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Dipartimento di Scienze Fisiologiche-Farmacologiche Cellulari-Molecolari, Sez. di Fisiologia Generale e Biofisica Cellulare, Università di Pavia, Pavia, Italy
Submitted 14 December 2005; accepted in final form 27 April 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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-agatoxin IVA (2 µM) and -conotoxin GVIA (3.2 µM) were added to the pipette solution. Our results show that types I and II hair cells express L-type Ca channels with similar properties. Moreover, they suggest that in vivo Ca2+ influx might occur at membrane voltages more negative than –60 mV. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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). Moreover, type I hair cells basolateral region is enveloped by a single calyx afferent nervous terminal, whereas type II hair cells are contacted by numerous bouton-like afferent and efferent terminals. Finally, type I hair cells alone express a low-voltage-activated outward rectifying K+ current, named IK,L, which is responsible for the low cell membrane input resistance in the voltage range close to the resting membrane voltage found in vitro compared with type II hair cells (Rennie and Correia 1994; Rüsch and Eatock 1996).
It is commonly accepted that during head rotations the stereocilia, which are located at the apex of the hair cells, are bent as a consequence of the cupula deflection. Bending of the stereocilia results in an increase of the open probability of mechano-sensory ion channels located at the tip of the stereocilia (Hudspeth 1982). The resulting inflow of K+ ions would depolarize the hair cell. The final shape of the receptor potential is also dependent on the specific array of basolateral ion channels expressed; among these, voltage-dependent Ca channels are responsible for afferent transmitter release (Moser and Beutner 2000; Parsons et al. 1994).
Whole cell recordings have shown that, in both type I and type II hair cells, Ca2+ inflow occurs mainly through L-type Ca channels (Almanza et al. 2003; Bao et al. 2003), which activate close to –60 mV, and show negligible inactivation when Ba2+ is the permeant ion. Aside from L-channels, the presence of additional Ca channel types has been reported in type II hair cells (Dou et al. 2004; Martini et al. 2000), whose properties, however, have not yet been characterized.
As far as single Ca channel properties are concerned, to date recordings have been performed in type II hair cells only. The results, obtained by using the cell-attached configuration, have shown the predominant expression of L-type Ca channels, plus that of an "N-like" Ca channel type (Rodriguez-Contreras and Yamoah 2001).
In the present paper we have investigated the single Ca channel properties in both type I and type II hair cells from the chick embryo semicircular canal. We found that the two hair cell types express L-type Ca channels with similar properties. Moreover, our results suggest that in physiological conditions these channels might be already active at voltages that encompass the resting membrane potential of type I and type II hair cells.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Slice preparation
Detailed procedures for semicircular canal dissection and slice preparation have been reported previously (Masetto et al. 2003). Briefly, fertilized chicken eggs of the Cobb variety were obtained at a local supplier and incubated at 38.3°C. Once removed from the eggs, embryos were decapitated and semicircular canals were dissected out. The ampullae were incubated in D-MEM (No. 31600–026, GIBCO BRL-Life Technologies), supplemented with 1.5% newborn calf serum (No. N-4637, Sigma, St. Louis, MO), NaHCO3 24 mM, piperazine- N,N'-bis (2-ethanesulfonic acid) 15 mM (Sigma), titrated to pH 7.4 with NaOH, and carboxygenated (95% O2-5% CO2) in a humidity-saturated chamber at 37°C. Osmolality was adjusted to 
320 mOsm. After an incubation period of 2–6 h, the organ was removed from the culture medium and embedded in 4% agar wt/vol (Sigma) in a slicing solution (see Table 1).
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Morphological criteria
Recordings were made from hair cells in selected regions, or zones, of the neuroepithelium of the two vertical (posterior and anterior) semicircular canals. The preparation consisted of a slice cut parallel to the longitudinal axis of the crista and passing in between the two eminentiae cruciatae. (see Fig. 1 in Masetto et al. 2003). This preparation allows recording from hair cells located at different distances from the planum semilunatum. According to previous nomenclature (Masetto et al. 2000, 2003), the two most peripheral regions of the sensory epithelium in the slice are called zone 1, each contacting one planum semilunatum (PS), which is a nonsensory epithelium; zone 3 is the most central region (which in the intact crista ampullaris would be flanked by the two eminentiae cruciatae); zone 2 is a region intermediate between zones 1 and 3. To increase our chances to record from type I hair cells, for which no data on single Ca channels are available in the literature, most of the recordings were made from zone 2 because in birds, it contains the highest ratio of type I versus type II hair cells (Kevetter et al. 2000; Masetto et al. 2000). Type I hair cells were distinguished from type II hair cells by their amphora shape, characterized by a very constricted region (neck) just below the dense apical plate bearing the stereocilia (see for example Fig. 1D in Masetto et al. 2003 and Fig. 1B in Masetto et al. 2005). The procedure for sealing type I hair cells was rather complicated because calyx remnants had often to be pulled out to reach the cell membrane. This could require more than one attempt.
