Journal of Physiology (1991), 434, pp. 369-398 369 With 16 figures Printed in Great Britain
THE ACTIONS OF CALCIUM ON THE MECHANO-ELECTRICAL TRANSDUCER CURRENT OF TURTLE HAIR CELLS BY A. C. CRAWFORD, M. G. EVANS* AND R. FETTIPLACEt$ From the Physiological Laboratory, University of Cambridge, Cambridge CB2 3EG (Received 25 July 1990)
SUMMARY 1. Mechano-electrical transducer currents evoked by deflections ofthe hair bundle were recorded in turtle isolated hair cells under whole-cell voltage clamp. The outcome of perfusing with solutions of reduced Ca2+ concentration was investigated. 2. The transducer current was roughly doubled by lowering the concentration of divalent cations from normal (2-2 mM-Mg2+, 2-8 mM-Ca2+) to 0 Mg2+, 0 5 mM-Ca2+. No significant effects on the current's kinetics or reversal potential, or on the current- displacement relationship, were noted. 3. If the Ca2+ concentration was lowered to 50 ,UM (with no Mg2+), there was about a threefold increase in the maximum current but other changes, including loss of adaptation and a decreased slope and negative shift in the current-displacement relationship, were also observed. As a result, more than half the peak transducer current became activated at the resting position of the hair bundle compared to about a tenth in the control solution. 4. The extra changes manifest during perfusion with 50 /LM-Ca2+ had also been seen when the cell was held at positive potentials near the Ca2+ equilibrium potential. This supports the view that some consequences of reduced external Ca2+ stem from a decline in its intracellular concentration. 5. With 20,tM-Ca2+, a standing inward current developed and the cell became unresponsive to mechanical stimuli, which may be explained by the transducer channels being fully activated at the resting position of the bundle. 6. The results are interpreted in terms of a dual action of Ca2+: an external block of the transducer channel which reduces the maximum current, and an intracellular effect on the position and slope of the current-displacement relationship; the latter effect can be modelled by internal Ca2+ stabilizing one of the closed states of the channel. 7. During perfusion with 1 gM-Ca2 , the holding current transiently increased but then returned to near its control level. There was a concomitant irreversible loss of sensitivity to hair bundle displacements which we suggest is due to rupture of the mechanical linkages to the transducer channel.
* Present address: Department of Physiology, Medical School, University Walk, Bristol BS8 lTD. t Present address: Department of Neurophysiology, University of Wisconsin Medical School, 273 Medical Sciences Building, 1300 University Avenue, Madison, WI 53706, USA. $ Names printed in alphabetical order. MS 8682 370 A. C. CRAWFORD, M. C. EVANS AND R. FETTIPLACE 8. Following treatment with 1 #uM-Ca2", single-channel currents with an amplitude of -9 pA at -85 mV were sometimes visible in the whole-cell recording. The probability of such channels being open could be modulated by small deflections of the hair bundle which indicates that they may be the mechano-electrical transducer channels of conductance about 100 pS. 9. Open- and closed-time distributions for the channel were fitted by single exponentials, the mean open time at rest being approximately 1 ms. The mean open time was increased and the mean closed time decreased for movements of the hair bundle towards the kinocilium. 10. The Ca2+ concentration in the endolymph of the turtle's cochlear duct was measured with a Ca2+-sensitive microelectrode and found to be 65 /M. The signifi- cance of the composition of endolymph for mechano-electrical transduction by hair cells is discussed.
INTRODUCTION The composition of endolymph, the fluid that bathes the transducing apparatus of hair cells in the inner ear, is unusual, since like an intracellular fluid it is rich in K+ but deficient in Na+ and other cations (Johnstone, Schmidt & Johnstone, 1963; Bosher & Warren, 1968). The divalent cation concentrations are kept particularly low, reported values being about 20 /SM for Ca2+ and 10/tM for Mg2+ (Bosher & Warren, 1978; Ikeda, Kusakari, Takasaka & Saito, 1987). The significance of the ionic composition has never been fully explained and the role ofCa21 ions is especially puzzling. It has been known for some time that in the absence of Ca2+ hair cells cease to function as mechano-electrical transducers (Sand, 1975; Jorgensen, 1983). As little as 20 /M can maintain transduction (Corey & Hudspeth, 1979; Ohmori, 1985), yet Ca2+ concentrations greater than 250 JSM diminish the flow of transducer current (Corey & Hudspeth, 1983). The mechanisms of these effects are unknown. It has recently been shown that an additional role for Ca2+ is to control adaptation of the transducer current during a maintained stimulus (Eatock, Corey & Hudspeth, 1987; Assad, Hacohen & Corey, 1989; Crawford, Evans & Fettiplace, 1989). The regulation probably occurs by Ca2+ entering the cytoplasm and resetting the range ofbundle displacements that are detected. Here we have extended these observations by recording transducer currents in isolated cells bathed in salines of reduced Ca2+ content. Most of our results are consistent with a dual action of Ca2+: a blocking of the transducer channel probably at its external surface, and an effect of intracellular Ca2+ on the gating process. An unexpected finding was that with 1 ,uM-Ca2+, almost all of the transducer channels vanished irretrievably, but occasionally a single channel remained long enough for its properties to be analysed.
METHODS Preparation and recording techniques Turtles (Pseudemys scripta elegans) were decapitated and hair cells isolated from the basilar papilla by methods described fully in Art & Fettiplace (1987) and Crawford et al. (1989). Cells were plated out onto glass cover-slips coated with 2-5 mg/ml concanavalin A which immobilized the cell bodies but allowed the ciliary bundles to be manipulated. Displacements of the bundles, which had a maximum height of about 6 ,um, were produced by a glass stylus attached to a piezo-electric CALCIUM AiNTD HAIR CELL TRAiNSDUCTION3371 bimorph. The glass probe was placed near the tip of the bundle behind the tallest row of stereocilia, and if clean it adhered to the bundle and so could push or pull the bundle towards or away from the kinocilium. Since displacements of the hair bundle towards the kinocilium activate the transducer conductance (Shotwell. Jacobs & Hudspeth, 1981), these will sometimes be referred to as positive displacements, whereas bundle stimuli in the opposite direction will be denoted as
TABLE 1. Composition of extracellular solutions containing various concentrations of calcium Solution NaCl KCI MgCl2 CaC12 Other A Normal 130 4 2-2 2-8 B Normal 0 Mg2+ 136 4 0 2-8 C 0 5 mM-Ca2l 136 4 0 0 5 D 50,uM-Ca2+ 136 4 0 0 05 E 20,uM-Ca2+ 136 4 0 0 F 1 /nM-Ca2+ 130 4 0 2-5 5 Na-HEDTA G Normal K+ 0 135 2 2 2-8 (5 CsCl) H K+, 0 5 mM-Caa2+ 0 135 2-2 0-5 I Endolymph 0 140 0 0 5 Concentrations are in mm. All solutions also contained 5 mM-Na-HEPES (or K-HEPES for G-I) and 4 mM-glucose. Solution A is artificial perilymph (Crawford & Fettiplace, 1980) and was usually used as the control solution. Ca2+ concentrations were measured with a calcium electrode. Solution E had no added Ca2+ but contained 14-28 ,UM, probably as contamination from NaCl. In some experiments with the K+-based solutions, a mm-CsCl was added to block the inward rectifier. negative. In some experiments where it was necessary to produce large negative displacements (i.e. away from the kinocilium) the glass probe was positioned on the kinocilial side of the bundle. A more detailed description of the stimulation equipment and its calibration is given in Crawford et al. (1989). The bundle could be stepped from one position to another in less than 100 ,us. Transducer currents were measured using the whole-cell patch-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981) with a List EPC-7 amplifier. Borosilicate recording electrodes (resistance 5-8 MQ) were usually filled with a solution containing (in mM): KCl, 125; MgCl2, 3; KHEPES, 5; K2EGTA, 5; Na2ATP, 2-5; pH adjusted to 7-2 with KOH. For experiments where the membrane potential was clamped to depolarized levels, the patch electrode was filled with a solution of composition (in mM): CsCl, 125; MgCl2, 3; NaHEPES, 5; Na2EGTA, 5; Na2ATP, 2-5; pH adjusted to 7 2 with NaOH. The time constant of the recording system, estimated from the product of the cell capacitance and the series resistance remaining after compensation, had a minimum value of 50-100 ,ts. Transducer currents were normally measured at a holding potential of -85 mV, 30-40 mV negative to where the voltage-sensitive currents are activated, so as to make it unlikely that variation in these currents, particularly the large Ca2+-activated K+ current, contributed to the effects observed. All membrane potentials were corrected for the junction potential (1-6 mV according to the solution) and for incomplete compensation of the series resistance. Experiments were performed at 18-23 'C. Solutions Experiments were normally begun in a control solution resembling perilymph which contained (in mm): NaCl, 130; KCI. 4; CaCl2, 2-8; MgCl2, 2-2; glucose, 4; NaHEPES, 5; pH adjusted to 7 6 with NaOH. The ionic environment of a cell was rapidly changed using a U-tube perfusion system (Krishtal & Pidoplichko, 1980; Fenwick, Marty & Neher, 1981). Under the best conditions the solution bathing a cell could be exchanged within a few hundred milliseconds, though positioning of the U-tube too close to a cell introduced turbulence and excess noise into the recording. The speed with which the Ca2" concentration at the cell's membrane could be lowered was verified by following the abolition of the Ca2+-activated K+ current in a low-calcium saline. The compositions of the various external solutions are given in Table 1. These fall into two broad categories: those with K+ as the major cation, and those containing reduced concentrations of Ca2+ which were calibrated with a TTPCA Ca2"-sensitive electrode (World Precision Instruments, Hastings). The solution found to contain 20 ,yM-Ca2+ was nominally calcium free when made up, 372 A. C. CRAWFORD, M. G. EVANS AND R. FETTIPLACE but the residue probably reflected contamination in the NaCl. That containing 1 /M-Ca2" was buffered with HEDTA (Sigma) which was calculated to have an effective dissociation constant of 0-8 /LM at pH 7-6 using stability constants from Martell & Smith (1974). Data analysis During an experiment, the membrane current and other signals were stored on FM tape (Racal Store 7, bandwidth 0-8 kHz) for later analysis. Subsequently the current was low-pass filtered (6 pole Bessel), digitized in a Cambridge Electronic Design 1401 Interface and analysed on an IBM- AT computer using a DAOS software package (Laboratory Software Associates, Fitzroy, Australia). In quantifying transducer currents, typically between four and ten responses were averaged under a given set of conditions, and the resulting trace was often smoothed with a three- point digital filter. Residual stimulus artifacts due to capacitative coupling between the recording electrode and the piezoelectric drivers have been removed from the final records (Crawford et al. 1989). For the analysis of the single-channel records, the current was filtered at between 1 and 2-5 kHz before being digitized at a sampling rate of 6 kHz. Channel open and closed times were determined by setting a threshold at half the amplitude of the open channel current (Colquhoun & Sigworth, 1983). A minimum resolvable interval of 320 ,ts was imposed on the data and values less than this minimum were ignored for construction of histograms of the open and closed times. Single-exponential probability density functions were fitted to the histograms using an algorithm in DAOS that performed a least-squares fit on the Chebyshev transform of the data. Owing to unresolved brief closures, the true open times will be shorter than those estimated (Marty & Neher, 1983), but no attempt has been made to apply a correction. For examining the effects of hair bundle stimulation on channel opening, step deflections of the bundle lasting 50 ms were applied at about 10/s. Open and closed times were determined during equal periods with the bundle in the resting and displaced positions and were then pooled from a number of consecutive stimuli. Calcium-sensitive microelectrodes Measurements of the Ca2+ concentration of endolymph in the intact cochlea were performed with Ca2+-sensitive single- or double-barrelled micropipettes constructed as described by Tsien & Rink (1980). The Ca2+ electrodes had tip diameters of approximately 1 ,tm and were filled with an ion exchanger (Fluka) based on the neutral carrier ETH 1001 (Oehme, Kessler & Simon, 1976), and an electrolyte consisting of 10 mM-CaCl2, 90 mM-XCl. Before and after a measurement the electrodes were calibrated in a series of Ca2+ buffers (Tsien & Rink, 1980), and had a mean response of 32 mV per tenfold change in Ca2+ concentration in the range 10/tM to 1 mm of this ion. The experiments were carried out on an isolated half-head preparation (Crawford & Fettiplace, 1980). The turtle was decapitated, the head split sagitally, and the scale tympani opened and filled with artificial perilymph (solution A). The Ca2+ electrode was introduced into the scale tympani and was advanced into the cochlear duct through the region of basilar membrane not covered by hair cells. In one experiment, the K+ concentration in endolymph was also determined with an electrode filled with a K+ ion exchanger (IE190, World Precision Instruments, Hastings) and 140 mM-KCl. The endocochlear potential, measured with a reference electrode filled with 3 M-KCI, was never more than a few millivolts with respect to the perilymph. (-1-5+ 2-2 mV, mean+s.D., n = 6), which agrees with our previous results (Crawford & Fettiplace, 1980).
RESULTS Transducer currents in artificial endolymph Hair cells constitute an epithelium that isolates two fluid compartments of dissimilar composition: the hair cell's basolateral aspect is bathed in plasma-like perilymph, whereas its apical facet and hair bundle are exposed to endolymph. Such fluid separation is not readily achievable in experiments on isolated hair cells which are normally immersed in an artificial perilymph. Since the mechano-electrical transducer channels are thought to reside in the hair bundle (Hudspeth, 1982; Ohmori, 1988), it is important to ask what advantages for the transduction process accrue from the special ionic composition of endolymph. CALCIUM AND HAIR CELL TRANSDUCTION 373 Figure 1A compares transducer currents recorded in perilymph (solution A in Table 1) with those in an artificial endolymph (solution I) which has K+ as the major cation, no Mg2' and 0.5 mM-Ca2+. (Although 0 5 mM-Ca2+ was chosen at the outset, subsequent measurements to be described later indicate that the Ca21 concentration in turtle's endolymph is nearly an order of magnitude lower.) The currents were measured under voltage clamp at a holding potential of -85 mV. There was a roughly threefold increase in the size of the currents in endolymph accompanied by an equivalent increase in current noise, suggesting that the main effect is an augmentation of the effective conductance of the transducer channel. However, other features of the responses, including their kinetics and the form of the current-displacement relationship, were not greatly affected. In particular, the currents developed rapidly, with onset time constants of 100-250 ,ts comparable to those in perilymph (Crawford et al. 1989), and still displayed adaptation. Figure 1B gives a plot of the peak currents against bundle displacement; the smooth curve through the control and recovery points has been scaled by 3-1 in order to fit the measurements in endolymph. There was no change in the fraction of current activated at the resting position of the bundle, in contrast to the behaviour seen with 50 4uM-Ca21 (Fig. 5) where there also developed a large standing inward current. Identical results to those of Fig. 1 were obtained in another cell, where the total current increased by 2-5 times but the proportion activated at rest was 0-14 in the control and 0'17 in the artificial endolymph. Families of transducer currents similar to those in Fig. 1A were obtained in a number of different ionic solutions in order to dissect the changes found in artificial endolymph. All such measurements were made at a holding potential of about -80 mV and the factor by which the control current-displacement relationship had to be magnified in order to match that in the test solution was ascertained. The collected results are given in Table 2. These may be summarized by saying that each of the three manipulations, exchanging K+ for Na+, removing Mg2+ and lowering Ca2+ to 0 5 mm, contributed to the increase in total transducer current observed in endolymph, though the largest effect stemmed from reduction in the divalent cations. An additional 50% increase in the peak current could be produced if the concentration of Ca2+ was lowered to 50 /tM (solution D), but in this solution there was also a loss of adaptation and a shift in the current-displacement relationship. The mechanisms underlying these extra effects are discussed in later sections. Reversal potentials Much of the increase in current observed in artificial endolymph is due to the reduced concentrations of divalent cations. This increase can be seen in the records of Fig. 2 during perfusion with solution C, which is sodium-based but has 0 5 mM-Ca2+ and no Mg2+. There were no shift in the current-displacement relationship and the response to a single large stimulus is displayed at different holding potentials. It is worth noting that the transducer current shows pronounced adaptation at the largest negative membrane potentials, but at membrane potentials positive to the current's reversal potential, the adaptation is lost and the tail currents are slowed. These changes have been noted previously and used as evidence that adaptation may be mediated by a rise in intracellular Ca2+ resulting from its influx via the transducer 374 A. C CRAWFORD, M. G. EVANS AND R. FETTIPLACE
A
m2EPM 4
0 r- nA Perilymph
-02 0i loVel
nA -021 Endolymph
-0.41
0 20 40 60 80 Time (ins)
B 0.5
0*4 nA
Endolymph
A
0 1 Perilymph
0 I I I -1 0 -0.5 0 0.5 1.0 1-5 Bundle displacement (pum)
Fig. 1. For legend see facing page. CALC,IUIM AND HAIR CELL TRANSDUCTION 375 channels (Assad et al. 1989; Crawford et al. 1989). It is to be expected that the influx wouhi be diminished at positive holding potentials due to a reduction in the electromotive driving force on the ion. Curiously, the adaptation is if anything more pronounced in the low-divalent cation saline, reasons for which will be discussed later.
