responded in a like manner.


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L- 10 ms

FIG. 1 1. Responses to voltage- and current-clamp protocols in the same cell. A: current responses are derived with the same prepulse protocol as in Fig. 1A. This is classified as a fast cell. B: cell is held at 0 current and then clamped to a depolarizing current level. The voltage of the cell at 0 current was -50 mV. C: voltage responses to similar current-clamp steps are changed by the presence of a hyperpolarizing current prepulse.

resulting raw data is shown in Fig. 10. Some of the non-A- type currents (middle column) are reduced or blocked, but none of the A-type inactivating currents (right-hand column) were affected by the lack of calcium in the external medium. As studied with this protocol, neither the fast nor the slow A-type channels (n = 6) appeared to be calcium dependent.

Current-clamp responses

The voltage-clamped current responses of a cell, which were typical of many of the fast cells studied, are shown in Fig. 11 A. This voltage-clamp protocol used the 80 ms, - 130 mV prepulse as described for Fig. 1 A.

In the current clamp responses of Fig. 11, B and C, the cell depolarized to a relatively constant potential at the low- est current steps. At the higher current steps, the cell contin- ued to depolarize very slowly until the voltage suddenly stepped to a much higher plateau. This plateau occurred earlier at higher current steps. The voltage responses to the depolarizing current clamp steps in Fig. 11 C have the same general shape as in Fig. 11 B except that the step to the higher plateau is significantly delayed. The most likely cause of this delay is the recruitment of the inactivating channels by the hyperpolarizing current pulse. Also the oscillatory-like wave at the beginning of the voltage-response trace is narrower and taller in Fig. 11 C as compared with Fig. 11 B. Similar findings were reported by Sugihara and Furukawa ( 1989). The sharpening and decreased

dampening of the response may also be due to the action of the inactivating currents. Similar current-clamp responses were seen in slow cells. The main difference was that the rise to the second plateau was much slower.

DISCUSSION

Two types of A-channels

We have attempted to demonstrate by kinetic analysis, steady-state activation and inactivation and susceptibility to 4-AP that there are two types of A-channels in hair cells isolated from the XC of R. pipiens. The following are used to describe A-channels: I ) voltage-sensitive, 2) rapidly activating, 3) rapidly inactivating, and 4) sensitive to 4-AP (Figs. 1,2, and 8). The separation into fast and slow catego- ries is first seen in the kinetic analysis of Fig. 2. In addition to the kinetic analysis, studies of the inactivating curves also show two different types of A-channels (Fig. 5 ). When the effect of increasing the amplitude and duration of the prepulse used to enable the inactivating channels was examined ( Figs. 6 and 7 ), we again found evidence for two types of channels. The differences found between the channels were in the steady-state inactivation properties and in the kinetics of inactivation.

The possibility that the apparent differences between the fast and slow channels was due to an error in the voltage protocol subtraction method was addressed pharmacologically. It is possible that a slowly inactivating delayed rectifier type of channel was contaminating the subtraction tech-


nique. This type of conductance was described in semicircular canal hair cells by Correia et al. ( 1989). 4-AP was used to selectively antagonize the rapidly inactivating A- type channels. The action of 4-AP (Fig. 8) on the cells rein- forces the original hypothesis of two A-type channels and validates the voltage protocol subtraction method. When the responses of the cells after 4-AP application were subtracted from those before 4-AP application, the currents blocked by 4-AP are revealed, and both types of A-currents can be seen with characteristics as previously described with a prepulse protocol (Fig. 8). Although further studies were planned with 4-AP, the nonspecificity of the drug, as re- viewed in Choquet and Korn ( 1992), made further investigations with 4-AP ambiguous.

Apparently, the reversal potential (Fig. 3) and the normalized activation conductance (Fig. 4) are the same for both fast and slow channels. However, the slower A-channels carry larger currents at a given membrane voltage as compared with the faster A-channels. It is not possible to tell from this data whether the greater current is the result of more channels per cell or more current per channel.

