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1 School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK
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1 mM. The block of this current by nifedipine suggests the direct involvement of Cav1.3 Ca2+ channels in its activation. On the basis of its high sensitivity to apamin (KD 360 pM) it was identified as a small-conductance Ca2+-activated K+ current (SK), probably SK2. A similar current was also found in outer hair cells (OHCs) from the beginning of the second postnatal week. In both cell types the appearance of the SK current coincided with their becoming responsive to acetylcholine (ACh), the main efferent neurotransmitter in the cochlea. The effect of ACh on IHCs was abolished when they were simultaneously superfused with strychnine, consistent with the presence of nicotinic ACh receptors (nAChRs). Extracellular Ca2+ either potentiated or blocked the nAChR current depending on its concentration, as previously reported for the recombinant 
910 nAChR. Outward currents activated by ACh were reduced by blocking the SK current with apamin or by preventing SK current activation with intracellular BAPTA ( 10 mM). The endogenous mobile Ca2+ buffer concentration was estimated to be equivalent to about 1 mM BAPTA, suggesting that in physiological conditions the SK channel is significantly activated by Ca2+ influx through both Cav1.3 Ca2+ channels and 910 nAChRs. Current clamp experiments showed that in IHCs the SK current is required for sustaining a train of action potentials and also modulates their frequency when activated by ACh.
(Received 29 July 2004;
accepted after revision 23 August 2004;
first published online 26 August 2004)
Corresponding author C. J. Kros: School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK. Email: c.j.kros{at}sussex.ac.uk
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910 nAChRs (Elgoyhen et al. 2001; Maison et al. 2002). In the mature cochlea, the main role of SK channels is in cholinergic inhibition by the medial efferent fibres of the auditory nerve, which contact the electromotile OHCs (Guinan, 1996). Efferent inhibition of auditory hair cells is achieved by Ca2+ influx through nAChRs activating hyperpolarizing SK currents (Fuchs & Murrow, 1992; Evans, 1996; Glowatzki & Fuchs, 2000; Oliver et al. 2000). A similar inhibitory mechanism has also been described for vestibular hair cells (for a review see Guth & Norris, 1996). In addition to the inhibitory responses, slow and rapid excitation have also been described for some vestibular hair cells, associated with the activation of muscarinic ACh receptors (Guth & Norris, 1996) and nAChRs different from those containing the 910 subunits (Holt et al. 2003), respectively. Rat IHCs respond to ACh at postnatal day 7 (P7) but no longer at P21 (Glowatzki & Fuchs, 2000) suggesting a specific role for the efferent neurotransmitter before their functional maturation that coincides with the onset of hearing at about P12 (Uziel et al. 1981). By contrast, OHCs of both rat and gerbil first become sensitive to ACh from around P8 (Dulon & Lenoir, 1996; He & Dallos, 1999) when they become electromotile (He et al. 1994; Marcotti & Kros, 1999) and this sensitivity is maintained in adult cells. The developmental maturation of synaptic connections within the organ of Corti is likely to be influenced by the shape and frequency of spontaneous or induced action potentials of immature IHCs (Kros et al. 1998; Marcotti et al. 2003a), analogous to the immature retina (Shatz & Stryker, 1988; Maffei & Galli-Resta, 1990; Meister et al. 1991). The timing of the expression of various K+ (Marcotti et al. 1999; Marcotti et al. 2003a) as well as Ca2+ and Na+ (Marcotti et al. 2003b) currents leads to changes in the properties of action potentials that occur during IHC maturation. It is also conceivable that spiking activity might be influenced by extracellular events such as the release of neurotransmitters or neuromodulators onto IHCs by a transient efferent innervation (Gil-Loyzaga, 1995). In IHCs of pre-hearing rats it has been shown that superfusion of ACh reduced the frequency of action potentials (Glowatzki & Fuchs, 2000). In mouse IHCs efferent fibre endings make initial contact from around birth (Sobkowicz, 1992; Bruce et al. 1997, 2000) and therefore the efferent system could conceivably play a role in the modulation of spontaneous or induced action potentials from as early as P0. The aims of this paper were to investigate the biophysical properties, the development and the functions of the SK current in mouse cochlear IHCs. For comparison some experiments were also carried out in OHCs.
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IHCs (n = 248) and OHCs (n = 45) were studied in acutely dissected organs of Corti from CD-1 mice (Swiss CD-1, Charles Rivers, Margate, UK) from embryonic day 14.5 (E14.5) to postnatal day 18 (P18) for IHCs and P0 to P25 for OHCs where the day of birth (P0) corresponds to E19.5. For embryonic experiments only, mice were paired overnight and checked for vaginal plugs the following morning. Assuming ovulation occurs midway through the dark cycle, the mid-point of the light cycle of the day following mating is considered to be E0.5. Adult and neonatal mice were killed by cervical dislocation and embryos by decapitation, in accordance with UK Home Office regulations.
