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Center for Neuroscience, Department of Otolaryngology, University of California at Davis, Davis, California 95616
Correspondence: Address reprint requests to Ebenezer N. Yamoah, University of California at Davis, Ctr. for Neuroscience, Dept. of Otolaryngology, 1544 Newton Court, Davis, CA 95616. Tel.: 530-754-6630; Fax: 530-54-7183/754-5046; E-mail: enyamoah{at}ucdavis.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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). For rapid and precise transmission of behaviorally significant stimuli, hair cells employ voltage-gated Ca2+ channels that are poised to mediate neurotransmitter release at the hair cell-afferent nerve synapse. The dihydropyridine-sensitive L-type voltage-gated calcium channel (L-VGCC) is the predominant channel in hair cells (Zidanic and Fuchs, 1995; Kollmar et al., 1997a,b; Rodriguez-Contreras and Yamoah, 2001). The channel activates following a mechanically induced generator potential and allows Ca2+ entry, thereby mediating neurotransmitter release (Roberts et al., 1990; Fuchs, 1996; Moser and Beutner, 2000; Spassova et al., 2001). The influx of Ca2+ also activates Ca2+-activated K+ currents, which underlie resonance of the membrane potential (Crawford and Fettiplace, 1981; Ashmore, 1983; Lewis and Hudspeth, 1983; Armstrong and Roberts, 1998; Smotherman and Narins, 1999; Fettiplace and Fuchs, 1999).
The VGCC in hair cells belongs to the L-type Ca2+ channel class (Kollmar et al., 1997a,b). However, within this class, the hair cell L-VGCC exhibits three distinct properties: 1) The channels activate at a potential which is 
20 mV more negative than the threshold of activation of similar cardiac dihydropyridine-sensitive Ca2+ currents (Ohmori, 1984; Tsien et al., 1988; Art and Fettiplace, 1987; Hudspeth and Lewis, 1988a,b; Zidanic and Fuchs, 1995). 2) The current activates and deactivates rapidly (time constants 0.5 ms). 3) The voltage- and current-dependent inactivation is slower than their cardiac counterparts (Lewis and Hudspeth, 1983; Fuchs et al., 1990; Zidanic and Fuchs, 1995). In a previous study, the properties of L-VGCC in hair cells were examined using varying concentrations of mostly Ba2+, and at times, Na+, as permeant ions to increase the magnitude of the whole-cell and single-channel currents (Art and Fettiplace, 1987; Art et al., 1995). The whole-cell Ca2+ current was evaluated with the implicit assumption that the total current was carried by the L-VGCC (Zidanic and Fuchs, 1995). Recently, several reports have demonstrated that although the predominant Ca2+ current is the L-VGCC, hair cells do express multiple Ca2+ channels that have distinct pharmacological and permeation properties (Su et al., 1995; Martini et al., 2000; Rodriguez-Contreras and Yamoah, 2001; Rodriguez-Contreras et al., 2002). Thus, single-channel recordings would be the most direct strategy to determine the properties of a single class of VGCC in hair cells.
Measurements of single Ca2+ channel currents have demonstrated that the unitary current magnitude and the conductance saturate at permeant ion concentrations above 20 mM, and thus, the sensitivity range of the channels lies between 1–20 mM (Yue and Marban, 1990; Church and Stanley, 1996; Rodriguez-Contreras et al., 2002). Because the voltage-dependent kinetics of ion channels are remarkably affected by the species and concentration of the permeant ions (Zhou and Jones, 1995; Hille, 2001), we hypothesized that the gating of L-VGCC in hair cells would be altered at different concentrations of Ca2+. By determining the kinetic properties of single L-type Ca2+ channels in hair cells, at concentrations within the dynamic range of the channel, the genuine features of the channel as Ca2+ transporters may then be elucidated.