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; Rüsch and Eatock 1996). In a few hair cells, we could confirm the hair cell identity electrophysiologically by achieving the ruptured whole cell configuration following cell-attached recording. Electrical recordings
Patch pipettes were pulled from borosilicate glass pipettes (Hilgenberg GmbH, Malsfeld, Germany), tips were fire-polished, and partially coated with silicone elastomer (Sylgard; Dow Corning 184, Midland, MI). For cell-attached experiments, micropipettes had a resistance in the bath of 5–10 M
when filled with Extra_Ba_70 or Extra_Ba_5 (see Table 1). To control the patch transmembrane potential, we zeroed the cell membrane voltage by perfusing the slice with a high_K+ extracellular solution (Extra_high_K, see Table 1). To verify that this was indeed occurring, in a few preliminary experiments, we measured the hair cell membrane voltage in current-clamp mode in ruptured whole cell configuration before and during perfusion with Extra_high_K; in these experiments, the pipette solution was a standard intracellular solution (Intra_K, Table 1), and micropipettes had a resistance in the bath of 2–3 M. We found that after 3 min of perfusion, the cell membrane resting potential was steadily shifted from –70, –60 mV to 0 mV. Therefore cell-attached recordings were started after 
3 min of perfusion with the Extra_high_K. In a few cells, following cell-attached recording, we were able to achieve the whole cell configuration. The value of the resting membrane potential, read in current-clamp mode during the first seconds in whole cell, i.e., before the cell content was washed out by the pipette solution, confirmed that during cell-attached recording the cell membrane potential was zeroed by the Extra_high_K solution. Furthermore, recording in whole cell voltage-clamp mode allowed us to confirm hair cell type I versus type II identity on the basis of the total ionic current expressed. Unfortunately, a good seal and cell access were only occasionally achieved during the passage from cell-attached to whole cell because of the small pipette tip; on the other hand these small pipette tips produced much better seals (>20 G) and lower noise than larger pipette tips. As a routine, the crista slice was changed after each recording, since perfusion with Extra_high_K accelerated deterioration of the preparation, likely as a consequence of the prolonged cells' depolarization. All recordings were made at room temperature (22–24°C).
In cell-attached experiments, BaCl2 was used instead of CaCl2 to emphasize currents through Ca channels and to block K channels. Pipette solutions included TEACl, 4-aminopyridine, and CsCl to block K channels (see Table 1), niflumic acid (Sigma) 50 µM to block chloride channels, and (±)-Bay K 8644 (5 µM) (Sigma) to better resolve L-channel openings, otherwise hardly detected (Hess et al. 1984). In a few experiments, Extra_Ba_5 included the P/Q, and N type Ca channels blockers -agatoxin IVA (2 µM) and -conotoxin GVIA (3.2 µM) (Alomone Laboratories).
The patch-clamp amplifier was an Axopatch 200B (Axon Instruments, Foster City, CA). The amplifier's filter bandwidth was set at 2 kHz. Current and voltage were measured and controlled through a DigiData 1322 interface (AD/DA converter; Axon Instruments) connected to a personal computer running pClamp software (V 9.2, Axon Instruments). Data were digitized at 10 times the filter rate (i.e., 20 kHz), and analysis was limited to events lasting >360 µs, i.e., longer than twice the dead time (166 µs) (see Colquhoun and Hawkes 1995). Openings were long enough and well resolved. Single-channel activity was recorded by applying 500-ms voltage pulses in the range from –80 to 20 mV, from –70 mV holding potential (Vh). Consecutive voltage steps were applied every 550 ms; each recording session consisted of 50 or 100 pulses.
To minimize voltage errors due to liquid junction potentials between the pipette solution and the bath solution, prior to each experiment session, the offset potential was electronically zeroed with the patch pipette tip immersed in the recording chamber containing the same solution as the pipette. The bath chamber was then rinsed and filled with standard extracellular solution (See Extra_std in Table 1). The resulting offset between the pipette solution and the bath solution was not adjusted because it would disappear following cell-attached establishment (Neher 1992). Usually the offset potential did not change by >1–2 mV when measured at the end of the experiment, indicating the absence of significant drifts of the circuit voltage offsets.