TABLE 2. Increase in transducer current in different ionic solutions Test solution It/Ic Na+/K' Ca2l (mM) Mg2" (mM) (mean+s.D.) (B) Na' 2-8 0 1-4+0-1 (n = 3) (C)t Na+ 05 0 1-9+0-1 (n = 3) (G) K+ 2-8 2-2 1 25 + 0-02 (n = 2) (I) K+ 05 0 2-8+0-3 (n = 2) (D) Na+ 0 05 0 3-1 (D)* Na+ 0-05 0 2-1 +0-4 (n = 3) It/Ic, ratio of maximum transducer current in test solution to that in control solution; values obtained at holding potentials between -75 and -85 mV. All controls measured in solution A (Na+, 2-8 mM-Ca2+, 2-2 mM-Mg2+) except for * where it was solution B which contained no Mg2+. The assumptions made for deriving values in 0 05 mM-Ca2+ are discussed in the text. All measurements were made with KCl-filled electrodes except t when CsCl electrodes were used.
From these records the relationship between peak transducer current and membrane potential is constructed in Fig. 3. Owing to rectification in the low- divalent cation solution, the increase in the current was greater at negative membrane potentials, causing the current to be nearly doubled at -80 mV. However there was little change in the current's reversal potential: the mean reversal potential in the control (solution A) was 4 6+1-9 mV (n = 5) and that in the low-divalent cation saline (solution C) was 4 0 + I 0 mV (n = 2), all measurements being made with Cs'-filled electrodes. These results argue that removal of the divalent ions increases the conductance of the transducer channel, and they are contrary to the conclusions of Jorgensen (1984) who postulated that the reversal potential shifted to more negative values as the Ca2+ concentration was lowered. The current-voltage relationship in the endolymph (solution I) also displayed some rectification when measured with a Cs+-filled electrode; however, the reversal
Fig. 1. Effects of an endolymph-like solution on the mechano-electrical transducer of an isolated hair cell. A, averaged transducer currents at a holding potential of -85 mV for displacements of the tip of the hair bundle. Movements towards the kinocilium, denoted as positive in the stimulus monitor, cause an increase in inward current given as negative relative to the current level without stimulation. The upper records (perilymph) were measured in solution A of Table 1 (Na+, 2-8 mM-Ca2+, 2-2 mM-Mg2+) and the lower records (endolymph) were obtained in solution I (K+, 0 5 mM-Ca2+, 0 Mg2+). Four responses averaged for each trace. In this and subsequent figures, currents were recorded with KCl- filled electrodes unless otherwise stated. B, current-displacement relationship derived from the experiment in A. Open circles and triangles are controls before and after measurements in endolymph which are plotted as filled symbols. The smooth curve drawn by eye through the filled circles was scaled by 0-32 to fit the open symbols. 376 A. C. CRAWFORD, M. G. EVANS AND R. FETTIPLACE potential was near that of the control, and in two experiments had a mean value of 5-5 mV. The reversal potentials in the low-divalent cation salines may be used to estimate the permeability ofthe channel to monovalent cations. With the assumption that the channels obey the constant field equation (Hodgkin & Katz, 1949), and
80 56
40 -1 0
'A,AA \_ 2 8mmM-Ca2+ -40 - -24 2 2 mM-Mg2+ -80 -64 mv
80-
40 - 1 X/ 57
-80 -43 0 ff -63 mV -120 0 20 40 60 80 Time (ms) Fig. 2. Averaged transducer currents at different holding potentials in a normal perilymph (solution A) and saline containing low concentrations of divalent cations (solution C). At the top is shown the timing ofthe 0 7 ,tm displacement step towards the kinocilium, which applies to all the traces. The holding potentials and the concentrations of Ca2+ and Mg2+ are given beside the traces. The records which were made with a CsCl-filled electrode have been superimposed so that current levels in the absence of bundle stimulation coincide. In this cell, deflections of the hair bundle away from the kinocilium elicited no transducer current under any of the experimental conditions. Note the loss of adaptation and change in kinetics at positive holding potentials. neglecting the contribution of the 0 5 mM-Ca2+, the relative permeabilities calculated were: PNa:PK:Pcs = 1:099:0-79. These are in good agreement with the published values of Ohmori (1985). The effects ofMg2+ ions In contrast to the results in Figs 1 and 2, where the predominant consequence of lowering the concentration of divalent cations is merely to amplify the responses, more complex effects were produced by reducing the Ca2+ concentration to 0 5 mm CALCIUM AND HAIR CELL TRANSDUCTION 377 without removal ofMg2+. These effects, which were entirely reversible, are illustrated in Fig. 4. The most obvious change visible in the lower set of responses in the test solution is the loss of adaptation; this would be expected if adaptation were mediated by Ca21 influx into the cell. A second feature of note is the slowing of the kinetics of
100 pA
50 -
-80 -60 -40 -20 I I I I 20 40 60 80 mV
-50 *^ OA 2.8 mM-Ca2+, 2.2 mM-Mg2+ 0 0.5 mM-Ca2+, 0 Mg2+ -100 _
-150 -
-200 _ Fig. 3. Current-voltage relationships for transducer in perilymphs containing normal and low divalent cation concentrations derived from the records of Fig. 2. The peak transducer current is plotted against membrane potential which has been corrected for uncompensated series resistance. The open circles and triangles are measurements in normal artificial perilymph (2-8 mM-Ca2+, 2-2 mM-Mg2+) before and after perfusion with saline containing low concentrations of divalent cations (05 mM-Ca2+, 0 Mg2+, @). Note that the reversal potential is unaffected by reduction in divalent cations. the current. A quantitative analysis of the records showed that for displacements of the hair bundle away from the kinocilium, the current relaxed with a principal time constant of 5-5 ms. For bundle displacements towards the kinocilium, the current developed with two distinct components; for the largest response, the time constant of the fast component was 0 3 ms and that of the slow component was 3-9 ms. A third effect of reducing the Ca2+ is also evident in Fig. 4. In the control, only about 15 % of the cell's transducer conductance is turned on at the resting position of the bundle, but in 0 5 mM-Ca2+ the proportion activated at rest has risen to nearly 40 %. The differences are quantified in the current-displacement relationships in Fig. 4B. The change in current in each solution has been measured relative to the steady current with a large negative displacement and it has been normalized to the maximum current excursion, Imax. The value ofImax increased from about 240 pA in the control to 350 pA in 05 mM-Ca2+, but in addition the current-displacement relationship was translated about 120 nm to negative displacements and the slope of 378 A. C. CRAWFORD, M. G. EVANS AND R. FETTIPLACE
A
I
50 0 pA -100 2 8 mM-Ca2+, 2-2 mM-Mg2+ -200'-
100 pA 0
-100 0.5 mM-Ca2+, 2 2 mM-Mg2+ -200 K I 8 0 20 40 60 80 Time (ms)
B
1t 0 r 0 0
x
°° 2 8 mM-Ca2+, 2 2 mM-Mg2 * 0 5 mM-Cat2+ 2 2 mM-Mg2+
05
