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Potassium current properties in apical and basal inner hair cells from guinea-pig cochlea
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Hearing Research 180 (2003) 85^90 www.elsevier.com/locate/heares
Potassium current properties in apical and basal inner hair cells from guinea-pig cochlea Takashi Kimitsuki , Kazuhiro Kawano, Keiji Matsuda, Atsushi Haruta, Takahiro Nakajima, Shizuo Komune Department of Otorhinolaryngology, Miyazaki Medical College, 5200 Kihara, Kiyotake-cho, Miyazaki 889-1692, Japan Received 31 August 2001; accepted 2 April 2003
Abstract Inner hair cells (IHCs) of guinea-pigs were separately isolated from the apical and basal turn and the potassium currents were measured by the whole-cell voltage-clamp technique. The potassium current flows through two types of membrane conductance: a fast (Ik;f ), tetraethylammonium (TEA)-sensitive conductance and a slow (Ik;s ), TEA-resistant conductance. Membrane conductance demonstrated no significant differences between apical IHCs and basal IHCs. Reversal potentials were 365 3 2 mV and 368 3 5 mV in apical and basal IHCs, respectively. The rate of outward current activation was voltage dependent and faster in basal IHCs than in apical IHCs. TEA effect was stronger on basal IHCs than on apical IHCs, suggesting that Ik;f is dominant in basal IHCs. 6 2003 Elsevier Science B.V. All rights reserved. Key words: Cochlea; Inner hair cell; Potassium current; Tetraethylammonium; Tonotopic
1. Introduction In the mammalian cochlea, there are two types of hair cells that subserve distinct functions and receive characteristic patterns of innervation. The single row of inner hair cells (IHCs) that receive nearly all the a¡erent innervation and are primary acoustic transducers. The three rows of outer hair cells (OHCs) receive e¡erent axons from neurons located in the superior olivary complex of the brainstem and form part of the feedback loop that regulates frequency selectivity and sensitivity (Guinan and Stankovic, 1996; Ulfendahl and Flock, 1998). Variations in the expression of OHC potassium conductances along the tonotopic gradient of the adult mammalian cochlea have been investigated by Housley and Ashmore (1992), Mammano and Ashmore (1996) and Raybould and Housley (1997). They dem-
* Corresponding author. Tel.: +81 (985) 85 2966; Fax: +81 (985) 85 7029. E-mail address: [email protected] (T. Kimitsuki).
onstrated a position-dependent increase in the basolateral membrane conductance from the apex to the base along the length of the cochlea. However, variation in the expression of IHC potassium conductance along the tonotopic axis of the cochlea is unknown. The two IHC potassium currents are distinguishable by their pharmacology and their activation kinetics (Kros and Crawford, 1990). The fast activating current, Ik;f , was blocked by tetraethylammonium (TEA) but was resistant to 4-aminopyridine (4-AP). This current has recently been implicated in developmental changes in IHCs during the postnatal days just preceding functional maturation of hearing in mice (Kros et al., 1998). Another potassium current, Ik;s , was activated more slowly on depolarization and was blocked by 4-AP but not by TEA. The physiological role of this current has not yet been elucidated. In this study, we obtained separately isolated IHCs from the apical turn and basal turn and measured the potassium currents by the whole-cell voltage-clamp technique. This study investigated and compared the two types of potassium currents in the apex and basal IHCs by their pharmacology and activation kinetics.
0378-5955 / 03 / $ ^ see front matter 6 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0378-5955(03)00109-6
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2. Materials and methods
2.3. Animal care
2.1. Preparation of isolated IHCs
The experimental design was reviewed and approved (Accession No. 1999-057-3) by the Committee for Ethics in Animal Experiments in Miyazaki Medical College. The experiments were carried out under the Guidelines for Animal Experiments in Miyazaki Medical College.
Adult albino guinea-pig (200^350 g) were killed by rapid cervical dislocation and both bullae were removed and the cochlea was exposed. The cochlea, fused to the bulla, was placed in a Ca2þ -free external solution (142 mM NaCl, 4 mM KCl, 3 mM MgCl2 , 2 mM NaH2 PO4 , 8 mM Na2 HPO4 , adjusted to pH 7.4 with NaOH). The otic capsule was opened, allowing removal of the organ of Corti attached to the modiolus. IHCs were isolated by micro-dissecting a selected turn of the organ of Corti, from turn 1^2 and turn 4. The organ of Corti was dissected as the inner portion as close to the bone (tympanic lip) as possible to obtain a higher number of IHCs. A selected turn of Corti was treated with trypsin (0.5 mg/ml, T-4665, Sigma) for 15 min. Gentle mechanical trituration was carried out and the enzyme was rinsed by superfusing with a standard external solution (142 mM NaCl, 4 mM KCl, 2 mM MgCl2 , 1 mM CaCl2 , 2 mM NaH2 PO4 , 8 mM Na2 HPO4 , adjusted to pH 7.4 with NaOH) at least 10 min before starting any experiments.