Analysis
Data analysis was performed using Clampfit (pClamp version 9.2, Axon Instrument), Microcal Origin (Version 6.0, Microcal Software, Northampton, MA), Microsoft Excel V. 5.0c (Microsoft, Redmond, WA), GraphPad Prism version 2.01 (GraphPad Software, San Diego, CA). For statistical analysis, F-test and Student's t-test were used when comparing two groups of hair cells. Data are presented as means ± SE or ± SD as specified in the text; n = number of cases. Fast capacitive transients were minimized on-line by the patch-clamp analogue compensation. Uncompensated capacitive currents were corrected by averaging sweeps with no channel activity (nulls) and subtracting them from the active sweeps. Event detection was performed with the 50% threshold detection method with each transition visually inspected before being accepted. This allowed us to reconstruct idealized traces for calculating single-channel amplitude distribution, open and closed time histograms, and open probability (Po) diagram. Po was calculated as the ratio: (total open time)/(total open time + total closed time). Analyses were performed from the beginning to the end of the sweeps because, consistent with previous whole cell measurements (Masetto et al. 2000, 2005), we found no evidence of Ca channel inactivation during the sweep.
The total number of Ca channels (N) was initially estimated as the largest number of simultaneously open channels seen in the record at most depolarized voltages, at which potentials the probability of channel opening was highest. Because, however, maximum Po was 
0.5, the following algorithm was applied to estimate the likelihood of single-channel activity in those patches without superimposed openings (Plummer et al. 1989): P2(T) = 1 – (1 – P2o)T/t, where P2(T) is the cumulative probability of observing superimposed openings due to the activity of two identical channels over the total observation time T, P2o is the overall probability of finding two simultaneous openings, and t is twice the mean open time. At most depolarized voltages P2(T) was >0.999 despite the absence of superimposed openings during the observation time T.
Histograms for amplitude distribution at each voltage were fitted with a single or second-order Gaussian distribution by using the Levenberg-Marquardt algorithm to obtain the mean amplitude and SD. Average Po values at each potential (Po-V diagrams; Fig. 4) were calculated by averaging the mean values obtained from each patch, excluding null sweeps, fitted with a Boltzmann function (Eq. 1)
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; Hess et al. 1984; Nowycky et al. 1985), it can be useful to separately analyze sweeps where the channel is actively gating (i.e., channel "mode" 1" or "2"), from null sweeps, where the channel is unavailable for opening (channel mode "0"). On the other hand, characterization of null sweeps distribution as well is important by itself because it can be affected by drugs, second messengers, cell metabolic state, hormones, etc. Nonetheless, to better compare present results with those reported in the literature, values for Po including null sweeps are also given in the text.
To calculate the open and closed time at each potential two different approaches were used: 1) mean open (to) and closed (tc) times obtained from each experiment were averaged; this method gives an estimate of the two parameters independently from the number of exponentials used for fitting the data in each experiment and was used because in many experiments, only few data could be obtained due to rare openings; 2) data from the analysis of all experiments were pooled together to build a distribution of the open and closed times on a log binned scale (20 bins/decade). The interpolation of the histograms with two or three exponentials with maximum likelihood method (Sigworth and Sine 1987) provided the time constant values for the open (
o) and closed (c) states (Eq. 2)
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i are the relative area and time constant of the ith component of the distribution.