I I 5I -1.0 -0.5 0 0-5 1-0 1-5 Bundle displacement (pM) Fig. 4. A, families of transducer currents recorded in normal perilymph (solution A) above and in K+, 0 5 mM-Ca2l (solution H) below. Each trace is the average of four responses at -85 mV holding potential. The stimulus monitor above applies to both sets of currents; ordinate scales are given relative to the current level in the absence of bundle stimulation. Note that in the K+, 0 5 mM-Ca2l saline, adaptation has disappeared and a larger fraction of the transducer current can be turned off by negative displacements of the bundle. B, current-displacement relationships for the experiment of Fig. 4A. All transducer currents were measured relative to the steady current level produced by the largest displacement away from the kinocilium. The currents, I, have been normalized to Im.x, their maximum CALCIUM AND HAIR CELL TRANSDUCTION 379 its steepest region was reduced. The negative shift of the current-displacement function accounts for the larger fraction of current turned on in the absence of stimulation. The assorted consequences of lowering the Ca2+ level to 0 5 mm in the presence of Mg2+, the disappearance of adaptation, the slowed kinetics and the shift in the current-displacement relationship, are seen under two other circumstances which may provide a clue to their mechanism. They occur in the absence of Mg2+ provided the Ca2+ concentration is further reduced extracellularly to 50-100 /tM. Note the striking similarity of the records in Fig. 5, which were obtained in the presence of 50 ,uM-Ca2+ but no Mg2+ with those in Fig. 4A. Moreover, an analogous transformation of the current can be achieved in the control solution by switching the holding potential from a normal negative value, -80 mV, to a positive value such as -70 mV (see Fig. 12 of Crawford et al. 1989). It is worth noting that in a given experiment, the various changes in the transducer current were not of equal strength. Thus while a given manipulation, such as depolarization, would abolish adaptation and slow the kinetics, this was not always accompanied by a significant shift in the current-displacement relationship (Crawford et al. 1989). An explanation that has been presented previously to account for these effects *(Crawford et al. 1989) is that Ca2+ ions normally enter the stereocilia along with the transducer current and induce adaptation and speed-up the current's kinetics by acting at an intracellular site. Depolarizing the cell reduces the driving force on Ca2+ entry, lowering the influx and hence the intracellular concentration of this ion. The differences in responses in the presence and absence of Mg2` suggests that Mg2+ may have a similar effect to depolarization, by either reducing Ca2+ entry or antagonizing its action at the intracellular site. This action of Mg2+ on the current-displacement relationship has also been reported by Hacohen, Assad, Smith & Corey (1989) based on measurements of microphonic currents in the bull-frog saccule. Effects of 50 puM-Ca2+ The changes observed on perfusing with 50 /zM-Ca2+, 0 Mg2+ were in general similar to those described in the previous section with 0 5 mm-Ca2+, 2-2 mM-Mg2+. Examples of experimental records are shown in Fig. 5, where it can be seen that the transducer currents in 2X8 mM-Ca2+ have rapid onsets and offsets and exhibit adaptation, whereas the currents in 50 #m-Ca2+ have lost their adaptation and the kinetics, particularly for negative-going bundle deflections, are slowed. Moreover, a change in the fraction of the conductance activated when the bundle was in its resting position was always observed. In Fig. 5, the responses in the control solution require only about 10% of the total conductance to be activated at rest, but in the low Ca2+ solution more than 30 % of the conductance was turned on at rest. Normalized current-displacement relationships for this cell are plotted in Fig. 6; the convention is again adopted that the currents were measured relative to the
values, which were: 0 (control), 253 pA; * (K+, 05 mM-Ca2+), 349 pA; O (recovery), 234 pA. The smooth curves were calculated from eqns (2), (3) and (4) in the Discussion, with a, = 4a2 = 11-43 ,um-, x2 = 0093 /,m, and xl = x2-dx, where dx = 0 (control) and dx = 0-23 ,um (test). Note the negative shift and diminished slope of measurements in the test solution; such changes were not seen when only the Na+/K+ substitution was made. 380 A. C. CRAWFORD, M. G. EVANS AND R. FETTIPLACE steady level with a large displacement away from the kinocilium which should close all the transducer channels. The low Ca2+ caused a reversible shift of the current- displacement relationship in a negative direction and reduced its maximum slope. In all five experiments it was found that treatment with 50,am-Ca2+ caused a substantial increase in the holding current equivalent to an extra inward current of
2
pm0 M-0 1I ------j------=-----
50 pM-Ca2+
-0 1_
nA 2 r,2 2.8 mM-Ca 2.8 mM-Ca2+ -0-2-
-0.3 - L_ 0 40 80 0 40 80 0 40 80 Time (ms) Fig. 5. Families of transducer currents measured in 0 Mg2` perilymph (solution B: control, left; recovery, right) and in 50 /LM-Ca2+, 0 Mg2` (solution D, middle). Each trace is the average of four responses at -85 mV holding potential. The ordinate scale is given relative to the current for a large negative displacement in the control solution. Note that in addition to the loss of adaptation and the change in the proportion of the current activated at the bundle's unstimulated position, a standing inward current develops in the low-Ca2+ solution. a few hundred picoamperes. This is visible in Fig. 5 where the resting level in the test solution is offset by about 140 pA relative to that in the control. One hypothesis is that all or part of this change in holding current reflects persistent activation of the transducer conductance at the resting position of the bundle. If this were the case, the conclusions inferred from the plots of Fig. 6 would be qualitatively correct, but the changes would be underestimated: in the low Ca2+ solution, the maximum current would be even larger and the slope of the current-displacement relationship would be less. One way of testing for the possibility that the change in holding current in low Ca2+ is largely due to an increased activation of the transducer channels would be to demonstrate that the holding current can be diminished by bundle motion. An experimental difficulty is that with the large negative displacements needed, the stimulatory glass probe, which is normally positioned behind the tallest row of. stereocilia, may become detached and not transmit the full extent of its motion to the bundle. To circumvent this problem and achieve larger negative deflections, a few experiments were performed with the probe placed on the kinocilial side of the bundle so that it could push the bundle away from the kinocilium. The results from two such experiments are shown in Fig. 7A and B. In the left-hand records in CALCIUM AND HAIR CELL TRANSDUCTION 381 Fig. 7A the transducer currents in normal Ca2+ are highly asymmetric in response to large positive and negative stimuli of equal amplitude. In the right-hand records, the responses in 50 1tm-Ca2+ to identical stimuli are now symmetrical and occur around a holding current which is 360 pA more negative. The step away from the kinocilium turns off about 60 % of the increased holding current.
1.0
X 0-0.5 ;/ ,O 2.8 mM-Ca2, @50pOM-Ca2+ oL -0.5 0 0.5 1.0 1.5 Bundle displacement (jum) Fig. 6. Current-displacement relationship for the experiment of Fig. 5. All transducer currents were measured relative to the current level produced by the largest displacement away from the kinocilium in the respective solution. The currents, I, have been normalized to Imax, their maximum values, which were: 0 (control), 115 pA; * (50 /M- Ca2l), 174 pA; O (recovery), 116 pA. Smooth curves drawn through points by eye.