In the present study, we determined the effects of permeant ions and their concentrations on the kinetic properties of L-VGCC in frog hair cells. We compared single-channel records of L-VGCC using varying concentrations of the charge carriers, Ba2+ and Ca2+. Our results show that L-VGCCs in hair cells have different modes of gating as revealed by the use of different charge carriers. In addition, surface charge screening effects of divalent cations alone were insufficient to account for the voltage-dependent shifts of activation of the channel using varying ion concentrations. We propose that ion permeation and gating of the channel may be linked.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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). Frogs were sacrificed, using a protocol approved by the University of California, Davis, Animal Research Services, and inner ears were removed quickly and placed in oxygenated low-Ca2+ frog Ringer solution (in mM): 110 NaCl, 2 KCl, 3 D-glucose, 0.1 CaCl2, and 5 n-2-hydroxyethylpiperazine-n'-2-2-ethanesulfonic acid (HEPES), pH 7.4 with NaOH. Macular tight junctions were disrupted by exposing the perilymphatic surface to 4 mM ethylene glycol-bis(ß-aminoethyl ether)-n,n,n',n'-tetraacetic acid (EGTA) for 15 min. After washing the preparation with fresh saline, the saccular macula was isolated and incubated in frog saline containing 50 µg/ml protease (type XXIV, Sigma Chemical, St. Louis, MO) for 20 min. The tissue was washed and transferred to frog saline containing 1 mg/ml bovine serum albumin (Sigma Chemical) and 2 mg/ml DNase I (Worthington, Lakewood, NJ) for 10 min. This procedure has been used previously (Chabbert, 1997) and differs from other more severe enzymatic treatments that can alter ionic conductances (Armstrong and Roberts, 1998). The otolithic membrane was excised and hair cells were dissociated from the macula using an eyelash. Hair cells were allowed to settle onto the bottom of the recording chamber (coated with the lectin concanavalin-A) for 20 min before patch-clamp recording.
Whole-cell current recordings
Whole-cell Ca2+ and Ba2+ currents were recorded from hair cells with length-to-apical diameter ratio (LAD) of 4 that expressed predominantly L-type currents (Rodriguez-Contreras and Yamoah, 2001). The tips of the electrodes were filled with a solution containing (in mM) 130 CsCl, 5 HEPES (pH 7.3 with CsOH). To gain electrical access to the cell, electrodes were backfilled with solution containing (in mM) 130 CsCl, 1 CaCl2, 5 HEPES, and amphotericin 200 µg/ml (pH 7.3 with CsOH; Rodriguez-Contreras and Yamoah, 2001). To ensure that recordings were in the perforated-patch mode instead of whole-cell mode, the backfilled solution of the patch electrode contained 1 mM Ca2+. A switch from the perforated-patch to whole-cell mode resulted in rapid cell death because of Ca2+ toxicity. Series resistance (5–10 M
) was compensated (nominally 70–80%). Liquid-junction potentials were recorded and adjusted as described previously (Rodriguez-Contreras and Yamoah, 2001). Ca2+ currents were amplified with an Axopatch 200B amplifier (Axon Instruments, Foster City, CA). Outward K+ currents were blocked with TEACl, 4-AP, and cesium (Cs+) ions. Bath solutions contained (in mM) 90 NaCl, 25 TEA-Cl, 5 4-AP, 1.6-5 CaCl2/BaCl2, 3 glucose, and 5 HEPES (pH 7.4). Current records were filtered at 2–5 kHz with a low-pass Bessel filter and digitized at 10 kHz with a Digidata interface (Axon Instruments) controlled by custom-written software. Data were stored and analyzed in a personal computer.
Single-channel recording
The patch-clamp technique in the cell-attached configuration was used (Hamill et al., 1981). Patch pipettes were made from quartz glass using a horizontal laser puller (P-2000, Sutter Instruments, Novato, CA). Patch electrodes were filled with divalent cation solutions (in mM): 5-70 Ca2+ and Ba2+, 20 tetraethyl ammonium chloride (TEACl), 5 4-aminopyridine (4-AP), 5 HEPES at pH 7.4 (adjusted with TEAOH). N-methyl-D-glucamine (NMG) was used to substitute for divalent cation and to maintain an osmolarity of 280 mosmol. To identify the L-type channel, the experiments were conducted in the presence of the agonist Bay K 8644 (Calbiochem, La Jolla, CA) in the bath solution, which was applied after control current traces have been recorded. Stock solutions of Bay K 8644 (100 mM) were made in DMSO, and a final concentration of 5 µM was used. The bath solution contained (in mM): 80 KCl, 3 D-glucose, 20 TEACl, 0.2 CaCl2, 5 4AP, 5 HEPES, and was adjusted to pH 7.4 with TEAOH, to shift the resting potential to 0 mV. Using the external recording solutions, the resting membrane potential of hair cells was -0.4 ± 2.1 mV (n = 32; Rodriguez-Contreras and Yamoah, 2001). All other chemicals were obtained from Sigma. Experiments were carried out at room temperature (21°C).