The organs of Corti were dissected and transferred to a microscope chamber and immobilized using nylon mesh fixed to a stainless steel ring. The chamber was perfused by means of a peristaltic pump at a flow rate of about 10 ml h–1, with extracellular solution composed of (mM): 135 NaCl, 5.8 KCl, 1.3 CaCl2, 0.9 MgCl2, 0.7 NaH2PO4, 5.6 D-glucose, 10 Hepes-NaOH, 2 sodium pyruvate. Amino acids and vitamins for Eagle's minimum essential medium (MEM) were added from concentrates (Invitrogen, Paisley, UK). The pH was adjusted to 7.5 and the osmolality was about 308 mosmol kg–1. The organs of Corti were observed with an upright microscope (Zeiss ACM, Germany or Olympus, Japan) with Nomarski differential interference contrast optics (x 40 water immersion objectives). Only healthy-looking cells (criteria included smooth surface of the cell membrane, absence of vacuoles in the cytoplasm and lack of Brownian motion of mitochondria) with well-preserved hair bundles were investigated. The position of cells along the cochlea was recorded as fractional distance from the extreme apex. In the immature cochlea, apical-coil cells were positioned at a fractional distance between 0.16 and 0.24 and basal-coil cells between 0.83 and 0.90. Mature apical-coil cells (> P12) were positioned between 0.07 and 0.24, corresponding to an approximate frequency range of 0.9–4.2 kHz (using eqn (13) in Ehret, 1975).
Electrical recording
Whole-cell voltage-clamp recordings were performed using an EPC-8 (HEKA, Lambrecht, Germany) or an Optopatch (Cairn Research Ltd, Faversham, UK) patch-clamp amplifier. Membrane currents under voltage clamp from IHCs and OHCs were studied at room temperature (22–25°C) apart from those shown in Fig. 1 that have been recorded near body temperature (35–37°C). To obtain realistic voltage responses, all current clamp experiments were performed at 35–37°C. Patch pipettes were pulled from soda glass capillaries (Harvard apparatus Ltd, Edenbridge, UK) and electrode resistances in extracellular solution were around 2–3 M
. The shanks of the pipettes were coated with surf wax (Mr Zogs SexWax, Carpinteria, CA, USA) to reduce their capacitance.
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Data acquisition was performed using pCLAMP software (Axon Instruments, Union City, CA, USA) connected to a LabMaster DMA Interface or a Digidata 1320 A. Data were filtered at 2.5 kHz (8 pole Bessel), sampled at 5 kHz and stored on computer for off-line analysis using Origin software (OriginLab, Northampton, MA, USA). For voltage-clamp experiments, current recordings were corrected offline for leak conductance (gleak) usually measured around –90 mV of 1.8 ± 0.1 nS (n = 237, E14.5–P10) for immature IHCs and 5.3 ± 0.7 nS (n = 8, P12–P16) for more mature IHCs after the onset of hearing. For immature OHCs gleak around –90 mV was 1.6 ± 0.2 nS (n = 27, P3–P7). In mature OHCs gleak was measured at very hyperpolarized potentials (typically between –114 and –124 mV) at which the large IK,n (Marcotti & Kros, 1999) was deactivated (1.6 ± 0.2 nS, n = 18, P9–P25). Membrane capacitance (Cm) was 8.3 ± 0.1 pF (n = 248) in IHCs and 7.2 ± 0.2 pF (n = 45) in OHCs. In whole-cell recordings, the residual series resistance (Rs) after compensation (50–90%) was 1.9 ± 0.1 M (n = 242, E14.5–P18, ranging from 0.5 to 7.1 M) in IHCs and 2.5 ± 0.2 M (n = 45, P3–P25, ranging from 1.0 to 6.6 M) in OHCs resulting in average voltage-clamp time constants of about 15 and 19 µs, respectively. Under perforated-patch conditions, RS after compensation (0–70%) was 10.8 ± 2.3 M (n = 6, P8–P9, ranging from 3.8 to 20 M) and the average voltage-clamp time constant was 83 µs. Membrane potentials were corrected for residual series resistance and for a liquid junction potential measured between pipette and bath solutions. The liquid junction potential was usually –4 mV for the KCl-based and –10 mV for the potassium aspartate-based intracellular solution. When the Ca2+ buffers in the KCl-based intracellular solution were increased to 10 or 30 mM it was –5 and –8 mV, respectively. For current-clamp experiments, offline series resistance correction was applied when the voltage drop exceeded 1 mV. When 10 or 30 mM of the Ca2+ buffers were used the holding potential was –85 and –88 mV, respectively. During perforated-patch recordings the holding potential was –80 or –90 mV. The holding currents were plotted as zero current unless otherwise stated.
