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Effect of cyclopiazonic acid on membrane currents in isolated inner hair cells from guinea-pig cochlea
Neuroscience Letters 323 (2002) 211–214 www.elsevier.com/locate/neulet
Effect of cyclopiazonic acid on membrane currents in isolated inner hair cells from guinea-pig cochlea Takashi Kimitsuki*, Takahiro Nakashima, Yuko Wada, Mitsuru Ohashi, Shizuo Komune Department of Otorhinolaryngology, Miyazaki Medical College, 5200 Kihara, Kiyotake-cho, Miyazaki 889-1692, Japan Received 17 December 2001; received in revised form 6 February 2002; accepted 8 February 2002
Abstract Cyclopiazonic acid (CPA) is a reticulum-like intracellular Ca 21 store depletory, which raises intracellular Ca 21 concentration. The effect of CPA on membrane currents in isolated inner hair cells (IHCs) from guinea-pig cochlea was investigated by the patch-clamp technique in the whole-cell configuration. Four out of eight IHCs showed an augmentation of the currents and the other four cells showed an inhibition of the currents by extracellular CPA application. The activation kinetics of outward currents were not changed by CPA. Three out of four IHCs obtained from the basal part of the cochlea demonstrated augmentation, whereas three out of four IHCs from the apical part demonstrated inhibition of the currents. This result suggests that Ca 21-activated currents were dominant in the basal IHCs of the cochlea. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Inner hair cell; Cochlea; Cyclopiazonic acid; Calcium-activated potassium current; Guinea-pig; Patch-clamp
The organ of Corti is composed of two clearly differentiated types of mechanoreceptive cells: the inner hair cells (IHCs); and the outer hair cells (OHCs). While IHCs are considered primary afferent transducers, performing a purely sensory function and transmitting auditory information to the central nerve system, OHCs are active mechanical vibrators, tuning the mechanical responses of the basilar membrane. Ca 21-activated K 1 currents (IK–Ca) have been observed in isolated mammalian OHCs [1,6] and OHCs in the intact organ of Corti [11]. In IHCs, Kros and Crawford [10] reported two identified potassium currents distinguishable by their pharmacology and their activation kinetics: the fast activating current, Ik,f; and the slow activating current, Ik,s. They concluded that no IK–Ca had existed in IHCs. However, a Ca 21 jump experiment using the photolable caged Ca 21 chelator, DM-nitrophen, confirmed the presence of IK–Ca in IHCs from guinea-pig cochlea [3]. We elevated the intracellular Ca 21 concentration by using cyclopiazonic acid (CPA). CPA is a reticulum-like intracellular Ca 21 store depletory, which passively depletes both * Corresponding author. Tel.: 181-985-85-2966; fax: 181-98585-7029. E-mail address: [email protected] (T. Kimitsuki).
caffeine- and inositol 1,4,5-triphosphate (IP3)-sensitive intracellular Ca 21 stores by specifically inhibiting the Ca 21-adenine triphosphatase (Ca 21-ATPase) store uptake mechanism [17]. Therefore, CPA elevated cytosolic Ca 21 concentration ([Ca 21]i) by inhibiting the Ca 21 re-uptake into the intracellular Ca 21 stores. We investigated whether the currents of IHCs were potentiated or inhibited by applying CPA extracellulary. The tonotopical differentiations along the cochlear axis were discussed. An adult albino guinea-pig (200–350 g) was 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 Ca 21-free external solution (142 mM NaCl, 4 mM KCl, 3 mM MgCl2, 2 mM NaH2PO4, 8 mM Na2HPO4, adjusted to pH 7.4 with NaOH). The otic capsule was opened, allowing removal of the organ of Corti attached to the modiolus. Basal IHCs were isolated by micro-dissecting 1–2 turns of the organ of Corti, and apical IHCs were obtained from turn 4. 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 out by superfusing with a standard external solution (142 mM NaCl, 4 mM KCl, 2 mM MgCl2, 1 mM CaCl2, 2 mM NaH2PO4, 8 mM Na2HPO4, adjusted to pH 7.4 with NaOH) at least 10 min before start-
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 2) 00 16 1- 1
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T. Kimitsuki et al. / Neuroscience Letters 323 (2002) 211–214