3. Results 3.1. Membrane currents under voltage-clamp in apical and basal IHCs Currents in response to depolarizing voltage steps from a holding potential of 380 mV were recorded from IHCs in apical and basal turns. Typical current records are shown in Fig. 1A. Both apical and basal IHCs had outwardly rectifying currents in response to depolarizing voltage pulses, with only a slight inward current when hyperpolarized. Fig. 1B represents the steady-state current^voltage relation (I^V) measured at the end of each 40 ms command step for all seven cells from apical turn and six cells from the basal turn.
2.2. Recording procedures Membrane currents were measured by conventional whole-cell voltage-clamp recordings using EPC-8 (HEKA, Lambrecht, Germany). Data acquisition was controlled by the software PULSE/PULSEFIT (HEKA, Lambrecht, Germany). Recording electrodes were pulled on a two-stage vertical puller (PP830 Narishige, Tokyo, Japan) using 1.2 mm o.d. borosilicate glass (GC-1.2, Narishige, Tokyo) ¢lled with internal solution (144 mM KCl, 2 mM MgCl2 , 1 mM NaH2 PO4 , 8 mM Na2 HPO4 , 2 mM ATP, 3 mM D-glucose, 0.5 mM EGTA, adjusted to pH 7.4 with KOH.). Pipettes showed a resistance of 4^7 M6 in the bath and were coated with ski wax (Tour-DIA, DIAWax, Otaru, Japan) to minimize capacitance. The capacitance of the cells was 11.0 3 3.2 pF (n = 7) and 10.0 3 2.0 pF (n = 6) in apical and basal IHCs, respectively. The series resistance was 25.6 3 12.7 M6 (n = 7) and 26.6 3 14.2 M6 (n = 6) in apical and basal IHCs, respectively. Neither capacitance nor resistance demonstrated any di¡erence between apical and basal IHCs. Twenty-¢ve mM TEA solution (replacing 25 mM NaCl in the standard external solution) was applied under pressure using pipets with a tip diameter of 2^4 Wm positioned around 50 Wm from the IHCs. Cells were continuously perfused with external saline and all experiments were performed at room temperature (20^ 25‡C).
Fig. 1. Membrane currents under the voltage-clamp for IHCs from the apical and basal turns. (A) The left panels show recordings from apical IHCs, the right panels show those from basal IHCs. The upper panels show the voltage step protocol. The holding potential was 380 mV. The test pulse duration was 40 ms. The lower panels show the currents elicited by steps to absolute membrane potentials shown by each step. (B) Steady-state current^voltage relationships for apical (closed circles) and basal (open circles) IHCs. The currents were measured at the end of the 40 ms test pulse as shown in A (b, a). Each vertical line indicates 3 S.D. of the value. The total number of cells used were seven (apex) and six (base).
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Both curves showed a pronounced outward recti¢cation with maximal slope conductance of 37.2 3 9.0 nS and 37.0 3 12.6 nS for cells from the apical turn and basal turn, respectively.
intracellular Ca2þ . These results indicate that the current is mainly carried by potassium ions.
3.2. Reversal potential of the membrane currents in apical and basal IHCs
A comparison of the outward current kinetics recorded from apical and basal IHCs is shown in Fig. 3. The half-activating time of the rising phase was measured from six apical cells and six basal cells, and was plotted against the membrane potentials. The outward current kinetics depends on the membrane potential, becoming faster at more depolarized levels. Although the rate of outward current activation appears to be more rapid in basal IHCs than in apical IHCs, the analysis of variance (ANOVA) statistical testing did not demonstrate any signi¢cant di¡erences between apical and basal cells (P 6 0.05). The rising phase was separately ¢tted by two-exponential curves in apical and basal IHCs at +30 mV. In the basal IHCs, the fast and slow time constants were 0.41 3 0.09 ms and 2.32 3 0.87 ms (n = 4), respectively. In the basal IHCs, fast and slow time constants were 0.49 3 0.09 ms and 2.04 3 0.16 ms (n = 5), respectively, showing that there were no di¡erences between apical and basal IHCs.