Figure 7B shows records from another cell which was stimulated with a series of negative steps to the bundle. Fifty micromolar Ca2+ induced an inward holding current of about 280 pA, but most of this holding current could be turned off by displacements of increasing magnitude. However, we were unable to deflect the bundle by a large enough amount to saturate the current in the negative direction and therefore be certain that we had turned off all the transducer channels. A maximum of 68% of the holding current in low Ca2+ could be turned off with the largest step of 1-7 4am. This fraction must constitute a lower limit and therefore the simplest conclusion is that virtually all of the increase in holding current represents transducer current. It should be stressed that a change in the current flowing through the Ca2+-activated K+ channels is very unlikely to account for any of the increase in holding current, since the holding potential was -85 mV, close to the K+ equilibrium potential (-89 mV) and well negative to where the Ca2+-activated K+ current would normally be turned on (-50 mV, Art & Fettiplace, 1987). To correct the plot of Fig. 6 and draw quantitative conclusions about the changes occurring in 50 /tM-Ca2 , it will be assumed that, to a first approximation, all of the increase in holding current is transducer current. The results of the cell of Figs 5 and 6 are replotted as the open and filled diamonds in Fig. 8. All currents have been measured from the steady level produced by a large negative displacement in the control solution. This level is indicated by the dashed line in Fig. 5 and subsequent figures. The currents have also been normalized to the maximum current, which 382 A. C. CRAWFORD, M. G. EVANS AND R. FETTIPLACE
A l 07,Jum
0 50 ,uM-Ca2+
-0.2 nA
-0.4 - 2.8 mM-Ca2,
-0.6 L L I I 1L J-J 0 40 80 0 40 80 Time (ms)
B 0 jim-2
0 --IL 50 gM-Ca2+
-0.11-
nA -0-2p 2.8 mM-Ca2,
-0.3L Il 0 40 80 0 40 80 Time (ms) Fig. 7. A, averaged transducer currents for ± 0 7 ,um steps in normal perilymph (solution A, left) and in 50 ,uM-Ca2+, 0 Mg2+ (solution D, right). The stimulating glass probe was placed at the tip of the bundle on its kinocilial face. Between two and ten responses averaged to produce each trace. Holding potential -85 mV. Note that the symmetrical, non-adapting responses in the low Ca2+ solution are superimposed on a standing inward current. B, displacement sensitivity of the standing inward current in a different cell. Averaged transducer currents for negative deflections of the hair bundle in 0 Mg2+ perilymph (solution B, left) and in 50 ,tM-Ca2+, 0 Mg2+ perilymph (solution D, right). The stimulating probe was positioned at the tip of the bundle on its kinocilial face so that negative deflections were produced by the probe pushing the bundle in a direction away from the kinocilium. Each trace is an average of between thirteen and forty-two responses. Note that much of the standing inward current in low Ca2+ can be turned off by negative displacements of the bundle. CALCIUM AND HAIR CELL TRANSDUCTION 383 increased from 115 pA in the control to 261 pA in 50 /,sM-Ca2+. In three cells, the mean increase in the maximum current in 50 /tM-Ca2+ was 241 when the control solution did not contain Mg2+ (see Table 2). If the control solution did contain Mg2+, there was a larger, roughly threefold, increase in maximum current. The proportion of this
x 0-5.5 X/ 2.8 mM-Ca2+ Lv50 pM-Ca2+
0 O -2 -1 0 1 2 Bundle displacement (pm) Fig. 8. Current-displacement relationships in normal perilymph, 0 Mg2+ (solution B, open symbols) and in 50 /iM-Ca2+, 0 Mg2+ (solution D, filled symbols). All transducer currents, I, were measured from the current level produced by the largest negative deflection in the control solution, and have been normalized to Imax' their maximum values, which were: O (control), 115 pA; * and * (50 /tM-Ca2+), 261 pA. K and * taken from experiment of Fig. 5; * corrected from experiment of Fig. 7B as described in the text. The smooth curves were calculated from eqns (2), (3) and (4) in the Discussion, with a, = 3a2 = 4-02 um-1, x2 = 0-033 /sm, and xl = X2- dx, where dx =-055,um (control ) and dx = 3-73 ,um (test). maximum current that was activated when the bundle was in its unstimulated position had a mean value of 0-71 in 50 ,tM-Ca2- compared to 0-14 in 2-8 muM-Ca2". Owing to the difficulties of switching the stimulating probe from one side of the bundle to the other, it was not possible to determine a complete current-displacement relationship for 50 /tM-Ca2+ in a single hair cell. The measurements from Fig. 7B, where the probe was placed in front of the tallest row of stereocilia, have been combined with the measurements of Fig. 5, where the probe was behind the tallest stereociliary rank, and are plotted as the filled circles in Fig. 8. Since a value for the maximum current was unavailable for the cell ofFig. 7B, it was further assumed that the proportion of transducer current turned on when the bundle was unstimulated was the same for both cells. Despite these assumptions, the measurements from the two cells are in reasonable agreement and reinforce the conclusion that the slope of the normalized current-displacement function has been reduced in the 50 /tM-Ca2+ saline by a factor of about three. However, since the total transducer current, ImaX' also increased in 50 ,sM-Ca2+ the overall current sensitivity for small displacements was little affected by reducing Ca2+. The smooth curves drawn through the points in Fig. 8 have been calculated from a model described fully in the Discussion. This model was previously introduced (see Fig. 17 of Crawford et al. 1989) to account for the changes in the shape of the transducer's current-displacement relationship both at positive membrane poten- 384 A. C. CRAWFORD, M. G. EVANS AND R. FETTIPLACE tials and after adaptation. The theoretical scheme, which provides a reasonable fit to the experimental points, can be interpreted in terms of intracellular Ca2+ stabilizing a closed state of the transducer channel. Reversible and irreversible actions of reduced Ca2+ Previous reports (Corey & Hudspeth, 1979; Ohmori, 1985) have indicated that a minimum concentration of Ca2+ of 10-20/4m is required to sustain a mechano-
2.8 mM-Ca2+ 20 ,uM-Ca 2 8 mM-Ca2+ a b cr
nA- -0.25[_
-05_ L Wl I L WI I l 0 20 0 20 0 20 Time (ms) Fig. 9. Reversible effects of 20 4uM-Ca2+ saline. Transducer currents for 0-75 ,um positive deflections of the bundle (towards kinocilium), in normal perilymph containing 2-8 mM- Ca2` (solution A; a, control and c, recovery), and in 20 #M-Ca2+ (solution E; b). Stimulus monitor shown above; holding potential -85 mV. Each trace is the average of twenty- five responses. electrical transducer current in hair cells. We have investigated the effects of Ca2+ concentrations comparable to or less than this minimum in an attempt to understand the mechanism underlying the apparent blocking of the transducer. If the Ca2+ concentration was reduced to 20 ftM, a large inward current developed and simultaneously the cell lost its responsiveness to displacements of the hair bundle. Figure 9 gives average records before, during and after perfusion with the 20 /tM-Ca2+ solution (solution E). The upper traces show the transducer current for a near-saturating displacement of 075,tm; during the low Ca2+ perfusion the response has declined to almost nothing but the holding current has increased by 490 pA. Similar results were obtained in another cell for which the increase in holding current was 650 pA. This behaviour may represent an extreme form of the shift in the transducer's current-displacement curve found with 50 Am-Ca2 . We envisage that all the channels will be turned on, even at the resting position of the bundle, which will account for the standing inward current. Moreover, if the channels are fully activated, further displacements of the bundle in the excitatory direction will necessarily be ineffective. In experiments with 20,M-Ca2+, each mechanical stimulus was followed by a depolarizing voltage-clamp step of sufficient magnitude to turn on the Ca2+-activated CALCIUM AND HAIR CELL TRANSDUCTION 385 K+ channels which are the major voltage-sensitive channels in many hair cells (Lewis & Hudspeth, 1983; Art & Fettiplace, 1987). Pairs of mechanical and voltage-clamp pulses were repeated at about 10/s. The purpose of the voltage-clamp step was to monitor the time course of the Ca2+ concentration change at the cell membrane. As
2.8 mM-Ca2+ 1 iM-a2+ 2.8 mM-Ca2+ a bcd
nA -04 L -0W8L | | E | E | ~~~~~LI 0 20 0 20 0 20 0 20 Time (ms) Fig. 10. Irreversible effects of exposure to 1 /sM-Ca2+ saline. Transducer currents for 1 #um positive deflections of the bundle in 0 Mg2+ perilymph (solution B; a and d) and in 1 /SM- Ca2+ (solution F; b and c). Stimulus monitor shown above; holding potential -85 mV. Traces b and c were obtained 0 5 and 18 s after beginning low-Ca2+ perfusion. Each trace is the average of between five and thirty responses. There was no measurable current throughout for negative displacements of the bundle. Note that in d the transducer current did not recover following a prolonged wash in normal-Ca2+ perilymph. the Ca2+ concentration at the membrane fell, the outward K+ current declined (over a few hundred milliseconds), but as far as we could discern the disappearance of the K+ current was concurrent with the development of the standing inward current. If the changes in the transducer stem from a drop in the intracellular concentration of Ca2+, then a more rapid perfusion system will be required to reveal the time course of equilibration. After treatment with 20 /iM-Ca2 , the transducer current could always be restored by replenishing the Ca2+ in the bathing medium (Fig. 9). However, if the Ca2+ was further lowered to 1 ,UM and buffered with HEDTA, more complex and irreversible changes in the transducer current occurred, as are illustrated in Fig. 10. Immediately following introduction of 1 ftM-Ca2+, a large inward current developed and the response to bundle stimulation was greatly diminished in a manner similar to that with 20 /M-Ca2+. However, if the exposure to the lower concentration of Ca2+ continued for more than a few seconds, the standing inward current returned to near its control level. This disappearance ofthe inward current was always associated with an irretrievable loss of sensitivity to hair bundle stimulation, and the transduction process could not be recovered even when the cell was returned to a solution containing normal Ca2+. In contrast to the transducer current, the CaS+-activated K+ current, which also disappeared in 1 jtM-Ca2+ solution, always recovered to its full amplitude on returning to 2-8 mM-Ca2+. Our interpretation of these complicated events is that superfusion with very low
13 PHY 434 386 A. C. CRAWFORD, M. C. EVANS AND R. FETTIPLACE Ca2+ concentration leads in the first instance to full activation of the transducer conductance, but if the activation is prolonged, a permanent change takes place in the gating mechanism so as to produce an irreversible loss of mechanical sensitivity. The Ca2+ concentration of turtle endolymph Our experiments indicate that the performance of the mechano-electrical trans- ducer in hair cells depends critically on the concentration of Ca2+ in which the