ing any experiments. The tight neck and the angle between the cuticular plate and the axis of the cell are the important landmarks for identifying IHCs from swelling basal OHCs. Membrane currents were measured by conventional whole-cell voltage-clamp recordings using an 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 outside diameter borosilicate glass (GC-1.2, Narishige, Tokyo) filled with an internal solution (144 mM KCl; 2 mM MgCl2; 1 mM NaH2PO4; 8 mM Na2HPO4; 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 MV in the bath and were coated with ski wax (TourDIA, DIAWax, Otaru, Japan) to minimize capacitance. The cell’s capacitance was 11.4 ^ 7.3 pF (mean ^ SD) and the residual series resistance was 15.6 ^ 7.1 MV (n ¼ 8). CPA (10 mM; C-1530, Sigma, USA) was applied under pressure using pipettes with a tip diameter of 2–4 mm positioned around 50 mm from the IHCs. Membrane currents were measured within 3 min after CPA application. Cells were continuously perfused with external saline and all experiments were performed at room temperature (20–25 8C). The experimental design was reviewed and approved (Accession number 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. Membrane currents in response to depolarizing voltage steps (130 mV) from a holding potential of 270 mV were recorded in IHCs and 10 mM CPA was applied under pressure using pipettes. A control current before CPA application, a current during CPA and a current after washing-out are superimposed in Fig. 1. The amplitude of the outward current was partially reduced by CPA and incompletely recovered by washing-out. The middle part of the rising
Fig. 1. Suppression of membrane currents in IHC by 10 mM CPA application. A bald trace shows the current during the CPA application. Upper trace shows the voltage protocol. Inset: activation of CPA current were shown in faster sweep to discern the time constant.
Fig. 2. Voltage-dependency of CPA suppression in IHCs. (A) Membrane currents in control (left part) and during CPA application (right part). The upper part shows a schematic representation of the voltage protocol. (B) Steady-state current–voltage relationships for control (W) and CPA application (X). The currents were measured at the end of the 10 ms test pulse as shown in A (W,X).
phase was fitted with a single-exponential curve in order to derive a time constant describing the activation rate of the outward current. The time constants from 270 mV to 130 mV of the control current, CPA current and washingout current were 0.41 ^ 0.09 (mean ^ SD, n ¼ 6), 0.35 ^ 0.09 (mean ^ SD, n ¼ 6) and 0.38 ^ 0.07 (mean ^ SD, n ¼ 4) ms, respectively, suggesting that activation kinetics were not changed by CPA. Four out of eight IHCs showed an inhibition of the currents by CPA. Fig. 2 shows the CPA suppression of currents on various depolarizing voltage steps from a holding potential of 270 mV. The control current showed a pronounced outward rectification in current–voltage relation (Fig. 2B). Outward rectification is compatible with the properties of potassium current in previous reports [3,10]. CPA suppression was stronger in the outward current than in the inward current. In seven cells, the amplitudes of the inward current at 2130 mV in control and CPA solution were 0.99 ^ 0.38 and 0.99 ^ 0.59 nA (mean ^ SD), respectively. On the other hand, Fig. 3 shows a CPA augmentation which was also stronger in the outward current than in the inward current. Four out of eight IHCs demonstrated an augmentation by CPA. IHCs were isolated separately from the apical and the basal part of the cochlea and the effect of CPA was compared. The ratio of current amplitude at 130 mV during CPA application to that of the control current was calculated and plotted individually in Fig. 4. The ratio was 1.1 ^ 0.15 (n ¼ 4) in the basal part and 0.97 ^ 0.20 (n ¼ 4) in the
T. Kimitsuki et al. / Neuroscience Letters 323 (2002) 211–214
Fig. 3. Voltage-dependency of CPA augmentation in IHCs. (A) Membrane currents in control (left part) and during CPA application (right part). (B) Steady-state current–voltage relationships for control (W) and CPA application (X), measured at the end of the test pulse as shown in A (W,X).