The half-amplitude threshold analysis method hitherto employed failed to separate the sublevel events from the main levels. This was an expected consequence of 1) the small elementary current amplitude, meaning a low signal-to-noise ratio (half-amplitude analysis method requiring this ratio to be >7) (Colquhoun and Sigworth 1983), and 2) the low occurrence of the sublevels. Therefore to provide quantitative measurements of the subconductance levels, we used the mean-variance analysis method, which allows to exclude from the analysis those sample points at the transitions between current levels (Patlak 1988, 1993). By this analysis, the different current levels are defined as periods of time in which the variance of the current is as low as when no current is flowing (the background noise at the closed state). The analysis consists of sliding a "window" of width N sample points over the sampled data, meanwhile calculating the mean current, m, and sample variance, s2, for the N points in the window. The window is then advanced one point at a time, and the m-s2 paired values are calculated each time. Each bin, m-s2, can then be plotted versus its frequency of occurrence (number of events) in three-dimensional histograms like the one showed in Fig. 6B. Histograms were generated with the mvMachine software program (version 4.0.0.1
[EC]
, kindly provided by Dr. JB Patlak website www.physiology.med.uvm.edu/patlak/, University of Vermont, Burlington, VT), converted through Histofilter (version 1.0.0.1
[EC]
, TODO), and plotted and analyzed by Microcal OriginPro (Version 7.5, Microcal Software). In the histogram, the closed level, the main open level, and the two subconductance levels were obvious as peak volumes in the low-variance region. We summed all the conductive events for each m value the s2 of which was less than the background noise (Gollasch et al. 1992; Patlak et al. 1993), and we fit the resulting three-peaks distribution with the sum of three Gaussians. This provided the numerical values for the mean current amplitude (±SD) and area for each conductive level.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Patch pipettes were sealed onto the hair cells basolateral surface, where Ca channels have been found either dispersed or clustered at the active (presynaptic) zones in both otolith and acoustic organs (Brandt et al. 2005; Issa and Hudspeth 1994; Roberts et al. 1990; Rodriguez-Contreras and Yamoah 2001). However, the majority of patches here investigated did not show any single-channel activity, and those showing detectable activity mostly expressed only one Ca channel. It is possible that, by randomly patching the basolateral region, we never encountered an active zone. Other alternative explanations may exist: for example, Ca channel densities at the synaptic zones of semicircular canal hair cells might be lower, or most Ca channels are normally inhibited (or were inhibited in our experimental conditions).
An outward single-channel current was sometimes detected. Such patches were excluded from the analysis.
Figure 1, A and B, shows representative unitary currents recorded from type I and type II hair cells respectively, with Extra_Ba_70 (top) or Extra_Ba_5 (bottom) in the pipette (see Table 1 for solutions' composition). The bath solution had a high content in K+ to set hair cells' resting membrane potential at 0 mV. Most recordings were from zone 2, which contains the higher proportion of type I hair cells (see METHODS). Type I and type II hair cells were classified based on their morphology (Masetto et al. 2000); moreover, in a few cases, we were able to confirm their identity electrophysiologically by shifting to the whole cell configuration (see Whole cell recordings).
With 70 mM Ba2+ in the pipette, single-channel inward currents first appeared at –60 mV as very brief and rare openings. Conversely, with 5 mM Ba2+ in the pipette, single-channel inward currents were already detectable at –80 mV. The amplitude of the elementary current decreased with depolarization; inward currents were clearly detectable 20 mV (data not shown). The majority of patches contained only one active channel, and a few patches two channels; data from the latter patches were included in the single-channel current-voltage relationship but excluded from the analysis of the mean open probability and open and closed time.
Figure 1C shows the amplitude distribution for single-channel current at –30 mV; data were obtained with Extra_Ba_70 (top) or Extra_Ba_5 (bottom) as the pipette solution. Mean amplitude was –1.3 ± 0.04 (SE) pA with Extra_Ba_70 and –0.5 ± 0.06 pA with Extra_Ba_5. Data from type I and type II hair cells were pooled because ANOVA (8 type I and 12 type II hair cells) showed no significant differences in the unitary current amplitude, slope conductance or voltage activation threshold (see Table 2). Present data are consistent with type I and type II hair cells expressing a similar population of Ca channels as already observed for whole cell Ca currents (Bao et al. 2003; Masetto et al. 2005).
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Figure 1D shows average current calculated from the same patch over 500 consecutive sweeps at –20 mV. No time-dependent inactivation of Ca channels was found, as single-channel activity did not change significantly during 500-ms depolarizing pulses.
Single-channel activity was unaffected by inclusion of -agatoxin IVA (2 µM) and -conotoxin GVIA (3.2 µM) in the pipette solutions (n = 2; see legend for Fig. 1A).