Extracellular superfusion
The K+ channel blocker apamin (Calbiochem) was superfused to abolish the SK current specifically. Acetylcholine and strychnine (Sigma, Gillingham, UK) were used to assess the presence of nAChRs in hair cells. The dependence of the outward K+ current on Ca2+ channels was investigated by extracellular application of nifedipine (Sigma) or a Ca2+-free solution containing 0.5 mM EGTA (Figs 2 and 3A), in which MgCl2 was increased to 3.9 mM to keep membrane charge screening approximately constant (Blaustein & Goldman, 1968).
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Statistical comparisons of means were made by the two-tailed t test or, for multiple comparisons, analysis of variance, usually one-way ANOVA followed by the Tukey test (Figs 2E, 7E, 8C-E and 9F). Mean values are quoted ± S.E.M. in text and figures. In Figs 2E, 7E and 8C–E statistically significant differences (P < 0.05) are indicated by an asterisk.
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Results |
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Immature IHCs exhibited, in addition to delayed rectifier K+ currents (Marcotti et al. 2003a), a slowly activating outward current (Fig. 1: P3 and P9, indicated by arrows) that was evident when they were depolarized for a period in the order of seconds from the holding potential of –84 mV using 1 mM EGTA as the intracellular Ca2+ buffer and 1.3 mM extracellular Ca2+. This current was first seen around P0 in basal and P2 in apical IHCs and was no longer evident upon maturation (Fig. 1: P17). When the extracellular Ca2+ concentration was increased from 1.3 mM to 5 mM, this novel current was already evident using shorter (160 ms) depolarizing voltage steps (n = 4, P4 apical IHCs, data not shown) indicating its direct dependence on extracellular Ca2+. By contrast, immature OHCs (P0–P7) did not exhibit this slowly activating current (Fig. 2G). When a Ca2+-free solution (containing 0.5 mM EGTA) was superfused onto immature IHCs the slowly activating outward current was selectively and reversibly abolished (Fig. 2A), providing further evidence for its Ca2+ sensitivity. This current was also blocked (Fig. 2B) when nifedipine (30 or 50 µM) was superfused onto apical IHCs (P9, n = 5) implicating the involvement of L-type Ca2+ channels in its activation. The Ca2+- and nifedipine-insensitive current (Fig. 2A and B, red traces) exhibits a decay time course similar to that of the total outward K+ current expressed by embryonic IHCs, i.e. before the appearance of the slowly activating current (Fig. 1, E18.5), and by immature (P0–P7) OHCs (Fig. 2G, black traces). The maximum size of the Ca2+-sensitive K+ current in apical IHCs, measured at the steady state (4 s) around –20 mV, was 981 ± 223 pA (n = 4, P3 and P7).
Since this novel K+ current was abolished in the absence of extracellular Ca2+, we investigated its sensitivity to cytosolic Ca2+ using different concentrations of the fast Ca2+ buffer BAPTA (Neher, 1998). Figure 2C shows current recordings from apical IHCs (P9) using 0.1 mM, 1 mM or 30 mM BAPTA as the internal Ca2+ buffer instead of 1 mM EGTA. The IHCs buffered with 0.1 mM and 1 mM BAPTA, but not that with 30 mM BAPTA, showed the slowly activating outward K+ current (arrows) that was completely and reversibly abolished during superfusion of a Ca2+-free solution (red traces). Increasing the BAPTA concentration from 0.1 to 1 mM slowed the activation of the Ca2+-sensitive current, suggesting that the kinetics of the current reflect the time course of the change in intracellular Ca2+ (Tucker & Fettiplace, 1996). To determine the contribution of this Ca2+-sensitive current under endogenous Ca2+ buffering conditions and in the presence of 1.3 mM extracellular Ca2+, recordings from five P8–P9 IHCs were obtained using the perforated-patch technique. Figure 2D shows that the slowly activating current was evident in the presence of the native mobile buffer and that it was abolished during superfusion of a Ca2+-free solution. The amplitudes of the Ca2+-sensitive K+ current in P8–P9 apical IHCs (n = 23), measured near the beginning (100 and 200 ms) and at the end (4 s) of the voltage step, using whole-cell (intracellular 1 mM EGTA or different BAPTA concentrations) or perforated-patch recording are shown in Fig. 2E. Relative to recordings with 1 mM EGTA, the size of the Ca2+-sensitive K+ current, measured near –26 mV and near the beginning of the voltage step, was significantly (P < 0.001) reduced in the presence of 1 mM or higher BAPTA concentrations but not by 0.1 mM BAPTA. The current amplitude was also reduced (P < 0.01) when recorded under perforated-patch conditions. However, 10 mM or 30 mM BAPTA was needed to reduce significantly (P < 0.001) the amplitude of the Ca2+-sensitive current when measured at 4 s. These results suggest that during long-lasting voltage steps (> 400 ms) increasingly higher BAPTA concentrations are required to buffer the larger influx of Ca2+ flowing through voltage-gated Ca2+ channels. The effects of BAPTA were evident within a minute from reaching the whole-cell configuration. An approximate concentration of the endogenous mobile Ca2+ buffer, expressed as an equivalent BAPTA concentration, can be estimated by comparing the size of the Ca2+-sensitive current in the perforated-patch recordings with that obtained using different intracellular BAPTA concentrations (Fig. 2E). The amplitude of the Ca2+-sensitive current, measured at 100 or 200 ms in perforated-patch recordings, corresponds to that which would be obtained in about 0.7 mM BAPTA (Fig. 2F). When measured at 4 s native Ca2+ buffering was more efficient, equivalent to about 4 mM BAPTA, possibly reflecting the recruitment of additional buffers or Ca2+ sequestration (Tucker & Fettiplace, 1996).