On the basis of the evidence presented here, we are suggesting that the major inactivating conductances are of the A-type. Two A-type conductances have been described, fast and slow. The differences between the two are the kinetics of inactivation and the steady-state inactivation properties that leave a higher proportion of the fast channels enabled at rest.

The idea that there can be two forms of 1A in one cell, one with a fast and one with a slow inactivating component, is not new. Rudy ( 1988) suggested that close examination of data presented by Gustafsson et al. ( 1982), Halliwell et al. ( 1986), and Boyett ( 198 1) will reveal two transient outward potassium currents that fit the classification of A-type. One has fast inactivation characteristics and the other ex- hibits slower inactivation characteristics. Greene et al. ( 1990) also clearly describe in detail fast and slow A- currents in histaminergic neurons from the rat hypothalamus.

The differences observed between the fast and slow channels are localized to the inactivation properties. The steady- state inactivation properties, including the voltage of half- maximal conductance as well as the slope function (a reflection of the gating charge), are dramatically different. The kinetics of inactivation is also different. Activation, ion selectivity, and pharmacological sensitivity were not different. These results might then suggest that the differences between the channels are localized to the inactivation gate or the portion of the protein responsible for inactivation. For Al-shaker channels this site has been localized to the NH,-terminal (Hoshi et al. 1990; Zagotta et al. 1989; Za- gotta and Aldrich 1990). A ball and chain type of mechanism has been described in which the amino terminus acts as a tethered inactivation particle that can block the internal mouth of the channel ( Hoshi et al. 199 1; Zagotta et al. 1989; Zagotta and Aldrich 1990). It is possible, although speculative, that the differences between the fast and slow channels are due to differences in the NH,-terminal of the protein. Investigation of what regulates the preferential expression of one type over the other may lead to important information regarding signal processing in the vestibular end organs.

A-current is dominant

In hair cells from the SCC of R. pipiens, the largest most dominant current is clearly of the A-type (Fig. 1). Although it is possible that a holding period at -60 mV followed by the prepulses used may not have completely removed inactivation (in the - 130 mV prepulse case) or completely inactivated the Z* (in the -20 mV prepulse case), the examples demonstrate that these protocols allow effective isolation of the 1* (Fig. 1). It is also clear that the amount and character of the inactivating currents varies among hair cells isolated from the SCC. The resting membrane potential for the cells studied in this paper was between -45 and -70 mV. This means that somewhere between 20% of the slow channels and 50% of the fast A-channels are enabled and can be activated from a resting potential. These values are based on the steady-state inactivation plots described earlier. Although these currents can be activated from rest, only 2- 10% may be on at the resting membrane potential, based on the steady-state activation properties. Therefore the inactivating currents do not appear to be involved in establishing the resting membrane potential of the semicircular canal hair cells.

Current-clamp responses

In the current-clamp responses of Fig. 11, the differences noted between Fig. 11, A and B, are apparently caused by the enabling of A-channels in Fig. 11 C. Sugihara and Furu- kawa ( 1989) have reported similar findings in hair cells of the goldfish sacculus, which they also attribute to the A- currents. Puil et al. ( 1989) also present evidence that the A-current can significantly alter membrane oscillations in trigeminal root ganglion neurons. Hair cells are capable of generating spike-like activity (Evans and Fuchs 1987; alligator cochlea), (Hudspeth and Corey 1977-frog), (Art and Fettiplace 1987; turtle cochlea), and (Fuchs and Mann 1986; chick cochlea). The delay between the start of the current step and the sharp voltage rise to a final plateau seen in Fig. 11 appears to be the hair cell equivalent of spike-latency interval in neurons. Angelaki and Correia ( 199 1) have suggested that the membrane oscillations of semicircular canal are generated by the interaction between a delayed rectifier type of conductance and an A-type conductance. Preliminary evidence here suggests that the frequency of oscillation is not regulated by the A-current, rather the damping function (Q) is modulated.