Kinetics of single-channel currents
Figure 2 shows the average single-channel current-voltage (I-V) plot for the inward current recorded with Extra_Ba_70 (
) or Extra_Ba_5 (
) as the pipette solution. The mean inward current was always larger with Ba2+ 70 mM relative to Ba2+ 5 mM in the pipette. A 20-mV shift in the voltage activation threshold appeared because the channel activity was first detectable at –60 mV in Ba2+ 70 mM and at –80 mV in Ba2+ 5 mM. This leftward shift in 5 mM Ba2+ versus 70 mM Ba2+ was also consistent with the shift in the mean open probability versus voltage curves (see Fig. 4, A and B). The voltage shift was likely due to the different patch-membrane surface charge screening effect by Ba2+ 70 mM versus Ba2+ 5 mM (see DISCUSSION). In both experimental conditions, data were well fitted with straight lines, indicating that the channel has a linear conductance in the voltage range investigated. The average slope conductance was 21 pS (n = 20) with Extra_Ba_70 and 11 pS (n = 9) with Extra_Ba_5 as the pipette solution.
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o1 = 2.4 (29.2%), o2 = 6.8 (68.4%), o3 = 24.2 (2.4%). The presence of time constants differing by one order of magnitude suggests the presence of at least two gating modes, "mode 1" (short openings, consistent with o1 and o2) and "mode 2" (long openings, consistent with o3) –see e.g., Fig. 5A, inset. In our recordings, the gating "mode 2" was likely enhanced by the agonist Bay K 8644 (Hess et al. 1984; Nowycky et al. 1985).
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c1 = 0.5 (41.8%), c2 = 8.8 (47.8%), c3 = 76.6 (10.37%). These results indicate that the channel can spend long periods in the closed state (see null sweep probability in Fig. 4B). The significant differences in closed time constants would reflect the presence of at least two closed states.
Values obtained by weighted means of the time constants were in close agreement with to and tc. o and c had similar values for type I and type II hair cells (see Table 2). L channels may exhibit an increased activity following repolarization from depolarized voltages (Jones 1998). We therefore checked for any effect of predepolarization on single-channel activity on repolarization to –70 mV with Extra_Ba_5 as the pipette solution. We found no significant effect on the mean open time or the mean open probability (Po); however, we found that for openings that persisted on repolarization (single-channel tail current, see for example arrow heads in Fig. 1, A and B), tail current duration increased significantly with predepolarization in the range from –60 to 30 mV (Fig. 3D).
Po was not significantly different between type I and type II hair cells, (P > 0.05 at all voltages with Extra_Ba_5 and Extra_Ba_70, except P > 0.01 at –20 and –40 mV with Extra_Ba_70). Po was strictly voltage dependent, increasing with depolarization (Fig. 4). As shown in Fig. 4A, with Extra_Ba_70 in the pipette, Po was 0.03 at –50 ± 0.01 (SE) mV (n = 6) and 0.28 at –20 ± 0.15 mV (n = 12) with half-maximum Po (V1/2) at –22.4 ± 2.6 (SD) mV; the maximum Po (Pomax) was 0.42 ± 0.26 (SE) mV (n = 10) at 0 mV; k = 4.7. With Extra_Ba_5 in the pipette, Po was 0.15 at –60 ± 0.12 mV (n = 9) and 0.47 at –30 ± 0.29 mV (n = 7), with V1/2 at –45.7 ± 6.15 (SD) mV; Pomax was 0.54 ± 0.22 (SE) mV (n = 3) at –20 mV (Fig. 4B); k = 6.9. Thus V1/2 shifted by approximately –23 mV in Ba2+ 5 mM relative to Ba2+ 70 mM. Figure 4B also shows the Po/V relationships obtained after nulls inclusion (). Po with nulls was 0.05 at –60 mV and 0.16 at –30 mV. Po appeared to asymptote to a nonzero value when null sweeps were excluded from the analysis, whereas it did show a tendency to zero for voltages more negative than –80 mV when null sweeps were included. This might suggest that the channel has a low intrinsic (i.e., voltage-independent) probability of being open when it is outside the quiescent mode, which would then increase up to Pomax following depolarization.
A high percentage of null sweeps (bars in Fig. 4, A and B) was found at all voltages in both Extra_Ba_70 (mean value = 0.51 ± 0.08 SD) and Extra_Ba_5 (mean value = 0.63 ± 0.01 SD). Because of the high variability among patches, it is difficult to judge if null sweeps percentage decreased with depolarization. Moreover, null sweeps percentage appeared to decrease by increasing Ba2+ concentration. One possible explanation is that the dwell time in the closed state is both ion and concentration dependent as suggested by Rodriguez-Contreras and Yamoah (2003), who also reported, in frog sacculus hair cells, an increased propensity for L-type Ca channels to enter into quiescent modes when Ba2+ or Ca2+ concentrations were decreased.