A p A A.Lsi Aa A. - A A pA [ v =
I. LAII*L 1- -A-A A...i- PIAL. AA4. AIk.. Aa. A1.&k M.
A Iq A7 -VA.ALA. J..-AVAVA\J.1. A\Jy "--n.ALA(llJO
-..IA. . . I IC 0 B 0 10ims 40
c
20 - E z
0 -10 0 10 20 Current (pA) Fig. Il. A, examples of single-channel currents recorded in the whole-cell mode in 0 Mg2+ perilymph (solution B) after exposure of a cell to 1 /tM-Ca2+. Superimposed on the records are pairs of lines separated by 9 pA in the top two traces and 3-5 pA in the bottom two traces. The small, long-lasting channel in lower records was seen much less frequently than the large channel. C and 0 denote the closed the open states of the channel. Holding potential -85 mV, data filtered at 1-2 kHz. B, amplitude histogram of current records from experiment in A. Increase in inward current plotted as positive on the abscissa so smaller bump corresponds to the open state of the channel. Bin width 0-29 pA. The fitted Gaussian distributions to the histogram gave a unitary amplitude of - 8-7 + 2-2 pA. cells are bathed. In our original formulation of an artificial endolymph (solution I), we judged that 05 mM-Ca2+ was appropriate since this concentration produced a similar fraction of total transducer current activated in the absence of bundle stimulation as was inferred for the intact cochlea (Fettiplace & Crawford, 1989). Nevertheless we felt it advisable to establish directly the Ca2+ content of turtle CALCIUM AND HAIR CELL TRANSDUCTION 387 endolymph. Measurements were made with Ca2+-sensitive microelectrodes in an isolated half-head preparation identical to that in which we originally recorded hair- cell receptor potentials (Crawford & Fettiplace, 1980). Thirteen determinations in seven preparations gave a free Ca2+ concentration ranging from 5 to 163 /tM, with a mean value of 65 fM. A feature of the measurements was that the Ca2± concentration always increased from the minimum obtained immediately after penetration of the basilar membrane, but could be reduced again by further advancement of the electrode. Such behaviour may be due to leakage around the electrode of Ca2+ from the perilymph. This leads us to suggest that 65 #M constitutes an upper limit for the Ca2+ concentration in the endolymph of the turtle's cochlear duct. To check that ion- sensitive electrodes were indeed being introduced into the scala media, we also performed an experiment with a K+-sensitive electrode. Two measurements of the K+ concentration of endolymph in a single preparation each gave 140 mm, which is somewhat higher than the value of 114 mm reported by Johnstone et al. (1963), also for the turtle. Single-channel currents Following treatment with 1 ftM-Ca2+ and restoration of normal saline, it was sometimes possible to observe single-channel currents in the whole-cell recording. The cell had to be exposed to 1 j#M-Ca2+ for long enough for the macroscopic transducer current to have just disappeared; if the exposure was too brief, a small noisy transducer current indicative of multiple-channel activity remained. Based on a number of the channel properties which are documented below, including their polarity, displacement sensitivity and kinetics, we believe them to be the mechano- electrical transducer channels. Figure 1IA shows a particularly striking example where the events correspond to rapid openings to an amplitude of about 9 pA at a holding potential of -85 mV. Figure 11 B gives an amplitude histogram from this recording which has been fitted by two Gaussians with means separated by 8-7 pA. In some traces a smaller, more long-lived channel could be distinguished with an amplitude (3 5 pA) about a third of that ofthe large event (see the bottom two traces of Fig. llA). The openings and closings of the smaller channel seemed to be independent of those of the larger one, making it unlikely that one channel is a substate of the other. Other examples of the large channel can be seen in Figs 12 and 15, each of which comes from a separate experiment. In six experiments, single- channel currents with amplitudes of about 9 pA were observed (mean+S.D. = 9-3 + 1-3 pA), and in one additional experiment a small 3 pA channel was found alone, all currents being measured at a holding potential of -85 mV. In two recordings, occasional double openings of the large channels were also seen. In several of the events in Fig. 11A brief closures are evident but the current fails to return to the baseline level; similarly a number of the shorter openings appear to be incomplete. Presumably rapid openings and closures are filtered by the limited bandwidth (here 1-2 kHz) of a recording system which thus fails to register their full amplitude. These undersized events tend to fill in the valley between the peaks in the amplitude histograms (see Figs llB and 13) and, as a consequence, the channel size may be underestimated by the fits to such histograms. As might be expected, the ability to discern such brief single-channel currents depended critically on the level
13-2 388 A. C. CRA WFORD, M. G. EVANS AND R. FETTIPLACE of the background noise whose spectrum in a whole-cell measurement rises with frequency (Marty & Neher, 1983). The recordings were optimized by low series resistances and by making measurements at a holding potential of -85 mV where all voltage-sensitive channels should be turned off. Nevertheless many recordings were
02
"C 0
pA PO
0 20 40 60 80 Time (ms) Fig. 12. Responses of a single channel to O 15 #um deflections of the hair bundle towards kinocilium. Below the stimulus monitor are three individual currents recorded under similar conditions to those in Fig. 11 A. Pairs of lines of 9 pA separation are superimposed on the traces. The ensemble-average current response for fifty-five stimuli is given at the bottom; assuming only one channel is active, the right-hand ordinate is po, the channel's opening probability. Holding potential -85 mV, data filtered at 1-2 kHz. Recordings in 0 Mg2+ perilymph (solution B). too noisy to reveal single-channel events in the appropriate amplitude. For a similar reason, the extra noise produced by the perfusion system made it difficult to observe channels while the cell was being superfused with low-Ca2+ saline. Displacement sensitivity of channels The probability of opening of the 9 pA channel, wherever tested, was found to be sensitive to small deflections of the hair bundle. As shown in Figs 12 and 15, the channel was activated by displacements towards the kinocilium. Figure 12 presents three examples of channel activity during bundle stimulation, and at the bottom of the figure is shown the ensemble-average current for fifty-five such stimuli. Taking the channel amplitude as 9 pA and assuming that there was only one active channel, its probability of being open increased from about 0-15 in the resting state to about 0-5 for a 0-15 ,um step. Confirmation that only the large channel contributed to the ensemble current can be derived from the amplitude histograms for this recording (Fig. 13). The CALCIUM AND HAIR CELL TRANSDUCTION 389 histograms on the left and right were constructed from segments of record when the bundle was in its resting and displaced positions respectively. They were fitted by eye with pairs of Gaussians yielding channel amplitudes of 7-7 and 8-3 pA in the two positions. Reasons why these values may be underestimates for the single-channel
A B 300 20 -0 7 200-
Z 100
0- 0- -10 0 10 20 30 -10 0 10 20 30 Current (pA) Fig. 13. Amplitude histograms constructed from the experiment of Fig. 12. A, current recording with hair bundle in its resting position; B, current recording of identical duration with bundle displaced by 015,m towards kinocilium. Increase in inward current plotted as positive on abscissa so that right-hand hump corresponds to the open channel. Note that the probability of opening increases during bundle displacement. The fitted Gaussian distributions to the histograms give unitary amplitudes of: A, -7-7+229 pA; and B, -8-3+2-9 pA. current have been discussed above. The open probability in the two states may be deduced from the fraction of the area under the histogram contributed by the right- hand Gaussian, i.e. the Gaussian corresponding to the open channel. Values obtained were 0-20 and 0 50 in the resting and stimulated conditions respectively which are consistent with values derived from the ensemble average in Fig. 12. Channel kinetics All large (9 pA) channels displayed rapid gating, with a mean open time in the region of 1-2 ms when the bundle was in its resting position. The effects of bundle displacement on the open- and closed-time distributions are shown in Fig. 14 for the same cell whose responses to mechanical stimulation have been given in Fig. 12. Owing to the limited bandwidth of the recording, measurements of brief events were considered to be unreliable (Colquhoun & Sigworth, 1983; Colquhoun, 1987), and so the first two bins of each histogram have been discarded. The histograms have then been fitted with single exponentials to yield mean open and closed times. Both the open and closed times were sensitive to bundle displacement. The mean open time was 11 ms in the resting state and it was increased by a factor of approximately two to 2-1 ms for a 0-15 #tm bundle displacement; the same stimulus decreased the mean closed time, also by about a factor of two. On a simple two-state (one open and one closed state) scheme for the channel, which is implied by the single-component distributions, the probability of being open, po, is related to the mean open time (ro) and mean closed time (ic) by Po = ITO/(TO +,Tc). (1) 390 A. C. CRAWFORD, M. G. EVANS AND R. FETTIPLACE Inserting into eqn (1) values for T. and Tr determined from the histograms gives the probabilities of being open in the resting and stimulated states as 0418 and 044 respectively. These numbers agree with those inferred from the ensemble current (Fig. 12). Only for this one cell was it possible to examine the effects of bundle
A Resting B Stimulated
60 -
I 40 X = 1.1 ms r=2.1 ms
z
5 10 15 5 10 15 a) Open time (ms) Open time (ms) C D
30
.0 20 E r = 5.0 ms r= 2.7 ms
z 10.