Consistent with the absence of the slowly activating current in immature OHCs (Fig. 2G, black traces) the total outward K+ current was unaffected when cells (P3–P6, n = 6) were superfused with a Ca2+-free solution (Fig. 2G, red traces). It is worth mentioning that in one apical P6 OHC a very small Ca2+-sensitive current (about 30 pA) was isolated. By P9 all OHCs exhibited a similar slowly activating Ca2+-sensitive current (109 ± 21 pA, measured at 4 s near –15 mV, P9 and P12, n = 5, data not shown), suggesting that a similar current to that expressed in immature IHCs is present in OHCs from the beginning of the second postnatal week.
The Ca2+-activated K+ current is carried by SK channels
A selective reduction of the Ca2+-activated K+ current was also obtained when immature IHCs were superfused with apamin, a selective blocker of SK channels (Sah & Faber, 2002). Extracellular application of 300 nM apamin (Fig. 3A, blue traces) selectively abolished the slowly activating outward current expressed in immature IHCs (P7, n = 3). To test whether the Ca2+-sensitive component of the outward K+ current was completely abolished by apamin, the same IHCs were additionally superfused with a Ca2+-free solution also containing 300 nM apamin. Figure 3A (red traces) shows that a Ca2+-free solution did not further reduce the total outward K+ current. Apamin proved to be a very effective blocker of the SK current in apical IHCs with a KD of 360 pM (Fig. 3B). Although the apamin-sensitive current could be most clearly seen using long lasting voltage steps (Fig. 3A), its presence has been demonstrated in immature IHCs during shorter voltage steps in the order of 100 ms when all other K+ currents were blocked (Marcotti et al. 2003b). A selective block of the Ca2+-sensitive current by apamin (300 nM, P13–P17, n = 3) was also observed in mature OHCs (data not shown). The effect of apamin was very difficult to wash out completely and therefore after each application of the toxin the preparation was discarded. Although the SK current expressed in hair cells is thought to be activated by Ca2+ flowing through nAChRs (Elgoyhen et al. 2001) our results (Fig. 2A and B: Ca2+ and nifedipine sensitivity) show that SK channels can also be activated by Ca2+ influx through L-type Ca2+ channels containing the Cav1.3 (1D) subunit, known to be present in mouse cochlear hair cells (Platzer et al. 2000; Marcotti et al. 2003b; Michna et al. 2003).
Blocking the SK channels impedes IHC action potential activity
Immature IHCs can generate Ca2+-dependent action potentials spontaneously or in response to the injection of small depolarizing currents from the resting potential (Marcotti et al. 2003b). The shape and the frequency of these action potentials are specifically modulated by different conductances expressed during development in immature IHCs (Marcotti et al. 1999, 2003a,b). To determine whether the SK current also affects the IHC voltage responses, eight cells were superfused with a fully blocking concentration of apamin (100–300 nM) under current clamp using short (1 s) and long-lasting (up to 15 s) recordings. From the resting membrane potential, when a depolarizing current step of 30 pA (Fig. 3C and G, left panel) was applied to apical IHCs repetitive action potentials were elicited. The inset in Fig. 3C (indicated by the arrow) shows a spontaneous hyperpolarizing transient on an expanded time scale that has been previously attributed to inhibitory postsynaptic potentials (IPSPs) (Glowatzki & Fuchs, 2000). These small spontaneous hyperpolarizing transients were evident in 28% of immature IHCs (P2–P12, 26 out of 94 IHCs investigated) and they sometimes prolonged the interspike interval. Application of 300 nM apamin gradually abolished the evoked action potentials in all cells investigated (Fig. 3C–E) by progressively reducing the extent of repolarization of the IHCs after an action potential upstroke, finally resulting in a steady depolarization. Before action potential activity ceased an increase in the firing frequency was observed in some cases (e.g. from 12 to 15 Hz in Fig. 3C). Recordings using shorter current steps show that in the presence of apamin the cells remained depolarized in between steps, again failing to produce action potentials (Fig. 3G, middle panel). A fully blocking concentration of apamin (300 nM) abolished action potentials even for large depolarizing current steps (up to +100 pA tested). Therefore it appears that the delayed rectifier K+ current, IK,neo, alone is not sufficient to maintain repetitive spikes, most likely due to its steady-state inactivation that occurs with depolarizing voltage steps (Marcotti et al. 2003a). After washout (Fig. 3F and G, right panel), although IHCs were once more capable of firing action potentials they did not usually regain the ability of producing a sustained regular train of repetitive spikes, probably due to apamin being difficult to remove completely.