In cells from the apical turn and basal turn with normal intracellular and extracellular solutions a depolarizing voltage step of constant magnitude and duration (330 mV for 40 ms) was followed by voltage steps to various depolarized levels. Such a voltage pulse protocol allowed the instantaneous current^voltage curve and reversal potential to be determined. The resulting tail currents are shown for one cell in Fig. 2Aa. An instantaneous current^voltage curve is plotted in Fig. 2Ab. The tail current reversed at 367 mV in this cell. Reversal potentials of tail currents were 365 3 2 mV (mean 3 S.D., n = 3) and 368 3 5 mV (n = 4) in cells from the apical turn and basal turn, respectively (Fig. 2B, control). The potassium equilibrium potential is 390 mV (at 20‡C). The discrepancy of the reversal potential and the potassium equilibrium cannot be explained by this study. However, if sodium is another ion transiting through this current, the permeability ratio of potassium (PK ) to sodium (PNa ) would be 0.995/ 0.005 according to the constant ¢eld theory by Goldman, Hodgkin and Katz. If chloride is another ion transiting through this current, the permeability ratio PK /PCl would be 0.96/0.04. The calcium ion permeability is much smaller because of the low concentration of
3.3. Outward current kinetics in apical and basal IHCs
3.4. TEA-sensitive component of the membrane currents in apical and basal IHCs Based on the reversal potential, it seems likely that the membrane currents were carried mainly by potassium ions. Further support for this was sought by exposing cells to a solution containing 25 mM TEA, a block-
Fig. 2. (A) Tail currents and the instantaneous current^voltage curve. (a) Membrane tail currents, after the membrane potential returned from 330 mV to various levels. The upper part shows a schematic representation of the voltage protocol, the lower part shows the tail currents. The horizontal dotted line shows the zero current level. (b) Instantaneous current^voltage curve. Currents at the instant of repolarization as in a were plotted against the absolute potentials. The straight line was ¢tted by a least-squares method. (B) Reversal potentials of tail currents in the control and TEA-resistant currents in apical and basal IHCs. The reversal potential was 365 3 2 mV, 363 3 7 mV, 368 3 5 mV and 369 3 8 mV in the control apical (n = 5), TEA-resistant apical (n = 2), control basal (n = 4), and TEA-resistant basal IHCs (n = 3), respectively. Each vertical line indicates 3 S.D. of the value.
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Fig. 3. Activation kinetics of membrane currents. Half-activation times as a function of membrane potentials in apical (closed circles, n = 6) and basal (open circles, n = 6) IHCs. Each vertical line indicates 3 S.D. of the value.
er of potassium channels. Fig. 4 demonstrates TEA-resistant membrane currents (panel B, TEA) and TEAsensitive currents (panel C, control3TEA) in apical and basal IHCs. In the presence of TEA, the activation rate of currents generated by depolarized stimuli were slower than that in control solutions, suggesting that TEA
Fig. 5. Voltage dependency of the TEA e¡ect on apical and basal IHCs. The TEA-resistant currents at various membrane potentials were normalized by the amplitude of the control current at +50 mV in apical (close circles) and basal (close triangles) IHCs, and plotted against the corresponding membrane potentials. A total of ¢ve cells for both apex and base were used. Each vertical line indicates 3 S.D. of the value.
blocked a fast activation component of outward currents. The rising phase could be ¢tted by the singleexponential curve. At +30 mV, the time constant of TEA-sensitive current in apical and basal IHCs was 0.47 3 0.11 ms (n = 4) and 0.41 3 0.13 ms (n = 4), respectively. The time constant of TEA-resistant current in apical and basal IHCs was 5.23 3 1.61 ms (n = 4) and 4.48 3 1.05 ms (n = 4), respectively. The amplitude of initial TEA-sensitive currents does not show signi¢cant di¡erences between apical and basal IHCs, but a sustained current at the end of a 40 ms depolarizing pulse was larger in basal IHCs than in apical IHCs (Fig. 4C). Reversal potentials of TEA-resistant currents measuring the tail currents in apical and basal IHCs were 363 3 7 mV and 369 3 8 mV, respectively, similar to those of control cells (Fig. 2B). The relationship between normalized TEA-resistant currents and membrane potentials is shown in Fig. 5. The normalized values were determined by measuring the ratio of each TEA-resistant current to the control current at +50 mV. Although ANOVA did not demonstrate any signi¢cant di¡erences between apical and basal cells (P 6 0.05), the normalized TEA-resistant current was 0.70 in basal IHCs and 0.82 in apical IHCs.
4. Discussion Fig. 4. E¡ect of superfusion with TEA. Membrane currents from apical (left panels) and basal (right panels) IHCs are shown before (A) and during (B) superfusion with 25 mM TEA. The upper panels in A show a schematic representation of the voltage protocol. Horizontal dotted lines show the zero current level. (C) TEA-sensitive currents obtained by subtraction of responses in 25 mM TEA (B) from controls (A).