0 5 10 15 0 5 10 15 Closed time (ms) Closed time (ms) Fig. 14. Effects of bundle displacement on channel open and closed times. A and B, histograms of apparent open times with the bundle in its resting position and displaced by 0 15 jtm towards kinocilium. C and D, histograms of closed times with bundle in its resting and displaced positions. Histograms constructed from equal periods with the bundle in the two positions. Data consisted of fifty-five sweeps each of 100 ms duration filtered at 1-2 kHz. Bin width 0 16 ms, first two bins discarded. Single-exponential curves were fitted as described in the Methods with time constants that are given beside the histograms. Note that bundle deflection increases the mean open time and decreases the mean closed time. Same cell as Fig. 12. stimulation on the open- and closed-time distributions. For other cells, either the measurements were incomplete or the recordings too noisy to be reliably analysed. Other channel properties Gating of individual channels by displacement of the hair bundle showed a number of similarities to activation of the macroscopic transducer current. Firstly the channel's probability of being open was graded with the degree of displacement of CALCIUM AND HAIR CELL TRANSDUCTION 391 the hair bundle. This is illustrated in Fig. 15 which gives the ensemble-average currents for three stimulus amplitudes up to 075 jtm. The channel was inactive when the bundle was in its resting position, but steps towards the kinocilium increased its probability of opening. An important conclusion from these records
2 m pA : 0 9 L L1
_.A A v-v -vgv Nky-4. t}A Ai -t - v4 w>iA pA v W
20 ms Fig. 15. Effects of bundle displacement on channel opening. Ensemble-average currents for three amplitudes of displacement are superimposed and the stimulus monitor is shown at the top. Between 146 and 215 traces averaged. A single current record for the 05 ,um step is given at the bottom, the timing of the stimulus being the same as for averages. Holding potential -83 mV; recordings in solution A. Note adaptation in the largest average and in the single record.
(and those of Fig. 12) is that the channel is gated by mechanical stimuli of comparable amplitude to those that activate the macroscopic current. In another cell, not illustrated, a small decrease in activity was found for displacements away from the kinocilium which argues that it is not the non-specific mechanical disturbance of the hair bundle that is causing the channel to open. A further similarity with the macroscopic current was the presence of adaptation. Such adaptation is evident in the ensemble average to the largest displacement in Fig. 15, and it could also be seen in some of the individual records; an example is shown in the bottom part of Fig. 15. These various points of similarity between the gating properties of the 9 pA channel and those of the macroscopic current strengthen the conclusion that they are the transducer channels. The majority of measurements on the channel were made at a holding potential of -85 mV. One reason for choosing this potential was that other types of channel, such as voltage-dependent Ca2+ or K+ channels, would not be turned on at such a negative potential. Moreover, the holding potential was close to the K+ equilibrium potential (-89 mV) and so if a large-conductance Ca2+-activated K+ channel happened to be active, the single-channel current would be small due to the minimal driving force. For example, if we assume a conductance of 120 pS for the Ca2+- activated K+ channel, the current flowing through a single channel would be less 392 A. C. CRAWFORD, M. G. EVANS AND R. FETTIPLACE than 0'5 pA. It is therefore unlikely that the 9 pA channel can be identified with any of the normal voltage-dependent channels that have been found in vertebrate hair cells (Lewis & Hudspeth, 1983; Ohmori, 1984; Art & Fettiplace, 1987; Evans & Fuchs, 1987).
A B Membrane potential (mV) -65 mV -100 -80 -60 -40 -20 0 0 2
-65 4 62
M AA., p 8 _- I- s-83 ~J'A 70_ 10 < 34wLVI0-97
20 ms Fig. 16. Effects of holding potential on channel amplitude. A, samples of single-channel currents at the holding potentials given beside the traces. The pairs of lines superimposed on the records correspond to the current amplitudes plotted in B. Measurements in normal perilymph (solution A). B, current-voltage relationship for channel; straight line drawn by eye through the points extrapolates to a reversal potential of -10 mV. Channel conductance 106 pS.
Figure 16A shows an experiment where the channel current was recorded at other holding potentials. The potential range that could be examined was limited due to the extra current noise introduced by activation of voltage-dependent conductances, notably the Ca2+-activated K+ conductance. The channel amplitudes were de- termined directly from the records by measuring the current excursions during the longer openings. The current-voltage relationship is plotted in Fig. 16B: the channel current decreased with depolarization and the extrapolated reversal potential was -10 mV. By comparison the expected reversal potential of the transducer current with K+ as the major intracellular cation is very close to 0 mV. A value of 106 pS is calculated for the single-channel conductance.
DISCUSSION Two sites of action of divalent cations While the action of divalent cations on the activation of the transducer current seems rather complicated, our results can be simply explained if calcium were to act at two separate sites in the hair cell. One site is envisaged as directly accessible from the extracellular solution and is most likely the channel pore itself; the other is CALCIUM AND HAIR CELL TRANSDUCTION 393 probably an intracellular site where calcium acts having entered the cytoplasm through transducer or other membrane channels. Qualitatively similar effects of calcium on microphonic currents in the bull-frog saccule, including both a change in the peak current and a shift in the current-displacement relationship, have been described by Corey & Hudspeth (1983) and recently by Hacohen et al. (1989). The notion of two sites of action is supported by several pieces of evidence. In the first place, when magnesium was removed or when calcium was lowered by only moderate amounts (from 2-8 to 05 mm in the absence of magnesium), the transducer's current-displacement relationship was simply scaled up without shifts or changes in slope (see Table 1). So external magnesium and calcium may both partially block the transducer channel. This idea is consistent with the observation that increases in transducer current occur as quickly as the extracellular solution can be changed, and with the fact that transducer's current-voltage relationship is approximately linear at extracellular calcium concentrations, [Ca2±]0, in the range 05-2-8 mm. If calcium ions were to block the transducer channel from the inside, we would expect the transducer's current-voltage curve to show outward rectification at positive membrane potentials, i.e. at positive potentials there would be less calcium influx and less block. The only rectification evident in our experiments is a weak inward rectification occurring at negative potentials. Extracellular calcium ions have also been reported to block stretch-activated channels in Xenopus oocytes with a half-blocking concentration of 033 mm (Yang & Sachs, 1989). We believe that the effects of more drastic reductions of the external calcium concentration involve an intracellular site of action because the shifts and slope changes in the transducer's current-displacement relationship which occurred when calcium was lowered below 05 mm could be reproduced by depolarizing the hair cell even at high concentrations of divalent cations. This observation is explicable if reduced external calcium causes the intracellular calcium concentration, [Ca2+]i, to fall (Assad et al. 1989; Crawford et al. 1989). The transducer: its set point and sensitivity It is generally believed that each transducer channel is opened by a gating force developed on the channel via the displacement of a compliant spring (Corey & Hudspeth, 1983). At present the most likely structures to be identified with the gating springs are the tip links connecting adjacent stereocilia (Pickles, Comis & Osborne, 1984). In the context of this hypothesis we must explain how, as the extracellular calcium concentration is lowered below 05 mm, there can be a negative shift in the transducer's current-displacement relationship and a fall in the transducer's sensitivity. The proposition that calcium directly affects the mechanical properties of the tip link is rather unpalatable, because this structure would somehow have both to shorten (to produce the negative shift) and to become more compliant (to produce the decline in sensitivity) as the calcium concentration was lowered. Moreover, both effects mentioned above can be duplicated by depolarizing a hair cell (Assad et al. 1989; Crawford et al. 1989) so the site of action of calcium must be intracellular. A related idea that stems from the model of adaptation introduced by Howard & Hudspeth (1987) is that the anchorage point of the channel in the stereociliary 394 A. C. CRA WFORD, M. G. EVANS AND R. FETTIPLACE membrane is itself a force generator that can drag the channel up the stereocilium as [Ca2+]i falls. While this hypothesis accommodates both the requirement for calcium to act at an intracellular site and the negative shift of the displacement curve, it cannot explain the decreased slope of this relationship. Nevertheless, it is possible to imagine how at very low [Ca2+]i, the mechanical links to the channel could be stretched to the point that the transducer channels were fully activated and how the motor might eventually rupture these links and abolish transduction. An alternative approach is to direct attention at the mechanism of the channel itself. The form of the transducer's current-displacement relationship can be described by a scheme where the channel has two closed states (C1 and C2) and one open state (0) (Corey & Hudspeth, 1983; Holton & Hudspeth, 1986; Crawford et al. 1989): k, a ci -C2 0. (2)