Single-channel currents were filtered at 1–2 kHz using a low-pass Bessel filter, sampled at 10–40 kHz, and stored in a personal computer. The channels were activated at a frequency of 0.2 Hz. Analysis was carried out using custom-written software, which was linked to Origin software (MicroCal, Northampton, MA). Leak and capacitative currents were corrected offline by fitting smooth templates to null traces and subtracting it from active traces. Open-close transitions were detected using half-height threshold analysis criteria. Idealized records were used to construct ensemble-averaged currents, open probability (Po), and to generate histograms for the distributions of open and closed time intervals. Po, voltage, and recording-time surface plots were generated using a built-in matrix function from Origin software (MicroCal). The time to reach the median of the cumulative first latency histograms was determined as described by Zei and Aldrich (1998). Unless indicated otherwise, all averaged and normalized data are presented as mean ± SD. The statistical significance of observed differences between groups of cells or between different parameters describing the properties of the currents were evaluated using a two-tailed Student's t-test; p values are presented in the text, and statistical significance was set at p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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). However, the gating properties of single L-type channel have been studied almost exclusively at saturating concentrations of Ba2+ or Ca2+ (Fox et al., 1987; Imredy and Yue, 1994; but see Smith et al., 1993). This section of the results compares the differences between the gating properties of the L-type channel using permeant ion concentrations at the dynamic (5 mM) and saturating (70 mM) levels. Fig. 1 shows characteristic cell-attached recordings of consecutive single L-VGCC current traces from isolated hair cells with 70 mM Ba2+ (Fig. 1 A) and Ca2+ (Fig. 1 B) as the charge carriers. Outward K+ channel currents were blocked with TEA and 4-AP, and the cell membrane potential was artificially shifted to 0 mV using high bath K+ (80 mM; see Methods). The L-type channel was distinguished from the non-L-type channel by differences in the pharmacology of the two channels (Rodriguez-Contreras and Yamoah, 2001). Single-channel activity was recorded for 4 min, and then the recording chamber was perfused with a high K+ solution containing 5 µM Bay K 8644. Only patches with channels that showed increased long openings and had conductances that were consistent with L-type channels are presented in this report (Rodriguez-Contreras and Yamoah, 2001; Rodriguez-Contreras et al., 2002). Patch depolarization from a holding potential of -70 mV to step potentials between -20 and +10 mV are shown. A close inspection of the Ba2+ current traces reveals short and long openings as well as brief and long closures. In addition, the gating of the channel included periods of quiescence. The patch contained only one channel, as there was no stacking of events. Furthermore, direct transitions from fast to slow kinetics with similar current amplitude, and vice versa, were observed in these recordings, indicating that these gating modes were derived from a single channel. The Ba2+ currents also showed frequent re-openings upon membrane repolarization, which were readily visible in the ensemble-averaged currents that showed well-resolved tail currents. The Ca2+ current had infrequent openings but the brief and long event characteristics persisted. However, when compared to Ba2+ currents, there was an increased frequency of closures (Fig. 1 B). The ensemble-averaged Ca2+ currents showed a slow decay, which is in sharp contrast to the sustained Ba2+ currents as shown in Fig. 1 A (also see Fig. 5). Furthermore, compared to Ba2+ single-channel fluctuations, the probability of openings significantly decreased when Ca2+ was the charge carrier (p < 0.05), and the long closures dominated the unitary Ca2+ current traces (Fig. 1 B).