The potassium current in IHCs £ows through at least two types of membrane conductance: a fast (Ik;f ), TEAsensitive conductance and a slow (Ik;s ), TEA-resistant conductance. Although membrane conductance demonstrated no di¡erences between apical IHCs and basal IHCs (Fig. 1), TEA-sensitive Ik;f is larger in basal
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IHCs than in apical IHCs (Figs. 4 and 5). The fast and slow time constants in the rising phase of control currents were consistent with those of TEA-sensitive Ik;f and TEA-resistant Ik;s , respectively. There are no di¡erences in the value of the time constants between apical and basal IHCs, suggesting that the di¡erences in halfactivation time between apical and basal IHCs (Fig. 3) is due to di¡erences in the contribution of Ik;f and Ik;s in apical and basal IHCs, not due to kinetic di¡erences in Ik;f and/or Ik;s . Faster activation kinetics in basal IHCs might contribute to higher frequency signals in the basal cochlear turn. A similar tonotopical gradient along the cochlea with the e¡ect of TEA was observed in guinea-pig OHCs (Mammano and Ashmore, 1996). In OHCs, three different potassium conductances (Ik , IkðCaÞ , Ik;n ) were identi¢ed (Housley and Ashmore, 1992) and the extent of channel expression changes between the apex and base of the cochlea (Mammano and Ashmore, 1996; Nenov et al., 1997). The potassium current in basal OHCs showed signi¢cantly faster onset kinetics than that in apical OHCs. In chick basilar papilla (Rosenblatt et al., 1997; Navaratnam et al., 1997) and in turtle cochlea (Jones et al., 1999), there is a variation in the channel isoform of calcium-activated potassium conductance (IkðCaÞ ) along the length of the cochlea. Kþ currents largely determine the negative resting potential of the IHCs and contribute to the driving force for the mechano-electrical transducer at the apical pole of the cell. Opening of the transducer channels can be simulated by injecting a constant current through the patch-pipet (Kros and Crawford, 1990). This procedure generated fast depolarization with an associated spike (within 10 ms) due to activation of the fast potassium current (Ik;f ), and subsequent slow relaxation of the membrane potential due to the action of the slow potassium current (Ik;s ). Dominance of Ik;f in the basal IHCs might contribute to the fast depolarization generated by transducer channel openings. Ik;f was absent in IHCs in cochlear cultures from neonatal mice but was always found in cells isolated from mice after the onset of hearing about 11 days after birth (Kros et al., 1991). Ik;f was ¢rst observed in IHCs from the basal coil 11 days after birth and matured rapidly over the next 3 days (Kros et al., 1998). Before this time, mouse IHCs have only slow activated Kþ current (Ik:neo similar to Ik;s ) and demonstrate slow voltage responses and ¢re spontaneous and evoked action potentials. An action potential and subsequent dampened oscillation were observed in hair cells from lower vertebrate animals: frogs (Hudspeth and Lewis, 1988; Armstrong and Roberts, 1998), turtles (Art and Fettiplace, 1987), alligators (Fuchs and Evans, 1988) and chicks (Fuchs et al., 1988). In mammalian IHCs, during development of auditory responsiveness, Ik;f is ex-
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pressed, greatly accelerating the membrane time constant and preventing the action potential (Kros et al., 1991). Ik;f dominance in the basal IHCs suggests that functional development starts from the basal turn towards the apex. Morphologically, various structural maturation processes start near the basal end in a variº nggard, 1965; Ruben, 1967; Tanaka et ety of species (A al., 1979; Mu et al., 1997).
Acknowledgements This work was supported by a Grant-in-Aid for Scienti¢c Research 13671789 from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
References º nggard, L., 1965. Electrophysiological study of the development of A the cochlea function in the rabbit. Acta Otolaryngol. (Stockh.) Suppl. 203, 64 pp. Armstrong, C.E., Roberts, W.M., 1998. Electrical properties of frog saccular hair cells: distortion by enzymatic dissociation. J. Neurosci. 18, 2962^2973. Art, J.J., Fettiplace, R., 1987. Variation of membrane properties in hair cells isolated from the turtle cochlea. J. Physiol. 385, 207^242. Fuchs, P.A., Evans, M.G., 1988. Voltage oscillations and ionic conductances in hair cells isolated from the alligator cochlea. J. Comp. Physiol. A 164, 151^163. Fuchs, P.A., Nagai, T., Evans, M.G., 1988. Electrical tuning in hair cells isolated from the chick cochlea. J. Neurosci. 8, 2460^2467. Guinan, J.J., Jr., Stankovic, K.M., 1996. Medial e¡erent inhibition produces the largest equivalent attenuations at moderate to high sound levels in cat auditory nerve ¢bers. J. Acoust. Soc. Am. 100, 1680^1690. Housley, G.D., Ashmore, J.F., 1992. Ionic currents of outer hair cells isolated from the guinea-pig cochlea. J. Physiol. 448, 73^98. Hudspeth, A.J., Lewis, R.S., 1988. A model for electrical resonance and frequency tuning in saccular hair cells of the bull-frog, Rana catesbeiana. J. Physiol. 400, 275^297. Jones, E.M., Geray-Keller, M., Fettiplace, R., 1999. The role of Ca2þ activated Kþ channel spliced variants in the tonotopic organization of the turtle cochlea. J. Physiol. 518, 653^665. Kros, C.J., Crawford, A.C., 1990. Potassium currents in inner hair cells isolated from the guinea-pig cochlea. J. Physiol. 421, 263^291. Kros, C.J., Ru«sch, A., Russell, I.J., 1991. Di¡erences between potassium currents of neonatal and mature mouse inner hair cells in vitro. J. Physiol. 434, 51 pp. Kros, C.J., Ruppersberg, J.P., Ru«sch, A., 1998. Expression of a potassium current in inner hair cells during development of hearing in mice. Nature 394, 281^284. Mammano, F., Ashmore, J.F., 1996. Di¡erential expression of outer hair cell potassium currents in the isolated cochlea of the guineapig. J. Physiol. 496, 639^646. Mu, M.Y., Chardin, S., Avan, P., Romand, R., 1997. Ontogenesis of rat cochlea, a quantitative study of the organ of Corti. Dev. Brain Res. 99, 29^37. Navaratnam, D.S., Bell, T.J., Tu, T.D., Cohen, E.L., Oberholtzer, C.J., 1997. Di¡erential distribution of Ca2þ -activated Kþ channel splice variants among hair cells along the tonotopic axis of the chick cochlea. Neuron 19, 1077^1085.