Comparison with other hair cells

Although the 1A recorded by Hudspeth and Lewis ( 1988) in frog saccular hair cells is unlikely to be active during depolarizing changes from physiological resting membrane potentials, these authors suggest “Z* may become activated by a rapid depolarization after a long hyperpolarization, such as might result from mechanical stimulation following a burst of activity of the cell’s efferent nerve supply” (after Art et al. 1984; Murrow and Fuchs 1990) or from depolarizing mechanical stimulation after a strong hyperpolarizing mechanical stimulation. Murrow and Fuchs ( 1990) have demonstrated that the A-type channels are found in short hair cells as opposed to tall hair cells in the basilar papilla.


No particular cell type in the frog appears to be dominated by the A-type conductance.

Studying vestibular hair cells isolated from the chick, Oh- mori ( 1984) did not record the presence of an A-current. In these cells, the major outward K current was an IK(ca). Like- wise, Lewis and Hudspeth ( 1983) reported that the largest current produced by depolarization of hair cells isolated from the saccule of the bullfrog was the Ca*’ activated K+ current. Lang and Correia ( 1989) recorded an A-type current in pigeon semicircular canal hair cells. However, in the pigeon, the A-type current was only weakly active when membrane depolarizations occurred after preconditioning pulses between -70 and -50 mV and was totally inactive after preconditioning pulses more positive than -40 mV. Rennie and Ashmore ( 199 1) also report on A-currents in type II semicircular canal hair cells isolated from the guinea pig. The current is a smaller proportion of the total current as compared with SCC cells from the frog and is only found in selected cells. Thus, semicircular canal hair cells of different species and from different organs may differ from one another in regard to important electrophysiological properties. The differences found between different species is inter- esting and future work will be geared toward determining the significance of these differences.

Although hair cells from the SCC will show some electrical resonance, that is, a membrane voltage oscillation results from the injection of current (Correia et al. 1989; Ren- nie and Ashmore 199 1 ), this response is not nearly as ro- bust as has been seen in other hair cell systems (i.e., Crawford and Fettiplace 198 1). Angelaki and Correia ( 199 1) demonstrated that the interaction between a delayed rectifier and the A-type current could result in a membrane voltage oscillation of low Q and low frequency. The properties of the fast inactivating current reported here suggest that it may be involved in the hair cell response to a current injection; however, the slow conductance would re- quire an initial hyperpolarization for it to be enabled. Fu- ture experiments are being directed toward characterizing the frog semicircular canal hair cell voltage response properties, with special emphasis on the role of the inactivating conductances.

Possible significance of the inactivating currents

Several roles may be suggested for the A-currents in vestibular sensory cells. They could act as a hyperpolarizing buffer that serves to rapidly oppose transductional depolarizations of the hair cell membrane potential. It is important to consider that this putative buffer then turns itself off by inactivating, thus allowing the cell to respond to prolonged stimuli such as are appropriate for vestibular organs. They can act to block out responses to sudden vibratory move- ments of the perilymph/endolymph fluids that might be important for auditory responses by other areas of the inner ear.

In summary, frog semicircular canal hair cells appear to have two distinct A-type conductances. The channel types differ in steady-state inactivation properties as well as in the kinetics of inactivation. Hair cells vary in the relative proportion of each channel type found. The physiological significance remains to be studied, but preliminary data would

suggest that the fast inactivating current is involved in modulating the damping function of the membrane voltage oscillation.

We acknowledge the helpful criticism of this manuscript by Drs. C. W. Clarkson and G. G. Schofield. A special thanks goes to A. Alexander for outstanding technical support of this research.

The authors are grateful for support provided by National Institutes of Health Grants NS-2205 1 and DC-OO303-05 and the Southern Hearing and Speech Foundation. The New Zealand Deafness Research Foundation and the Medical Research Council of New Zealand are also thanked for assistance given to G. D. Housley.

Present address of G. D. Housley: Dept. of Physiology, University of Aukland, Private Bag, Aukland, New Zealand.

Address for reprint requests: C. H. Norris, Dept. of Otolaryngology, Head and Neck Surgery, Tulane University School of Medicine, 1430 Tu- lane Ave., New Orleans, LA 70112.

Received 7 May 199 1; accepted in final form 18 June 1992.

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