The developmental onset of ACh sensitivity differs between IHCs and OHCs
Since IHCs do respond to ACh at P7 (rat: Glowatzki & Fuchs, 2000) we investigated whether the onset of their sensitivity to ACh matched the appearance of the SK current just after birth. A typical example of the effect of ACh on an apical IHC (P8) is shown in Fig. 4A–E. The ACh-activated current (Fig. 4C) was obtained by subtracting the control currents (Fig. 4A) from the currents in the presence of 100 µM ACh (Fig. 4B). During superfusion of ACh the holding current measured at –84 mV (Control: –10 ± 2 pA, n = 11) rapidly became more negative (100 µM ACh: –462 ± 73 pA, n = 11, P < 0.001) and then, in the continued presence of ACh, it slowly (in the order of 10 s) and partially relaxed back to a value of –330 ± 51 pA (n = 11). This is likely to be due to the desensitization of nAChRs known to occur in both native (Fuchs & Murrow, 1992; Housley et al. 1992; Dulon & Lenoir, 1996; Evans, 1996) and recombinant 910 (Weisstaub et al. 2002) nAChRs. As shown in Fig. 4C, ACh evoked an instantaneous current that declined with time, reaching a steady-state level after about 80 ms, during both hyperpolarizing and depolarizing voltage steps from the holding potential of –84 mV. The effect of ACh on IHCs was rapid and completely reversible.
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ACh responses are mediated by the colocalization of nAChRs and SK channels
To confirm the presence of nAChRs in immature IHCs we superfused apical cells (n = 12, P3–P7) with 70 or 100 µM ACh with and without 1 µM strychnine, a known antagonist of both the homomeric 9 and the heteromeric 910 receptors (Elgoyhen et al. 1994, 2001). Figure 6A shows an example of membrane currents from a P3 IHC in control conditions, in the presence of 70 µM ACh and during the simultaneous superfusion of 70 µM ACh and 1 µM strychnine. When ACh was superfused alone (red traces), depolarizing and hyperpolarizing voltage steps from the holding potential of –84 mV elicited an instantaneous outward and inward current, respectively. In the presence of strychnine (blue traces) the effects of ACh were abolished suggesting the direct involvement of 9 or 910 nAChRs. The block of the ACh-activated current by strychnine can be better appreciated from the steady-state I–V curves shown in Fig. 6B. We examined the involvement of SK channels in the ACh responses by the combined superfusion of 100 µM ACh and 300 nM apamin. As shown in Fig. 6C and D (P9 apical IHC) the simultaneous superfusion of ACh and apamin onto immature IHCs inhibited the additional outward current that activated near –26 mV when ACh was applied alone (Fig. 6A, arrowhead), showing that SK channels are directly involved in ACh responses. The large inward relaxing current remaining in the presence of apamin (Fig. 6C, arrowhead) is likely to be due to cations entering the cell through nAChRs activated by ACh. The same effects were seen in two additional P7 IHCs.