apical part. Although analysis of variance did not demonstrate any significant differences between apical and basal cells (P , 0:05), three cells out of four showed augmentation in the basal site and three cells out of four showed inhibition in the apical site. CPA, an indole tetramic acid metabolite of Aspergillus and Penicillium, inhibits the Ca 21-stimulated ATPase and Ca 21 transport activity of sarcoplasmic reticulum [5]. CPA depletes both caffeine- and IP3-sensitive intracellular stores, by specifically inhibiting the Ca 21-ATPase store re-uptake mechanism without effecting plasma membrane ATPase [17]. Basal [Ca 21]i increase by CPA has been observed by using Ca 21 indicator (fura-2, indo-1) in smooth muscle [4,18,20] and astrocytes [2]. CPA caused a transient increase of [Ca 21]i and then decreased within 200 s to a plateau level [4]. We measured the membrane currents within 3 min after CPA application, therefore [Ca 21]i should have been elevated when we measured the currents. Four out of eight cells showed an augmentation of the currents by the extracellular CPA application. No Ca 21-activated conductance had been observed in the first report of K 1 currents of IHCs by Kros and Crawford [10], but they did not use Ca 21 jump experiments. They concluded that the absence of Ca 21-activated conductance in IHCs was from the lack of reduction of the outward currents in free Ca 21 extracellular solutions. However, Dulon et al. demonstrated the presence of IK–Ca in IHCs by a Ca 21 jump experiment using the photolable caged Ca 21 chelator, DM-nitrophen [3]. In our experiment, Ca 21-potentiated K 1 currents are dominant in the basal part of cochlea (Fig. 4). The fast
213
activating current (Ik,f) in IHCs, whose kinetics are similar to those of IK–Ca, is dominant in the basal part of cochlea (paper submitting). In OHCs, the extent of three different potassium channels’ (Ik, IK–Ca, Ik,n) expression changes between the apex and base [6,11,13]. Although IK–Ca in OHCs does not contribute greatly to the total currents [8], Ik,n, which is more sensitive to [Ca 21]i than Ik, is dominant in the basal turn [11]. In the chick’s basilar papilla (cochlea) [12,16] and the turtle’s basilar papilla [9], there is a variation in the channel isoform of Ca 21-activated K 1 conductance along the length of the basilar papilla. The magnitude of Ca 21-activated K 1 conductance increases from apex to base [14]. However, Ca 21-activated K 1 channels were twofold less Ca 21-sensitive in the basal turn than in apical cells in turtle basilar papilla [15]. Four out of eight cells showed an inhibition of the currents by the extracellular CPA application. In smooth muscle, reduction of IK–Ca by CPA has been reported [7,19]. They concluded that CPA decreases Ca 21-uptake by the inhibition of the Ca 21 pump in the sarcoplasmic reticulum, and the subsequent Ca 21 release from those storage sites thereby reduces the amplitude of IK–Ca. Suzuki et al. [19] reported a biphasic CPA response on IK–Ca by the low concentration application (1–3 mM), a potentiation of IK–Ca prior to the CPA-induced suppression. We observed different CPA responses to IK–Ca between apical and basal IHCs. These differentiations might be related to the biphasic effect observed in smooth muscle [19]. IK–Ca is responsible for the afterhyperpolarization which reset the membrane potential at the resting level after depolarized stimulation. The dominance of IK–Ca in the basal part of the cochlea is favorable to the fast resetting of membrane potential preparing for the next fast frequency stimulation.
Fig. 4. CPA effects on basal and apical IHCs. The ratio of current amplitude during CPA application to that of the control was calculated and plotted separately in basal (O, n ¼ 4) and apical (X, n ¼ 4) IHCs.