The equilibrium probability, p, of finding the channel in the open state is given by p = (1 +K2(1 +K1))-', (3) where K1 and K2 are the equilibrium constants for the two transitions. Both equilibrium constants are assumed to be regulated by displacement of the bundle, x, according to the Boltzmann equation (Holton & Hudspeth, 1986): K1 = -llkl = exp (al(xl-x)), (4) and K2= ,/a = exp (a2(X2-x)), (5) where a1, a2, x1 and x2 are constants. x1 and x2 correspond to the set-points for the two displacement-sensitive steps, i.e. the positions to which bundle displacement, x, is referred. For turtle hair cells bathed in artificial perilymph the ration al/a2 is between 3 and 4, which means that the first transition, C1 - C2, is three to four times more displacement-sensitive than the opening transition C2 -O0. If the constant x1 is now made to fall as [Ca2+]i is lowered, and conversely to increase as [Ca2+]i increases during adaptation (Crawford et al. 1989), then the equilibrium constant K1 becomes a function of both displacement and [Ca2+]i. Equations (3), (4) and (5) have been used to fit the current-displacement relation- ships in Figs 4B and 8. Most of the features of these plots, including the effects of reducing the external calcium concentration from 2-8 to 0 5 mm in Fig. 4B and 2-8 mm to 50 /aM in Fig. 8, are well described by reductions in xl. Note that both the shift in the position of the current-displacement curve and the change in its slope are reproduced. Thus we suggest that a scheme in which the transition C2 -> C1 is promoted by an increase in [Ca2+]i will largely account for the changes in the current-displacement relationship observed under conditions of reduced [Ca2+]. or adaptation. However, this model will not explain the behaviour at very low calcium concentrations (Fig. 10), where the transducer channel can become fully activated before mechanical responsiveness is lost irreversibly. These effects may require the mechanical linkages to the channel to be stretched and finally severed when [Ca2+]i falls below a minimum level. CALCIUM AND HAIR CELL TRANSDUCTION 395
The functions of the endolymph Our results show the benefits which accrue from surrounding the ciliary bundle with endolymph. The transducer current is about three times larger in artificial endolymph than in artificial perilymph, a difference mainly due to the reduced concentrations of divalent cations. Since the maximum transducer conductance (Gmax) is about 5 nS in artificial perilymph (Crawford et al. 1989) we would expect values of Gmax to be at least three times larger in vivo. These effects would give an expected Gmax of about 15 nS, which is closer to the value of 25 nS obtained from intracellular recording with micropipettes in the intact turtle cochlea (Fettiplace & Crawford, 1989). In these measurements in the intact preparation, the fraction of the receptor conductance turned on in the absence of acoustic stimulus was about 5% even though the hair bundle was surrounded by endolymph with a calcium content estimated at no more than 65 /tM. Thus in vivo the transducer's current-displacement curve is not greatly shifted to negative values of displacement in contrast to isolated cells when the [Ca21]0 is lowered to 50 JtM. This implies that the intracellular calcium in vivo is higher than in our experiments even though the calcium concentration of the endolymph is low. A plausible explanation for the difference is that in the intact cochlea the basolateral membranes of the hair cell will be exposed to a high-calcium perilymph and leakage across these membranes may well maintain a higher level of calcium throughout the cell. Our finding of a low calcium concentration, 65 /M, in endolymph raises a question about the physiological significance of adaptation in the transduction process. Adaptation is absent in saline containing 50 jtM-calcium (Figs 5 and 7A), and in a few other experiments we demonstrated that it was also absent in 100 /tM-calcium, with or without added magnesium. Therefore it seems likely that calcium entry through the transducer channels of turtle cochlear hair cells is insufficient in vivo to cause adaptation. A way out of this dilemma would be to postulate that in cells that were not voltage clamped, calcium entry, possibly via voltage-gated calcium channels in the basolateral membrane, could achieve the same effect. Thus an increase in the transducer conductance would generate a depolarizing receptor potential leading to activation of the voltage-sensitive calcium channels; the subsequent influx of calcium, if it gained access to the transducer channels, could cause them to adapt.
Transducer channels After hair cells had been bathed in saline containing 1 /kM-calcium, the macroscopic transducer current vanished and for a short while single-channel events became visible in the current record. Several lines of evidence suggest that these events represent individual transducer channels: (1) they could be activated by deflections of the hair bundle of the appropriate polarity (towards the kinocilium) and with amplitudes within the physiological range; (ii) they were gated rapidly, with open times of 1-2 ms, which accords with the swift onset of the macroscopic transducer current; (iii) their gating could display adaptation similar to that observed in the macroscopic current; (iv) they were only seen under circumstances where most of the transducer current had been abolished; (v) their size, -9 pA at -85 mV, 396 A. C. CRAWFORD, M. G. EVANS AND R. FETTIPLACE distinguishes them from other channels normally found in hair cells, including voltage-dependent Na+ or Ca2+ channels or any species of K+ channel. Although we cannot entirely eliminate an anomalous stretch-sensitive channel, the weight of evidence favours the view that they are the mechano-electrical transducer channels. We suggest that, following exposure to a very low Ca2+ concentration, the transducer channels are lost over an extended time period so that there will exist an interval when only a single channel remains active. There have been two prior estimates of the amplitude of transducer channels in hair cells, one based on unitary events in chick cells (Ohmori, 1985) and the other inferred from current fluctuations in bull-frog saccular hair cells (Holton & Hudspeth, 1986). The smallest step size observed in the chick was about 50 pS, though some data (e.g. Fig. 5 of Ohmori, 1985) clearly display a channel of twice that size, comparable to the 106 pS channel measured in the present work. In contrast the amplitude deduced from the variance of the current noise in bull-frog hair cells was only 17 pS when corrected to room temperature (Holton & Hudspeth, 1986). If the channels show strong flickering, such fluctuation measurements may underestimate the true channel size since high-frequency components in the current (corresponding to fast openings and closures of the channel) may be filtered by the recording system and cause the current variance to be underestimated. The properties of the hair cell's transducer channel documented here differ somewhat from those of the stretch-activated channels found in many tissues. The latter channels are smaller, typically about 50 pS (Sachs, 1990), and only their closed times are sensitive to stress (Guharay & Sachs, 1984; Sachs, 1990). By contrast, our limited evidence suggested that both the closed and open times of the transducer channel were altered by bundle stimulation. Owing to the restricted recording bandwidth, we cannot be confident that both open- and closed-time distributions do not have additional short-lived components, and indeed such components may be required to explain the sigmoidal onset kinetics for the macroscopic current (Crawford et al. 1989). Thus it would be unwise to construct a kinetic scheme solely on the basis of the histograms in Fig. 14. Nevertheless it is worth commenting on the significance of the stimulus-dependent open time. Taken at face value this observation indicates that the closing rate constant is stimulus-dependent. However if there were a second open state and if the transitions between the two open states were mechanically sensitive, this could also appear as a stimulus-dependent open time. We have previously postulated an additional open state in order to account for unusual offset kinetics (Crawford et al. 1989), but better recordings are needed to establish its presence.
This work was supported by grants from the Medical Research Council and the Howe Fund of the Royal Society. We thank Peter McNaughton for commenting on the manuscript.
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