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Nenov, A.P., Norris, C., Bobbin, R.P., 1997. Outwardly rectifying currents in guinea pig outer hair cells. Hear. Res. 105, 146^158. Raybould, N.P., Housley, G.D., 1997. Variation in expression of the outer hair cell P2X receptor conductance along the guinea-pig cochlea. J. Physiol. 498, 717^727. Rosenblatt, K.P., Sun, Z.-P., Heller, S., Hudspeth, A.J., 1997. Distribution of Ca2þ -activated Kþ channel isoforms along the tonotopic gradient of the chicken’s cochlea. Neuron 19, 1061^1075.
Ruben, R.J., 1967. Development of the inner ear of the mouse: a radioautographic study of terminal mitosis. Acta Otolaryngol. (Stockh.) Suppl. 240, 44 pp. Tanaka, K., Sakai, N., Terayama, Y., 1979. Organ of Corti in the human fetus: scanning and transmission electron microscopic studies. Ann. Otol. Rhinol. Laryngol. 88, 749^758. Ulfendahl, M., Flock, A., 1998. In vitro studies of cochlea excitation. Curr. Opin. Neurobiol. 8, 475^479.
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Potassium current properties in apical and basal inner hair cells from guinea-pig cochlea Takashi Kimitsuki , Kazuhiro Kawano, Keiji Matsuda, Atsushi Haruta, Takahiro Nakajima, Shizuo Komune Department of Otorhinolaryngology, Miyazaki Medical College, 5200 Kihara, Kiyotake-cho, Miyazaki 889-1692, Japan Received 31 August 2001; accepted 2 April 2003
Abstract Inner hair cells (IHCs) of guinea-pigs were separately isolated from the apical and basal turn and the potassium currents were measured by the whole-cell voltage-clamp technique. The potassium current flows through two types of membrane conductance: a fast (Ik;f ), tetraethylammonium (TEA)-sensitive conductance and a slow (Ik;s ), TEA-resistant conductance. Membrane conductance demonstrated no significant differences between apical IHCs and basal IHCs. Reversal potentials were 365 3 2 mV and 368 3 5 mV in apical and basal IHCs, respectively. The rate of outward current activation was voltage dependent and faster in basal IHCs than in apical IHCs. TEA effect was stronger on basal IHCs than on apical IHCs, suggesting that Ik;f is dominant in basal IHCs. 6 2003 Elsevier Science B.V. All rights reserved. Key words: Cochlea; Inner hair cell; Potassium current; Tetraethylammonium; Tonotopic
1. Introduction In the mammalian cochlea, there are two types of hair cells that subserve distinct functions and receive characteristic patterns of innervation. The single row of inner hair cells (IHCs) that receive nearly all the a¡erent innervation and are primary acoustic transducers. The three rows of outer hair cells (OHCs) receive e¡erent axons from neurons located in the superior olivary complex of the brainstem and form part of the feedback loop that regulates frequency selectivity and sensitivity (Guinan and Stankovic, 1996; Ulfendahl and Flock, 1998). Variations in the expression of OHC potassium conductances along the tonotopic gradient of the adult mammalian cochlea have been investigated by Housley and Ashmore (1992), Mammano and Ashmore (1996) and Raybould and Housley (1997). They dem-
* Corresponding author. Tel.: +81 (985) 85 2966; Fax: +81 (985) 85 7029. E-mail address: [email protected] (T. Kimitsuki).
onstrated a position-dependent increase in the basolateral membrane conductance from the apex to the base along the length of the cochlea. However, variation in the expression of IHC potassium conductance along the tonotopic axis of the cochlea is unknown. The two IHC potassium currents are distinguishable by their pharmacology and their activation kinetics (Kros and Crawford, 1990). The fast activating current, Ik;f , was blocked by tetraethylammonium (TEA) but was resistant to 4-aminopyridine (4-AP). This current has recently been implicated in developmental changes in IHCs during the postnatal days just preceding functional maturation of hearing in mice (Kros et al., 1998). Another potassium current, Ik;s , was activated more slowly on depolarization and was blocked by 4-AP but not by TEA. The physiological role of this current has not yet been elucidated. In this study, we obtained separately isolated IHCs from the apical turn and basal turn and measured the potassium currents by the whole-cell voltage-clamp technique. This study investigated and compared the two types of potassium currents in the apex and basal IHCs by their pharmacology and activation kinetics.