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This study was supported by a Grant-in-Aid for Scientific Research 13671789 from the Ministry of Education, Culture, Sports, Science and Technology of Japan. [1] Ashmore, J.F. and Meech, R.W., Ionic basis of membrane potential in guinea pig outer hair cells, Nature, 322 (1986) 368–371. [2] Carmignoto, G., Pasti, L. and Pozzan, T., On the role of voltage-dependent calcium channels in calcium signaling of astrocytes in situ, J. Neurosci., 18 (1998) 4637–4645. [3] Dulon, D., Sugasawa, M., Blanchet, C. and Erostegui, C., Direct measurement of Ca 21-activated K 1 currents in inner hair cells of the guinea-pig cochlea using photolable Ca 21 chelators, Pflug. Arch., 430 (1995) 365–373. [4] Ethier, M.F., Yamaguchi, H. and Madison, J.M., Effects of cyclopiazonic acid on cytosolic calcium in bovine airway smooth muscle cells, Am. J. Physiol. Lung Cell Mol. Physiol., 281 (2001) L126–L133. [5] Goeger, D.E., Riley, R.T., Dorner, J.W. and Cole, R.J., Cyclopiazonic acid inhibition of the Ca 21-transport ATPase in rat skeletal muscle sarcoplasmic reticulum vesicles, Biochem. Pharmacol., 37 (1988) 978–981. [6] Housley, G.D. and Ashmore, J.F., Ionic currents of outer hair cells isolated from the guinea-pig cochlea, J. Physiol., 448 (1992) 73–98. [7] Imaizumi, Y., Torii, Y., Ohi, Y., Nagano, N., Atsuki, K., Yamamura, H., Muraki, K., Watanabe, M. and Bolton, T.B., Ca 21 images and K 1 current during depolarization in smooth muscle cells of the guinea-pig vas deferens and urinary bladder, J. Physiol., 510(3) (1998) 705–719. [8] Jagger, D.J. and Ashmore, J.F., Regulation of ionic currents by protein kinase A and intracellular calcium in outer hair cells isolated from the guinea-pig cochlea, Pflug. Arch., 437 (1999) 409–416. [9] Jones, E.M., Geray-Keller, M. and Fettiplace, R., The role of Ca 21-activated K 1 channel spliced variants in the tonotopic organization of the turtle cochlea, J. Physiol., 518(3) (1999) 653–665. [10] Kros, C.J. and Crawford, A.C., Potassium currents in inner
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hair cells isolated from the guinea-pig cochlea, J. Physiol., 421 (1990) 263–291. Mammano, F. and Ashmore, J.F., Differentiation of outer hair cell potassium currents in the isolated cochlea of the guinea-pig, J. Physiol., 496(3) (1996) 639–646. Navaratnam, D.S., Bell, T.J., Tu, T.D., Cohen, E.L. and Oberholtzer, C.J., Differential distribution of Ca 21-activated K 1 channel splice variants among hair cells along the tonotopic axis of the chick cochlea, Neuron, 19 (1997) 1077–1085. Nenov, A.P., Norris, C. and Bobbin, R.P., Outwardly rectifying currents in guinea pig outer hair cells, Hear. Res., 105 (1997) 146–158. Pantelias, A.A., Monsivais, P. and Rubel, E.W., Tonotopic map of potassium currents in chick auditory hair cells using an intact basilar papilla, Hear. Res., 156 (2001) 81–94. Ricci, A.J., Geray-Keller, M. and Fettiplace, R., Tonotopic variations of calcium signaling in turtle auditory hair cells, J. Physiol., 524(2) (2000) 423–436. Rosenblatt, K.P., Sun, Z.-P., Heller, S. and Hudspeth, A.J., Distribution of Ca 21-activated K 1 channel isoforms along the tonotopic gradient of the chicken’s cochlea, Neuron, 19 (1997) 1061–1075. Seidler, N.W., Jona, I., Vegh, M. and Martonosi, A., Cyclopiazonic acid is a specific inhibitor of the Ca 21-activated ATPase of sarcoplasmic reticulum, J. Biol. Chem., 264 (1989) 17816–17823. Shmigol, A.V., Eisner, D.A. and Wray, S., Properties of voltage-activated [Ca 21]i transients in single smooth muscle cells isolated from pregnant rat uterus, J. Physiol., 511(3) (1998) 803–811. Suzuki, M., Muraki, K., Imaizumi, Y. and Watanabe, M., Cyclopiazonic acid, an inhibitor of the sarcoplasmic reticulum Ca 21 pump, reduces Ca 21-dependent K 1 currents in guinea-pig smooth muscle cells, Br. J. Pharmacol., 107 (1992) 134–140. Tao, L., Huang, Y. and Bourreau, J.P., Control of the mode of excitation–contraction coupling by Ca 21 stores in bovine trachealis muscle, Am. J. Physiol. Lung Cell Mol. Physiol., 279 (2000) L722–L732.