The distance between nAChRs and SK channels was investigated by recording membrane currents before and during superfusion of 100 µM ACh when the fast Ca2+ buffer BAPTA was used instead of EGTA in the intracellular solution (Naraghi & Neher, 1997). In the presence of 0.1 mM BAPTA (Fig. 7A and B) the superfusion of 100 µM ACh onto a P9 IHC activated instantaneous inward and outward currents (Fig. 7A) similar to those observed when 1 mM EGTA was used (Fig. 4B). When BAPTA was increased to 10 mM, ACh still produced a large inward current that relaxed back during hyperpolarizing voltage steps, but had no effect on the outward current (Fig. 7C and D), similar to when the SK channels were blocked by apamin (Fig. 6C and D). With 1 mM BAPTA only a partial reduction of the outward K+ current was seen (data not shown). Block of the ACh-activated SK current by BAPTA (5 or 10 mM) has previously been reported in short (outer) hair cells of the chick (Fuchs & Murrow, 1992) and guinea-pig OHCs (Evans, 1996). Figure 7E shows the size of the isolated steady-state (at 160 ms) ACh-activated currents obtained by subtracting the control currents from the currents in the presence of 100 µM ACh. Currents were measured at a membrane potential near –26 mV, a value corresponding to the peak outward ACh-activated current, using the following experimental conditions: 1 mM and 10 mM EGTA in the intracellular solution (as in Fig. 4C for 1 mM EGTA), 1 mM EGTA with the simultaneous superfusion of 300 nM apamin, perforated-patch recording and four different intracellular BAPTA concentrations. When 300 nM apamin was applied or the intracellular solution contained 10 or 30 mM BAPTA, the superfusion of 100 µM ACh did not elicit any significant outward K+ current. Compared with 1 mM EGTA, the size of the ACh-activated current was not significantly different when whole-cell recordings were obtained using 0.1 mM BAPTA, 1 mM BAPTA or 10 mM EGTA, or when perforated-patch was used. These results suggest that the SK current was effectively uncoupled from the nAChRs when 10 mM or higher BAPTA concentrations were used. The amplitude of the ACh-activated SK current during perforated-patch recording points to an endogenous buffer concentration equivalent to 1.1 mM BAPTA (Fig. 7F), a value close to that obtained when the SK current activated by Ca2+ influx through the Cav1.3 Ca2+ channels was investigated (0.7 mM, Fig. 3F).
ACh activates 910 nAChRs in immature IHCs
Recent findings have shown that the current flowing through recombinant 910 nAChRs was potentiated by the divalent cations Ca2+ and Ba2+ at concentrations up to 500 µM and blocked at higher concentrations (Weisstaub et al. 2002). By contrast, the ACh-activated current through the homomeric 9 receptor was reduced but not potentiated by Ca2+ regardless of the Ca2+ concentration (Katz et al. 2000). Therefore we investigated whether the native nAChRs expressed in immature IHCs showed biophysical properties of 9 alone or of the heteromeric 910 receptors. To test the sensitivity of native nAChRs to extracellular Ca2+ we superfused apical IHCs (n = 27, P7) with a Ca2+-free solution or with solutions containing 100 µM, 500 µM, 1 mM or 5 mM Ca2+. The isolated ACh-activated currents (Fig. 8A), in the presence of different Ca2+ concentrations, were obtained by subtracting the control currents from the currents during the superfusion of ACh. As previously shown for the recombinant nAChRs (Weisstaub et al. 2002), extracellular Ca2+ concentrations up to 500 µM (Fig. 8A, top panels) increased both the instantaneous and steady-state inward nAChR currents, whereas a reduction occurred when higher concentrations of the ion were used (Fig. 8A, bottom panels). The remaining small current in the presence of a Ca2+-free solution is likely to be carried by Na+ flowing through nAChRs as previously described (McNiven et al. 1996; Weisstaub et al. 2002). Note the absence of outward SK current. A small ACh-activated current was also recorded when 5 mM Ca2+ was used as Ca2+ acts as a permeant blocker of heteromeric 910 nAChRs (Weisstaub et al. 2002). In this condition a small SK current was present. Single exponential fits to the current relaxation near –144 mV indicate that the time constant became significantly (P < 0.0001) faster as extracellular Ca2+ was increased (Ca2+-Free: 29.6 ± 1.4 ms, n = 5; 100 µM: 21.9 ± 1.4 ms, n = 8; 500 µM: 18.3 ± 3.1 ms, n = 5; 1 mM: 10.7 ± 1.0 ms, n = 5; 5 mM: 1.1 ± 0.3 ms, n = 4). These results suggest that the observed current relaxation, at hyperpolarized test potentials, is likely to be due to extracellular Ca2+ partially blocking the nAChRs. The average instantaneous I–V curves for the different experimental conditions in Fig. 8A are shown in Fig. 8B.
Figure 8C shows the amplitude of the instantaneous inward current at a membrane potential near –144 mV as a function of extracellular Ca2+, where both potentiating and blocking effects exerted by Ca2+ can be appreciated. The changes in current amplitude were significant at P < 0.0001. Both potentiation and block were also evident from the steady-state currents measured at the same potential (data not shown). In contrast to the inward nAChR current, the size of the instantaneous and steady-state outward current, mainly carried by the SK channel, was only significantly reduced (P < 0.0001) when extracellular Ca2+ was absent (Fig. 8D, for the instantaneous current). The similar sizes of the SK current over the range of 500 µM to 5 mM Ca2+ suggests that 500 µM Ca2+ is already sufficient for maximally activating the SK current. Increasing the extracellular Ca2+ concentration shifted (P < 0.0001) the reversal potential of the total ACh-activated current towards more hyperpolarized potentials (Fig. 8E), probably due to a progressively increased contribution from the SK current.