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1.6 mM Ca2+, the signal-to-noise ratio of the unitary Ca2+ currents recorded under these conditions was low and not suitable for detailed kinetic analyses (Rodriguez-Contreras and Yamoah, 2001). Since the apparent dissociation constant (KD) of the channel is 5 mM (Rodriguez-Contreras et al., 2002), we crosschecked the properties of the Ca2+ channels using 1.6 versus 5 mM charge carrier. Shown in Fig. 2 are whole-cell Ca2+ (Fig. 2 A) and Ba2+ (Fig. 2 B) current traces measured using 1.6 and 5 mM of the charge carriers. The corresponding current-voltage relationship is shown in Fig. 2 C. The steady-state activation of whole-cell Ca2+ currents in hair cells, which expressed mostly the L-type current (see Methods), was also examined. The rightward shift in the steady-state activation curve, as external Ca2+ (1.6 mM) and Ba2+ (1.6 mM) were increased to 5 mM, was modest (Fig. 2, D and E), providing credible assurance that 5 mM Ca2+ or Ba2+ would be a reasonable concentration to determine the kinetics of the Ca2+ channel.
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Concentration-dependent shift in the activation of single L-type channel currents
The effect of charge carrier concentration on the activation of L-VGCC was assessed from the Po versus voltage plots shown in Fig. 6, where the solid lines represent fits to the Boltzmann function. Although the data were derived from Bay K 8644-treated channels, the open-probability of the channels was invariably less than 1 (Fig. 6, A and B). For comparisons of the activation curves, the data in Fig. 7, A and B, have been normalized. Whereas the activation threshold of the channel was -50 mV and the half-activation voltage (V1/2) was -30 mV for 70 mM Ba2+ currents, the activation voltage and V1/2 for Ca2+ currents were -45 and -10 mV, respectively. Moreover, the slope factor (k) for current carried by the two ions remained fairly constant (5 mV). The effects of reduced concentrations (5 mM) of permeant ions were modest compared to the predicted decline in the surface charge screening of divalent cations (Zhou and Jones, 1995). The V1/2 of Ca2+ current shifted by 16 mV in the hyperpolarizing direction (Fig. 7, B and C). By contrast, the negative potential shift of the V1/2 for 5 mM-Ba2+ current was fairly small (7 mV). In sharp contrast to the observed shifts in the activation curves, if one assumes that there were no binding of the permeant ions to the surface charge, a voltage shift of 25 mV is expected from a 70 to 5 mM drop in divalent ion concentration (Zhou and Jones, 1995; Zamponi and Snutch, 1996). Further, the slope factors (in VM-1) were 0.23 and 0.13 for Ca2+ and Ba2+, respectively, representing an approximate twofold difference between these ions (Fig. 7 C).
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). Taken together, these data suggest that ion permeation is linked tightly to the gating of the channel. Alternatively, if one of the permeant ion increases the tendency of the channel to enter into a nonconducting state, then the Po at any given step voltage is expected to be low for that ion, and should shift the activation curve to the right. Thus, Ca2+-dependent inactivation of the channel can potentially yield the present findings as well (Imredy and Yue, 1994).
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). The appearance of multiple null sweeps as shown in Figs. 1 and 3 ruled out the possibility that the L-VGCC exhibited a single closed state. The normalized cumulative first latency distributions was voltage-dependent as shown in Fig. 9 A, which suggested that the contribution of transitions before first openings to the voltage-dependence of channel activation, and the time to reach the median first latency should decrease with increasing membrane depolarization (Zei and Aldrich, 1998). For a channel that exhibits Ca2+-dependent inactivation (see Fig. 5), we expected that the first latency distribution would be faster in Ba2+ than in Ca2+. Fig. 9 B depicts the time course of the first latency distribution of the channel in 5 mM Ca2+ and Ba2+. Using charge carrier concentrations (in mM) of 4 Ca2+ and 1 Ba2+, the first latency distribution became brief when compared to Ca2+ alone, and shifted toward the distribution produced by 5 mM Ba2+ (Fig. 9, B and C). However, the median of the first latency distribution decayed exponentially with voltage as shown in Fig. 9 C. Furthermore, the leftward shift in the curve as the charge carrier was changed from 5 mM Ca2+ to Ba2+ indicated that at least one of the transition rates from the closed states before first opening may be altered by the permeant ion.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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7 mV more positive than Ba2+ currents. However, in mixtures of Ca2+ and Ba2+ ions (molar ratio, 4:1), L-VGCC currents had activation profiles similar to those of Ba2+ currents, indicating that the interactions between the charge carriers and the channel shape the kinetics of the currents. Overall, we propose that ion-channel interactions involving the permeation pathway are able to modify the gating properties of single L-VGCC in hair cells.