0378-5955 / 03 / $ ^ see front matter 6 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0378-5955(03)00109-6
HEARES 4692 19-5-03
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T. Kimitsuki et al. / Hearing Research 180 (2003) 85^90
2. Materials and methods
2.3. Animal care
2.1. Preparation of isolated IHCs
The experimental design was reviewed and approved (Accession No. 1999-057-3) by the Committee for Ethics in Animal Experiments in Miyazaki Medical College. The experiments were carried out under the Guidelines for Animal Experiments in Miyazaki Medical College.
Adult albino guinea-pig (200^350 g) were killed by rapid cervical dislocation and both bullae were removed and the cochlea was exposed. The cochlea, fused to the bulla, was placed in a Ca2þ -free external solution (142 mM NaCl, 4 mM KCl, 3 mM MgCl2 , 2 mM NaH2 PO4 , 8 mM Na2 HPO4 , adjusted to pH 7.4 with NaOH). The otic capsule was opened, allowing removal of the organ of Corti attached to the modiolus. IHCs were isolated by micro-dissecting a selected turn of the organ of Corti, from turn 1^2 and turn 4. The organ of Corti was dissected as the inner portion as close to the bone (tympanic lip) as possible to obtain a higher number of IHCs. A selected turn of Corti was treated with trypsin (0.5 mg/ml, T-4665, Sigma) for 15 min. Gentle mechanical trituration was carried out and the enzyme was rinsed by superfusing with a standard external solution (142 mM NaCl, 4 mM KCl, 2 mM MgCl2 , 1 mM CaCl2 , 2 mM NaH2 PO4 , 8 mM Na2 HPO4 , adjusted to pH 7.4 with NaOH) at least 10 min before starting any experiments.
3. Results 3.1. Membrane currents under voltage-clamp in apical and basal IHCs Currents in response to depolarizing voltage steps from a holding potential of 380 mV were recorded from IHCs in apical and basal turns. Typical current records are shown in Fig. 1A. Both apical and basal IHCs had outwardly rectifying currents in response to depolarizing voltage pulses, with only a slight inward current when hyperpolarized. Fig. 1B represents the steady-state current^voltage relation (I^V) measured at the end of each 40 ms command step for all seven cells from apical turn and six cells from the basal turn.
2.2. Recording procedures Membrane currents were measured by conventional whole-cell voltage-clamp recordings using EPC-8 (HEKA, Lambrecht, Germany). Data acquisition was controlled by the software PULSE/PULSEFIT (HEKA, Lambrecht, Germany). Recording electrodes were pulled on a two-stage vertical puller (PP830 Narishige, Tokyo, Japan) using 1.2 mm o.d. borosilicate glass (GC-1.2, Narishige, Tokyo) ¢lled with internal solution (144 mM KCl, 2 mM MgCl2 , 1 mM NaH2 PO4 , 8 mM Na2 HPO4 , 2 mM ATP, 3 mM D-glucose, 0.5 mM EGTA, adjusted to pH 7.4 with KOH.). Pipettes showed a resistance of 4^7 M6 in the bath and were coated with ski wax (Tour-DIA, DIAWax, Otaru, Japan) to minimize capacitance. The capacitance of the cells was 11.0 3 3.2 pF (n = 7) and 10.0 3 2.0 pF (n = 6) in apical and basal IHCs, respectively. The series resistance was 25.6 3 12.7 M6 (n = 7) and 26.6 3 14.2 M6 (n = 6) in apical and basal IHCs, respectively. Neither capacitance nor resistance demonstrated any di¡erence between apical and basal IHCs. Twenty-¢ve mM TEA solution (replacing 25 mM NaCl in the standard external solution) was applied under pressure using pipets with a tip diameter of 2^4 Wm positioned around 50 Wm from the IHCs. Cells were continuously perfused with external saline and all experiments were performed at room temperature (20^ 25‡C).
Fig. 1. Membrane currents under the voltage-clamp for IHCs from the apical and basal turns. (A) The left panels show recordings from apical IHCs, the right panels show those from basal IHCs. The upper panels show the voltage step protocol. The holding potential was 380 mV. The test pulse duration was 40 ms. The lower panels show the currents elicited by steps to absolute membrane potentials shown by each step. (B) Steady-state current^voltage relationships for apical (closed circles) and basal (open circles) IHCs. The currents were measured at the end of the 40 ms test pulse as shown in A (b, a). Each vertical line indicates 3 S.D. of the value. The total number of cells used were seven (apex) and six (base).
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Both curves showed a pronounced outward recti¢cation with maximal slope conductance of 37.2 3 9.0 nS and 37.0 3 12.6 nS for cells from the apical turn and basal turn, respectively.
intracellular Ca2þ . These results indicate that the current is mainly carried by potassium ions.