ACh modulates the action potential frequency of immature IHCs
An inhibitory role of ACh on evoked action potential activity has been reported for P7 rat IHCs (Glowatzki & Fuchs, 2000). To gain more insight into the modulation of the spontaneous and evoked action potential frequency by nAChRs, we investigated voltage responses before and during superfusion of different ACh concentrations in P2–P4 IHCs (n = 32), results for one of which are shown in Fig. 9A–C. The application of ACh onto IHCs at concentrations of 3 µM or 10 µM caused a hyperpolarizing shift in the resting potential and reduced the frequency of induced action potentials (Fig. 9B). Spontaneous spikes were abolished using 3 µM ACh, the lowest concentration tested. In the presence of 30 µM (Fig. 9C) or higher ACh concentrations, evoked spikes were abolished at all current injection levels tested (up to +220 pA) and the resting potential became progressively hyperpolarized. The effects of 10 µM ACh on action potentials were abolished when 100 nM strychnine was simultaneously superfused (Fig. 9D), providing support for the nicotinic nature of the AChRs. A similar effect of strychnine was also observed in seven other P2–P4 IHCs. The superfusion of strychnine alone did not affect the spike frequency when compared with control values (n = 5). Figure 9E shows that the reduction in spike frequency was dependent on both ACh concentration and the current injection amplitude. Increasing the extracellular concentration of ACh also resulted in a significant hyperpolarization (P < 0.0001) by up to –11 mV of the resting membrane potential (Fig. 9F) compared with that in control conditions (–62.2 ± 1.1 mV, n = 32, P2–P4).
Effects of ACh on voltage responses in the absence of the SK current
As shown in Fig. 3C–F IHCs were unable to sustain repetitive action potentials when the SK current was blocked by apamin and the action potentials acquired long depolarized plateaus. Similar results were also obtained when 30 mM BAPTA was included in the intracellular solution (Fig. 10A) which prevents the activation of the SK current (Fig. 7E). In the presence of BAPTA, the application of 100 µM ACh (Fig. 10B) onto apical P6 IHCs now caused a depolarizing shift in the resting membrane potential (from –79.6 ± 1.5 to –48.2 ± 9.7 mV, n = 4, significant at P < 0.05) instead of the membrane hyperpolarization observed in Fig. 9F when the SK current was available. This depolarization led to a lowering of the threshold for firing action potentials with depolarized plateaus.
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Discussion |
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Here we show that immature IHCs express a Ca2+-activated K+ current from just after birth that disappears after the onset of hearing. This transient expression suggests that this current might contribute, together with other currents (Marcotti et al. 1999, 2003a,b), to the maturation of IHCs. The novel current was still present when intracellular K+ was substituted by Cs+ (see Fig. 5A in Marcotti et al. 2003b) and was abolished during the superfusion of a Ca+-free solution, nifedipine or the bee venom apamin. These results are consistent with the presence of an SK-type current (Sah & Faber, 2002) activated by Ca2+ influx into the cell through Cav1.3 voltage-gated Ca2+ channels known to be present in immature IHCs (Platzer et al. 2000; Marcotti et al. 2003b). An SK current was also isolated in OHCs from around the beginning of the second postnatal week, but contrary to IHCs its expression was not transient as it remained in adult cells. A current with pharmacological properties similar to those described above has also been reported in hair cells of various vertebrates (Fuchs & Murrow, 1992; Doi & Ohmori, 1993; Evans, 1996; Nenov et al. 1996b; Tucker & Fettiplace, 1996; Yamamoto et al. 1997; Yuhas & Fuchs, 1999).
The very low KD for block by apamin (360 pM) in immature IHCs suggests that the most likely gene candidate responsible for the SK current is that expressing SK2 channels (Köhler et al. 1996). SK2 channels have been cloned from the mature mouse cochlea (Nie et al. 2004) and in situ hybridization and immunolabelling techniques have shown that in the mature rat cochlea these channels are expressed in OHCs but not in IHCs (Dulon et al. 1998; Oliver et al. 2000). The absence of mRNA encoding SK2 channels in mature IHCs (Dulon et al. 1998) is consistent with electrophysiological experiments showing the insensitivity of the total K+ current to apamin (Kros & Crawford, 1990; Dulon et al. 1995; Marcotti et al. 2004).