Several inferences that are independent of any model-based predictions of the gating and permeation properties of single L-VGCC in hair cells can be made from the present study. These include: 1), The activation and deactivation kinetics, as assessed from the ensemble-averaged currents, were exceedingly fast (<300 µs). This is in sharp contrast to the relatively slow kinetics of the L-type channels in cardiac myocytes and sensory neurons (Fox et al., 1987; Tsien et al., 1988). The activation time constants were similar to those obtained for L-type channels in mouse pancreatic ß-cells (Smith et al., 1993), and was consistent with the findings that hair cells and pancreatic ß-cells express the L-type channel variety of CaV1.3 (Kollmar et al., 1997a; Ertel et al., 2000; Rodriguez-Contreras and Yamoah 2001). 2), Multiple closed states of the channel are traversed from the resting to at least two kinetically discernible open states via voltage-dependent transitions, similar to those in K+ channels (Hess et al., 1986; Zagotta et al., 1994; Zei and Aldrich, 1998). Using an extension of the Hodgkin and Huxley (1952) formalism for ionic conductances, previous reports described the activation state variable (m) of the whole cell Ca2+ current in hair cells with a second-order function (m2; Hudspeth and Lewis, 1988b; Zidanic and Fuchs, 1995). This is consistent with the observation that L-type channels in hair cells have more than one closed state. Furthermore, a significant number of studies on the kinetics of single Ca2+ channel gating have been carried out using 110 mM Ba2+ as the charge carrier to increase the current amplitude. Since the proportion of channels in the different closed states and the dwell times of the open states vary from high to low concentrations of the permeant ions, such conditions do not reveal the true features of the channel as a Ca2+ transporter. 3), Because there was no marked shift in the activation curve for currents recorded in 1.6 versus 5 mM divalent cations, and because the single-channel currents were better resolved in the latter, 5 mM appears to be a suitable experimental concentration to study the kinetics of Ca2+ channels. Still, the present findings may fall short of revealing the physiological properties of the channel, since the current analyses were performed on a Bay K8644-modified channel. It would be interesting to use a gating modifier, such as kurtoxin, to determine whether these findings persist (Sidach and Mintz, 2002). 4), The L-VGCC in hair cells displays weak Ca2+-dependent inactivation. The modest inactivation and the increased occurrence of null sweeps necessitates the inclusion of an absorbing closed state, where the channel switches reluctantly toward openings (Imredy and Yue, 1994). Moreover, Ca2+ may favor some of the nonconducting state since Ba2+ currents do not exhibit pronounced inactivation. Previous studies on hair cell Ca2+ currents have shown the absence of a voltage-dependent inactivation mechanism (Zidanic and Fuchs, 1995), and a weak Ca2+-dependent inactivation process (Ohmori, 1984; Armstrong and Roberts, 1998; Smothermann and Narins, 1999; Platzer et al., 2000; Martini et al., 2000). Our present results demonstrate the presence of a Ca2+-dependent inactivation of the dihydropyridine-sensitive Ca2+ channels. The existence of a clear inactivation from the ensemble-averaged Ca2+ current traces generated from single-channel patches, implies that the inactivation observed at the whole-cell level may be a genuine biological phenomenon. Because the inactivation of the L-type Ca2+ current in hair cells is slow, long-duration stimuli may be required to study the phenomenon in detail. Thus, the apparent lack of strong inactivation in the whole-cell current traces (Fig. 2 A) as compared to the ensemble-averaged current (Fig. 5 B) may stem from varying stimulus protocols that were employed to elicit the current. Alternatively, the 
-shaped configuration of cell-attached patches may have dislodged the channels from synaptic proteins, which may impede the inactivation of the channel (Song et al., 2003). And 5), For the L-VGCC current in hair cells, ion permeation and gating may be linked closely. The molar ratio of Ba2+:Ca2+ mixtures that produced robust anomalous mole fraction effects in the channels' conductance (Rodriguez-Contreras et al., 2002) also showed striking alterations in the voltage-dependent activation of the current. Ca2+ was the predominant ion in a 4:1 ratio (Ca2+:Ba2+) solution, and thus, it is unlikely that Ca2+-dependent inactivation plays a dominant role in the shifts in first latency distribution curves. Therefore, Ca2+-dependent inactivation of the channel would be an unlikely explanation for the shift in first latency distribution as well as the activation curves described earlier, using the mole fraction of 4:1 (Ca2+:Ba2+). Instead, the data support the notion that permeation and gating are linked.