3.2. Reversal potential of the membrane currents in apical and basal IHCs
A comparison of the outward current kinetics recorded from apical and basal IHCs is shown in Fig. 3. The half-activating time of the rising phase was measured from six apical cells and six basal cells, and was plotted against the membrane potentials. The outward current kinetics depends on the membrane potential, becoming faster at more depolarized levels. Although the rate of outward current activation appears to be more rapid in basal IHCs than in apical IHCs, the analysis of variance (ANOVA) statistical testing did not demonstrate any signi¢cant di¡erences between apical and basal cells (P 6 0.05). The rising phase was separately ¢tted by two-exponential curves in apical and basal IHCs at +30 mV. In the basal IHCs, the fast and slow time constants were 0.41 3 0.09 ms and 2.32 3 0.87 ms (n = 4), respectively. In the basal IHCs, fast and slow time constants were 0.49 3 0.09 ms and 2.04 3 0.16 ms (n = 5), respectively, showing that there were no di¡erences between apical and basal IHCs.
In cells from the apical turn and basal turn with normal intracellular and extracellular solutions a depolarizing voltage step of constant magnitude and duration (330 mV for 40 ms) was followed by voltage steps to various depolarized levels. Such a voltage pulse protocol allowed the instantaneous current^voltage curve and reversal potential to be determined. The resulting tail currents are shown for one cell in Fig. 2Aa. An instantaneous current^voltage curve is plotted in Fig. 2Ab. The tail current reversed at 367 mV in this cell. Reversal potentials of tail currents were 365 3 2 mV (mean 3 S.D., n = 3) and 368 3 5 mV (n = 4) in cells from the apical turn and basal turn, respectively (Fig. 2B, control). The potassium equilibrium potential is 390 mV (at 20‡C). The discrepancy of the reversal potential and the potassium equilibrium cannot be explained by this study. However, if sodium is another ion transiting through this current, the permeability ratio of potassium (PK ) to sodium (PNa ) would be 0.995/ 0.005 according to the constant ¢eld theory by Goldman, Hodgkin and Katz. If chloride is another ion transiting through this current, the permeability ratio PK /PCl would be 0.96/0.04. The calcium ion permeability is much smaller because of the low concentration of
3.3. Outward current kinetics in apical and basal IHCs
3.4. TEA-sensitive component of the membrane currents in apical and basal IHCs Based on the reversal potential, it seems likely that the membrane currents were carried mainly by potassium ions. Further support for this was sought by exposing cells to a solution containing 25 mM TEA, a block-
Fig. 2. (A) Tail currents and the instantaneous current^voltage curve. (a) Membrane tail currents, after the membrane potential returned from 330 mV to various levels. The upper part shows a schematic representation of the voltage protocol, the lower part shows the tail currents. The horizontal dotted line shows the zero current level. (b) Instantaneous current^voltage curve. Currents at the instant of repolarization as in a were plotted against the absolute potentials. The straight line was ¢tted by a least-squares method. (B) Reversal potentials of tail currents in the control and TEA-resistant currents in apical and basal IHCs. The reversal potential was 365 3 2 mV, 363 3 7 mV, 368 3 5 mV and 369 3 8 mV in the control apical (n = 5), TEA-resistant apical (n = 2), control basal (n = 4), and TEA-resistant basal IHCs (n = 3), respectively. Each vertical line indicates 3 S.D. of the value.
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Fig. 3. Activation kinetics of membrane currents. Half-activation times as a function of membrane potentials in apical (closed circles, n = 6) and basal (open circles, n = 6) IHCs. Each vertical line indicates 3 S.D. of the value.
er of potassium channels. Fig. 4 demonstrates TEA-resistant membrane currents (panel B, TEA) and TEAsensitive currents (panel C, control3TEA) in apical and basal IHCs. In the presence of TEA, the activation rate of currents generated by depolarized stimuli were slower than that in control solutions, suggesting that TEA
Fig. 5. Voltage dependency of the TEA e¡ect on apical and basal IHCs. The TEA-resistant currents at various membrane potentials were normalized by the amplitude of the control current at +50 mV in apical (close circles) and basal (close triangles) IHCs, and plotted against the corresponding membrane potentials. A total of ¢ve cells for both apex and base were used. Each vertical line indicates 3 S.D. of the value.
blocked a fast activation component of outward currents. The rising phase could be ¢tted by the singleexponential curve. At +30 mV, the time constant of TEA-sensitive current in apical and basal IHCs was 0.47 3 0.11 ms (n = 4) and 0.41 3 0.13 ms (n = 4), respectively. The time constant of TEA-resistant current in apical and basal IHCs was 5.23 3 1.61 ms (n = 4) and 4.48 3 1.05 ms (n = 4), respectively. The amplitude of initial TEA-sensitive currents does not show signi¢cant di¡erences between apical and basal IHCs, but a sustained current at the end of a 40 ms depolarizing pulse was larger in basal IHCs than in apical IHCs (Fig. 4C). Reversal potentials of TEA-resistant currents measuring the tail currents in apical and basal IHCs were 363 3 7 mV and 369 3 8 mV, respectively, similar to those of control cells (Fig. 2B). The relationship between normalized TEA-resistant currents and membrane potentials is shown in Fig. 5. The normalized values were determined by measuring the ratio of each TEA-resistant current to the control current at +50 mV. Although ANOVA did not demonstrate any signi¢cant di¡erences between apical and basal cells (P 6 0.05), the normalized TEA-resistant current was 0.70 in basal IHCs and 0.82 in apical IHCs.