Effect of cyclopiazonic acid on membrane currents in isolated inner hair cells from guinea-pig cochlea Takashi Kimitsuki*, Takahiro Nakashima, Yuko Wada, Mitsuru Ohashi, Shizuo Komune Department of Otorhinolaryngology, Miyazaki Medical College, 5200 Kihara, Kiyotake-cho, Miyazaki 889-1692, Japan Received 17 December 2001; received in revised form 6 February 2002; accepted 8 February 2002
Abstract Cyclopiazonic acid (CPA) is a reticulum-like intracellular Ca 21 store depletory, which raises intracellular Ca 21 concentration. The effect of CPA on membrane currents in isolated inner hair cells (IHCs) from guinea-pig cochlea was investigated by the patch-clamp technique in the whole-cell configuration. Four out of eight IHCs showed an augmentation of the currents and the other four cells showed an inhibition of the currents by extracellular CPA application. The activation kinetics of outward currents were not changed by CPA. Three out of four IHCs obtained from the basal part of the cochlea demonstrated augmentation, whereas three out of four IHCs from the apical part demonstrated inhibition of the currents. This result suggests that Ca 21-activated currents were dominant in the basal IHCs of the cochlea. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Inner hair cell; Cochlea; Cyclopiazonic acid; Calcium-activated potassium current; Guinea-pig; Patch-clamp
The organ of Corti is composed of two clearly differentiated types of mechanoreceptive cells: the inner hair cells (IHCs); and the outer hair cells (OHCs). While IHCs are considered primary afferent transducers, performing a purely sensory function and transmitting auditory information to the central nerve system, OHCs are active mechanical vibrators, tuning the mechanical responses of the basilar membrane. Ca 21-activated K 1 currents (IK–Ca) have been observed in isolated mammalian OHCs [1,6] and OHCs in the intact organ of Corti [11]. In IHCs, Kros and Crawford [10] reported two identified potassium currents distinguishable by their pharmacology and their activation kinetics: the fast activating current, Ik,f; and the slow activating current, Ik,s. They concluded that no IK–Ca had existed in IHCs. However, a Ca 21 jump experiment using the photolable caged Ca 21 chelator, DM-nitrophen, confirmed the presence of IK–Ca in IHCs from guinea-pig cochlea [3]. We elevated the intracellular Ca 21 concentration by using cyclopiazonic acid (CPA). CPA is a reticulum-like intracellular Ca 21 store depletory, which passively depletes both * Corresponding author. Tel.: 181-985-85-2966; fax: 181-98585-7029. E-mail address: [email protected] (T. Kimitsuki).