In a previous study of the kinetics of VGCC in hair cells, four properties of the channel were found (Zidanic and Fuchs, 1995): 1), low efficacy of current blockade by dihydropyridine antagonists; 2), low threshold of activation; 3), rapid kinetics of activation/deactivation; and 4), lack of current- and voltage-dependent inactivation. The cloning of a CaV1.3 subunit from the chick basilar papillae and the identification of alternative spliced variants in the same tissue (Kollmar et al., 1997a,b) may provide an explanation for the unique features of the L-type VGCC in hair cells. Moreover, studies of native channels at the whole cell and single-channel levels have shown that hair cells express different types of VGCC (Su et al., 1995; Martini et al., 2000; Rodriguez-Contreras and Yamoah, 2001; Rodriguez-Contreras et al., 2002). Whereas the expression of multiple Ca2+ channels may explain the incomplete block by dihydropyridines, we have demonstrated that the effects of voltage-dependent block of the dihydropyridines can also contribute toward the apparent weak sensitivity of hair-cell Ca2+ currents to the drug (Rodriguez-Contreras and Yamoah, 2001). Heterologous expression of CaV1.3 in mammalian cell lines has demonstrated that the ensuing current was less sensitive to nimodipine and nifedipine (Koschak et al., 2001; Safa et al., 2001; Song et al., 2003; Xu and Lipscombe, 2001). Although the expression of the 
1 subunit of L-type channel has provided traces of the properties of the L-type current in hair cells, the contribution of auxiliary subunits of the channel may be required to confer the native current phenotype (Song et al., 2003). In addition, emerging evidence suggest that synaptic proteins may also modulate the 1 subunit to confer native channel properties (Song et al., 2003).
Effects of charge carriers and concentrations on the properties of L-VGCC currents
The permeation and gating properties of VGCC depend on the species and concentration of ions (Hille, 2001). The screening of negative surface charges by divalent cations alters the surface potential, which in turn affects the gating properties of the channels (Hille, 2001; Zhou and Jones, 1995). This is consistent with the results shown in Figs. 6 and 7, since the V1/2 is proportional to the increase in ion concentration. Similar reports have been presented for L-type VGCC in pancreatic ß-cells (Smith et al., 1993), in other native Ca2+ channels (Hagiwara and Ohmori, 1982), and in heterologous systems expressing different VGCC subunits (Mangoni et al., 1997; McNaughton and Randall, 1997; Wakamori et al., 1998). Moreover, our findings demonstrate that the species of the permeant ion determine the magnitude of the shift in the voltage-dependent activation. If charge screening were the only factor determining the effects on gating properties, we expected that different kinetic components of L-VGCC currents would be affected equally by the increase in charge carrier concentration. However, this was not the case in the present study. An alternative explanation for the effects of charge carriers on the gating properties of L-type VGCC would be the role of ion-channel interactions. Increasing the concentration of charge carriers has a direct effect on the permeation properties of VGCC, as observed by the saturation of single-channel current amplitude or conductance (Guia et al., 2001; Church and Stanley, 1996; Smith et al., 1993; Zhou and Jones, 1995). We have shown previously that hair cell L-type channel conductance saturates with a KD 5 mM (Rodríguez-Contreras et al., 2002), a value consistent with various studies on unitary L-type currents (Church and Stanley, 1996; Smith et al., 1993). In addition, the properties of L-type VGCC in the concentration range of 2–5 mM are distinct compared to the properties of the channel at higher concentrations (Rodriguez-Contreras et al., 2002). The data presented in Fig. 8 suggest that interaction of ions and the channel may have direct effects on the kinetics of L-type VGCC, thereby affecting the activation of the unitary currents.