4. Discussion Fig. 4. E¡ect of superfusion with TEA. Membrane currents from apical (left panels) and basal (right panels) IHCs are shown before (A) and during (B) superfusion with 25 mM TEA. The upper panels in A show a schematic representation of the voltage protocol. Horizontal dotted lines show the zero current level. (C) TEA-sensitive currents obtained by subtraction of responses in 25 mM TEA (B) from controls (A).
The potassium current in IHCs £ows through at least two types of membrane conductance: a fast (Ik;f ), TEAsensitive conductance and a slow (Ik;s ), TEA-resistant conductance. Although membrane conductance demonstrated no di¡erences between apical IHCs and basal IHCs (Fig. 1), TEA-sensitive Ik;f is larger in basal
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IHCs than in apical IHCs (Figs. 4 and 5). The fast and slow time constants in the rising phase of control currents were consistent with those of TEA-sensitive Ik;f and TEA-resistant Ik;s , respectively. There are no di¡erences in the value of the time constants between apical and basal IHCs, suggesting that the di¡erences in halfactivation time between apical and basal IHCs (Fig. 3) is due to di¡erences in the contribution of Ik;f and Ik;s in apical and basal IHCs, not due to kinetic di¡erences in Ik;f and/or Ik;s . Faster activation kinetics in basal IHCs might contribute to higher frequency signals in the basal cochlear turn. A similar tonotopical gradient along the cochlea with the e¡ect of TEA was observed in guinea-pig OHCs (Mammano and Ashmore, 1996). In OHCs, three different potassium conductances (Ik , IkðCaÞ , Ik;n ) were identi¢ed (Housley and Ashmore, 1992) and the extent of channel expression changes between the apex and base of the cochlea (Mammano and Ashmore, 1996; Nenov et al., 1997). The potassium current in basal OHCs showed signi¢cantly faster onset kinetics than that in apical OHCs. In chick basilar papilla (Rosenblatt et al., 1997; Navaratnam et al., 1997) and in turtle cochlea (Jones et al., 1999), there is a variation in the channel isoform of calcium-activated potassium conductance (IkðCaÞ ) along the length of the cochlea. Kþ currents largely determine the negative resting potential of the IHCs and contribute to the driving force for the mechano-electrical transducer at the apical pole of the cell. Opening of the transducer channels can be simulated by injecting a constant current through the patch-pipet (Kros and Crawford, 1990). This procedure generated fast depolarization with an associated spike (within 10 ms) due to activation of the fast potassium current (Ik;f ), and subsequent slow relaxation of the membrane potential due to the action of the slow potassium current (Ik;s ). Dominance of Ik;f in the basal IHCs might contribute to the fast depolarization generated by transducer channel openings. Ik;f was absent in IHCs in cochlear cultures from neonatal mice but was always found in cells isolated from mice after the onset of hearing about 11 days after birth (Kros et al., 1991). Ik;f was ¢rst observed in IHCs from the basal coil 11 days after birth and matured rapidly over the next 3 days (Kros et al., 1998). Before this time, mouse IHCs have only slow activated Kþ current (Ik:neo similar to Ik;s ) and demonstrate slow voltage responses and ¢re spontaneous and evoked action potentials. An action potential and subsequent dampened oscillation were observed in hair cells from lower vertebrate animals: frogs (Hudspeth and Lewis, 1988; Armstrong and Roberts, 1998), turtles (Art and Fettiplace, 1987), alligators (Fuchs and Evans, 1988) and chicks (Fuchs et al., 1988). In mammalian IHCs, during development of auditory responsiveness, Ik;f is ex-
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pressed, greatly accelerating the membrane time constant and preventing the action potential (Kros et al., 1991). Ik;f dominance in the basal IHCs suggests that functional development starts from the basal turn towards the apex. Morphologically, various structural maturation processes start near the basal end in a variº nggard, 1965; Ruben, 1967; Tanaka et ety of species (A al., 1979; Mu et al., 1997).
Acknowledgements This work was supported by a Grant-in-Aid for Scienti¢c Research 13671789 from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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Ruben, R.J., 1967. Development of the inner ear of the mouse: a radioautographic study of terminal mitosis. Acta Otolaryngol. (Stockh.) Suppl. 240, 44 pp. Tanaka, K., Sakai, N., Terayama, Y., 1979. Organ of Corti in the human fetus: scanning and transmission electron microscopic studies. Ann. Otol. Rhinol. Laryngol. 88, 749^758. Ulfendahl, M., Flock, A., 1998. In vitro studies of cochlea excitation. Curr. Opin. Neurobiol. 8, 475^479.
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