caffeine- and inositol 1,4,5-triphosphate (IP3)-sensitive intracellular Ca 21 stores by specifically inhibiting the Ca 21-adenine triphosphatase (Ca 21-ATPase) store uptake mechanism [17]. Therefore, CPA elevated cytosolic Ca 21 concentration ([Ca 21]i) by inhibiting the Ca 21 re-uptake into the intracellular Ca 21 stores. We investigated whether the currents of IHCs were potentiated or inhibited by applying CPA extracellulary. The tonotopical differentiations along the cochlear axis were discussed. An adult albino guinea-pig (200–350 g) was 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 Ca 21-free external solution (142 mM NaCl, 4 mM KCl, 3 mM MgCl2, 2 mM NaH2PO4, 8 mM Na2HPO4, adjusted to pH 7.4 with NaOH). The otic capsule was opened, allowing removal of the organ of Corti attached to the modiolus. Basal IHCs were isolated by micro-dissecting 1–2 turns of the organ of Corti, and apical IHCs were obtained from turn 4. 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 out by superfusing with a standard external solution (142 mM NaCl, 4 mM KCl, 2 mM MgCl2, 1 mM CaCl2, 2 mM NaH2PO4, 8 mM Na2HPO4, adjusted to pH 7.4 with NaOH) at least 10 min before start-
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 2) 00 16 1- 1
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T. Kimitsuki et al. / Neuroscience Letters 323 (2002) 211–214
ing any experiments. The tight neck and the angle between the cuticular plate and the axis of the cell are the important landmarks for identifying IHCs from swelling basal OHCs. Membrane currents were measured by conventional whole-cell voltage-clamp recordings using an 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 outside diameter borosilicate glass (GC-1.2, Narishige, Tokyo) filled with an internal solution (144 mM KCl; 2 mM MgCl2; 1 mM NaH2PO4; 8 mM Na2HPO4; 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 MV in the bath and were coated with ski wax (TourDIA, DIAWax, Otaru, Japan) to minimize capacitance. The cell’s capacitance was 11.4 ^ 7.3 pF (mean ^ SD) and the residual series resistance was 15.6 ^ 7.1 MV (n ¼ 8). CPA (10 mM; C-1530, Sigma, USA) was applied under pressure using pipettes with a tip diameter of 2–4 mm positioned around 50 mm from the IHCs. Membrane currents were measured within 3 min after CPA application. Cells were continuously perfused with external saline and all experiments were performed at room temperature (20–25 8C). The experimental design was reviewed and approved (Accession number 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. Membrane currents in response to depolarizing voltage steps (130 mV) from a holding potential of 270 mV were recorded in IHCs and 10 mM CPA was applied under pressure using pipettes. A control current before CPA application, a current during CPA and a current after washing-out are superimposed in Fig. 1. The amplitude of the outward current was partially reduced by CPA and incompletely recovered by washing-out. The middle part of the rising
Fig. 1. Suppression of membrane currents in IHC by 10 mM CPA application. A bald trace shows the current during the CPA application. Upper trace shows the voltage protocol. Inset: activation of CPA current were shown in faster sweep to discern the time constant.
Fig. 2. Voltage-dependency of CPA suppression in IHCs. (A) Membrane currents in control (left part) and during CPA application (right part). The upper part shows a schematic representation of the voltage protocol. (B) Steady-state current–voltage relationships for control (W) and CPA application (X). The currents were measured at the end of the 10 ms test pulse as shown in A (W,X).
phase was fitted with a single-exponential curve in order to derive a time constant describing the activation rate of the outward current. The time constants from 270 mV to 130 mV of the control current, CPA current and washingout current were 0.41 ^ 0.09 (mean ^ SD, n ¼ 6), 0.35 ^ 0.09 (mean ^ SD, n ¼ 6) and 0.38 ^ 0.07 (mean ^ SD, n ¼ 4) ms, respectively, suggesting that activation kinetics were not changed by CPA. Four out of eight IHCs showed an inhibition of the currents by CPA. Fig. 2 shows the CPA suppression of currents on various depolarizing voltage steps from a holding potential of 270 mV. The control current showed a pronounced outward rectification in current–voltage relation (Fig. 2B). Outward rectification is compatible with the properties of potassium current in previous reports [3,10]. CPA suppression was stronger in the outward current than in the inward current. In seven cells, the amplitudes of the inward current at 2130 mV in control and CPA solution were 0.99 ^ 0.38 and 0.99 ^ 0.59 nA (mean ^ SD), respectively. On the other hand, Fig. 3 shows a CPA augmentation which was also stronger in the outward current than in the inward current. Four out of eight IHCs demonstrated an augmentation by CPA. IHCs were isolated separately from the apical and the basal part of the cochlea and the effect of CPA was compared. The ratio of current amplitude at 130 mV during CPA application to that of the control current was calculated and plotted individually in Fig. 4. The ratio was 1.1 ^ 0.15 (n ¼ 4) in the basal part and 0.97 ^ 0.20 (n ¼ 4) in the
T. Kimitsuki et al. / Neuroscience Letters 323 (2002) 211–214
Fig. 3. Voltage-dependency of CPA augmentation in IHCs. (A) Membrane currents in control (left part) and during CPA application (right part). (B) Steady-state current–voltage relationships for control (W) and CPA application (X), measured at the end of the test pulse as shown in A (W,X).