A nonpermeant ion, NMG, was used to substitute for Ba2+ and Ca2+ under 5 mM permeant ion conditions. Zhou and Jones (1995) showed that NMG may produce partial blockade of Ca2+ channels, which would provide an alternative explanation for the change in gating kinetics observed in our study. However, the maximal concentration of NMG used in this study is much smaller (65 mM) than the reported IC50 of NMG (300 mM; Zhou and Jones, 1995). The 5–10% block of the channel is insufficient to explain our findings. Thus, it is unlikely that the results obtained in this study can be attributed to the blocking effects of NMG.
Functional implications for hair cells
The findings that the probability of channel openings increases with decreasing concentrations of Ca2+ is reminiscent of previous data from hippocampal neurons, where membrane repolarization-induced openings of the L-type Ca2+ channel may confer Ca2+-dependent afterhyperpolarization (Thibault et al., 1993). In hair cells, this phenomenon would be essential for sustaining membrane oscillation and preventing excessive hyperpolarization induced by multiple outward K+ currents. A common feature among vertebrate hair cells is the presence of small amplitude voltage-gated Ca2+ currents (Art and Fettiplace, 1987; Fuchs et al., 1990; Lang and Correia, 1989; Lewis and Hudspeth, 1983; Masetto et al., 2000; Nakagawa et al., 1991; Ohmori, 1984; Platzer et al., 2000; Smotherman and Narins, 1999), wherein the kinetic properties have been evaluated previously at the whole cell level (Hudspeth and Lewis, 1988a; Ohmori, 1984; Zidanic and Fuchs, 1995). Implicit in such analysis is the assumption that there is a homogeneous population of VGCC, an assumption which is not supported by recent evidence showing the presence of multiple VGCC in vertebrate hair cells (Su et al., 1995; Martini et al., 2000; Rodriguez-Contreras and Yamoah, 2001). In addition, previous studies used relatively higher concentrations of charge carriers to measure the current (Ohmori, 1984; Zidanic and Fuchs, 1995). By comparing the kinetic profiles of Ca2+ and Ba2+ currents at the single-channel level, we obtained qualitative results that allow inference of the properties of L-VGCC as physiological Ca2+ transporters. First, the species of charge carrier determines the Po of the channels with the Po being larger for Ba2+ than for Ca2+. This suggests that the functional impact of Ca2+ domain formation in hair cells may be determined by the number of VGCCs that are in close proximity to each other (Roberts et al., 1990), or, by the relative distance of the channels to the Ca2+ binding sites (Hall et al., 1997). Second, crucial differences were observed between the time-dependent properties of Ba2+ currents and those of Ca2+ currents at 70 mM, clearly showing that the species of ion affects the kinetics of L-VGCC current. Moreover, since the L-VGCC in hair cells showed Ca2+-dependent inactivation, it raises the possibility that damped oscillations in the membrane potential may be derived from recovery kinetics of inactivation.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Submitted on October 8, 2002; accepted for publication January 17, 2003.
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REFERENCES |
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) current at 70 (A) and 5 mM (B). The Po was determined from consecutive depolarizing steps to different potentials as described in 
, molar ratio 4:1). The half-activation potentials (V1/2) and the slope factor, k, for the examples shown are: (Ba2+:Ca2+ ratio, 5:0) -34.8 mV and 4.5 mV (Ba2+:Ca2+ ratio, 0:5); -23.2 mV and 6.7 mV, and (Ba2+:Ca2+ ratio, 1:4) -32.9 mV and 7.0 mV. The data were obtained from three different cells. (B) Bar plots of the V1/2 and the molar ratio of Ba2+ and Ca2+ are shown, where the number of patches (n), are indicated in parentheses. The mean V1/2 were (Ba2+:Ca2+): 5:0 = -34.7 ± 2.9 mV; 1:4 = 34.7 ± 1.5 mV; and 0:5 = -26.4 ± 2.0 mV. The activation curve of single L-type channels in the mixture of Ba2+/Ca2+ at molar ratio of 1:4 is very similar to Ba2+-alone solution.