Immature IHCs are transiently sensitive to the efferent neurotransmitter ACh
The SK channels expressed in hair cells are also thought to be activated by Ca2+ flowing through 910 nAChRs (Elgoyhen et al. 2001; Maison et al. 2002). The presence of heteromeric 910 nAChRs in immature IHCs was confirmed by experiments in which extracellular Ca2+ at micromolar concentrations potentiated and at millimolar concentrations reduced the ACh-activated current (Fig. 8), as previously described for the recombinant 910 nAChR (Weisstaub et al. 2002). Similar results, using a constant concentration of Mg2+, have also been shown for native nAChRs in chick short hair cells (McNiven et al. 1996) and in a preliminary study on immature rat IHCs (Gómez-Casati et al. 2004). While the concentration-dependent block of the nAChR current (Fig. 8A) can be simply explained by Ca2+ acting as a permeant blocker of these receptors, the potentiation may be caused by Ca2+ enhancing the probability of the channel being open, as previously described in neurones (Mulle et al. 1992; Amador & Dani, 1995).
ACh is the main efferent neurotransmitter in the mammalian cochlea (Eybalin, 1993) and has been shown to affect hair cells of various vertebrates (Fuchs & Murrow, 1992; Doi & Ohmori, 1993; Dulon & Lenoir, 1996; Nenov et al. 1996a; Evans, 1996; Yamamoto et al. 1997; He & Dallos, 1999; Yuhas & Fuchs, 1999; Glowatzki & Fuchs, 2000). In the mouse, efferent fibres synapse directly with IHCs or with their afferents from around birth although some developmental variability exists between strains and cochlear regions (Sobkowicz, 1992; Bruce et al. 1997, 2000). At about the same time, ACh appears to be present in the efferent endings below IHCs (Sobkowicz & Emmerling, 1989; Merchan Perez et al. 1994). By about P12 axosomatic contact between efferent fibres and IHCs no longer occurs (Shnerson et al. 1982; Bruce et al. 2000). Consistent with these morphological observations our results show that apical IHCs respond to ACh from around P2 (Fig. 5A), matching the appearance of the SK current in these cells, until just after the onset of hearing. Although the slow activation time course of the SK current (Fig. 2A–D) is reminiscent of the IK(Ca) found in mature IHCs, the latter was insensitive to apamin and blocked by intracellular Cs+ which permeates through SK channels (Fig. 8 in Marcotti et al. 2004). Also, in the guinea-pig, apamin was ineffective in mature IHCs (Kros & Crawford, 1990; Dulon et al. 1995). These results suggest that the SK current is down-regulated in mature IHCs, perhaps simultaneously with the nAChRs.
The disappearance of ACh responses in mature IHCs is likely to be related to developmental changes in the expression of the nAChRs. The period during which mRNAs for heteromeric 910 nAChRs are present around the first 2 weeks of postnatal development (Morley & Simmons, 2002) coincides with the occurrence of axosomatic efferent innervation of IHCs and the cells responding to direct application of ACh. Interestingly, a low level of expression of 9 mRNA alone in IHCs both before and after this period does not correlate with functional ACh receptors.
Contrary to IHCs, apical-coil OHCs begin to respond to ACh only from around the beginning of the second postnatal week (Fig. 5B), when the SK current was also detected for the first time in these cells in the absence of ACh. Previous studies on the rat and gerbil have shown that basal OHCs start to respond to ACh from P6 (Dulon & Lenoir, 1996; He & Dallos, 1999). Our findings are consistent with these results, considering that the maturation of apical OHCs is delayed by about 2 days compared with basal cells (He et al. 1994). The temporal discrepancy in the responsiveness to ACh between IHCs and OHCs is likely to be related to the later formation of efferent synaptic connections with OHCs (Pujol et al. 1998), which also corresponds to the expression of the heteromeric 910 nAChRs in these cells (Morley & Simmons, 2002).
Roles of the SK current and of efferent innervation in immature IHCs
Although it is known that efferent connections with OHCs reduce mechanical amplification in the mature cochlea (Guinan, 1996), their role in immature IHCs is less clear. Previous studies have shown that different current types expressed by immature IHCs can specifically modulate the shape and frequency of spontaneous or induced action potentials (Marcotti et al. 1999, 2003a,b). We were interested in determining whether the SK current has a well-defined role in modulating the action potentials in immature IHCs. Although these cells express both Ca2+ and Na+ currents, only the former is required for the generation of spontaneous and induced action potentials since they are reversibly abolished during superfusion of a Ca2+-free solution but not by TTX (Marcotti et al. 2003b). While the Ca2+ current together with the K+ current IK,neo determines the rate of rise and fall and the amplitude of the spike (Fig. 11), the Na+ current reduces the interspike interval (Fig. 11 and Marcotti et al. 2003b). Finally, the resting membrane potential of immature IHCs is mainly determined by both IK,neo and the inward K+ current IK1 (Fig. 11; Marcotti et al. 1999, 2003a). The increase in size of IK1 in the second postnatal week hyperpolarizes the resting potential and is the main factor in the disappearance of spontaneous, but not induced, action potentials from P6 onwards (Marcotti et al. 2003a).
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Vm) as a function of ACh concentration. Number of measurements shown above averaged data points in F also apply to E. All recordings at body temperature.