apical part. Although analysis of variance did not demonstrate any significant differences between apical and basal cells (P , 0:05), three cells out of four showed augmentation in the basal site and three cells out of four showed inhibition in the apical site. CPA, an indole tetramic acid metabolite of Aspergillus and Penicillium, inhibits the Ca 21-stimulated ATPase and Ca 21 transport activity of sarcoplasmic reticulum [5]. CPA depletes both caffeine- and IP3-sensitive intracellular stores, by specifically inhibiting the Ca 21-ATPase store re-uptake mechanism without effecting plasma membrane ATPase [17]. Basal [Ca 21]i increase by CPA has been observed by using Ca 21 indicator (fura-2, indo-1) in smooth muscle [4,18,20] and astrocytes [2]. CPA caused a transient increase of [Ca 21]i and then decreased within 200 s to a plateau level [4]. We measured the membrane currents within 3 min after CPA application, therefore [Ca 21]i should have been elevated when we measured the currents. Four out of eight cells showed an augmentation of the currents by the extracellular CPA application. No Ca 21-activated conductance had been observed in the first report of K 1 currents of IHCs by Kros and Crawford [10], but they did not use Ca 21 jump experiments. They concluded that the absence of Ca 21-activated conductance in IHCs was from the lack of reduction of the outward currents in free Ca 21 extracellular solutions. However, Dulon et al. demonstrated the presence of IK–Ca in IHCs by a Ca 21 jump experiment using the photolable caged Ca 21 chelator, DM-nitrophen [3]. In our experiment, Ca 21-potentiated K 1 currents are dominant in the basal part of cochlea (Fig. 4). The fast
213
activating current (Ik,f) in IHCs, whose kinetics are similar to those of IK–Ca, is dominant in the basal part of cochlea (paper submitting). In OHCs, the extent of three different potassium channels’ (Ik, IK–Ca, Ik,n) expression changes between the apex and base [6,11,13]. Although IK–Ca in OHCs does not contribute greatly to the total currents [8], Ik,n, which is more sensitive to [Ca 21]i than Ik, is dominant in the basal turn [11]. In the chick’s basilar papilla (cochlea) [12,16] and the turtle’s basilar papilla [9], there is a variation in the channel isoform of Ca 21-activated K 1 conductance along the length of the basilar papilla. The magnitude of Ca 21-activated K 1 conductance increases from apex to base [14]. However, Ca 21-activated K 1 channels were twofold less Ca 21-sensitive in the basal turn than in apical cells in turtle basilar papilla [15]. Four out of eight cells showed an inhibition of the currents by the extracellular CPA application. In smooth muscle, reduction of IK–Ca by CPA has been reported [7,19]. They concluded that CPA decreases Ca 21-uptake by the inhibition of the Ca 21 pump in the sarcoplasmic reticulum, and the subsequent Ca 21 release from those storage sites thereby reduces the amplitude of IK–Ca. Suzuki et al. [19] reported a biphasic CPA response on IK–Ca by the low concentration application (1–3 mM), a potentiation of IK–Ca prior to the CPA-induced suppression. We observed different CPA responses to IK–Ca between apical and basal IHCs. These differentiations might be related to the biphasic effect observed in smooth muscle [19]. IK–Ca is responsible for the afterhyperpolarization which reset the membrane potential at the resting level after depolarized stimulation. The dominance of IK–Ca in the basal part of the cochlea is favorable to the fast resetting of membrane potential preparing for the next fast frequency stimulation.
Fig. 4. CPA effects on basal and apical IHCs. The ratio of current amplitude during CPA application to that of the control was calculated and plotted separately in basal (O, n ¼ 4) and apical (X, n ¼ 4) IHCs.
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