Figure 5 shows a plot of Po versus recording time. Ca-channel activity persisted unaltered for the whole recording period (260 s) without evidence of channel run-down. Similar results were found for recordings lasting 10–20 min (data not shown). The recordings' sweeps with null or low activity (Po < 0.02) appeared clustered. During these sweeps, Ca channel openings were very brief (see e.g., Fig. 5, inset, b). During sweeps' clusters with higher Po, Ca channels could both open in mode 1 (e.g., a) or mode 2 (e.g., c). However, we did not distinguish clusters of channel activity in mode 1 or mode 2 (i.e., sweeps dominated by mode 1 vs. sweeps dominate by mode 2).
Transitions from the open state to sub-conductance levels were sometimes detectable at voltages between –40 and –10 mV (see e.g., Fig. 6A). Following careful inspection of all traces, these events appeared to occur very seldom. In those sweeps where sub-conductance levels were found, we performed the mean-variance (MV) analysis (Patlak 1993) to generate MV histograms like the one shown in Fig. 6B. At least two sub-conductance levels were clearly detectable, showing a mean amplitude at –30 mV of –0.5 ± 0.2 and –0.9 ± 0.1 (SD) pA. The contribution of the sub-conductance levels to the conductive state was low even for selected sweeps where sublevel events were most frequent. For recordings at –20 and –30 mV for example, the sum of the two sublevel events accounted for little <1% of the conductive events, that is the channel spent almost 99% of the conductive state in the main open level. Multiple conductance levels of L-type Ca channels were previously reported (Kunze and Ritchie 1990).
Whole cell recordings
Occasionally, following cell-attached recording, we were able to achieve the ruptured whole cell configuration without losing the seal. Figure 7, A and B, show such two examples for a type I and a type II hair cell, respectively. A large inward current was present in both hair cells at the holding voltage (–60 mV). This is consistent with the high-monovalent cations' concentration in the bath solution (135 mM K+, see Extra_high_K in Table 1) versus the internal solution (1 mM Cs+, see Extra_Ba_70). However, hyperpolarizing steps elicited inward currents the kinetics of which was significantly different in the two cell types. In the type I hair cell, a large instantaneous inward current was present on hyperpolarization, which progressively deactivated. At the end of the –120 mV pulse, the steady-state inward current was almost zero, indicating that almost all ion channels were closed at this membrane voltage. This behavior is consistent with IK,L expression by this cell (Masetto et al. 2000; Rennie and Correia 1994; Rüsch and Eatock 1996).
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). In both hair cells, little or no outward current was present at the test potential (40 mV) due to the small monovalent cations concentration in the internal solution.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Ca channel classification
Ca channel activity was still present and not affected when P/Q, and N type Ca channels blockers were added to the pipette solution. Because R-type Ca channels have a significantly lower slope conductance (13 pS in Ba2+ 110 mM) than found here and because T-type Ca channels show rapid ( 20–50 ms) (Hille 2001) and complete inactivation, we identified type I and type II hair cells Ca channels as L-type.
L-type Ca channels are multisubunit complexes constituted by different isoforms of the pore-forming 
1 subunit, named 1S (CaV1.1), 1C (CaV1.2), 1D (CaV1.3), and 1F (CaV1.4). Most literature agrees that hair cell L-type Ca channels activate close to –60 mV. This feature would distinguish them from classical neuronal L-type Ca channels, which activate 20–30 mV less negative (Ertel et al. 2000). However, more recently it has been reported that the CaV1.3 (1D) subunit of L-type Ca channels activates at significantly more negative voltages than the other L-type CaV1 subunits (Koschak et al. 2001; Mangoni et al. 2003; Xu and Lipscombe 2001; Zhang et al. 2002 –reviewed in Lipscombe et al. 2004). The expression of the CaV1.3 subunit has been reported in cochlear hair cells (Green et al. 1996; Kollmar et al. 1997; Platzer et al. 2000) as well as in mammalian vestibular epithelia (Bao et al. 2003). Also consistent are the observations that chick cochlear hair cell L-type Ca channels (Zidanic and Fuchs 1995) and the CaV1.3 subunit (Xu and Lipscombe 2001) are incompletely inhibited by dihydropyridines. Finally, genetic ablation of the CaV1.3 subunit resulted in cochlear hair cell exocytosis dysfunction (Brandt et al. 2003) and congenital deafness (Dou et al. 2004), although vestibular function did not appear compromised, which might be explained by the expression of other Ca channel types in vestibular hair cells (see following text). Given the hyperpolarized activation range found here, the most reasonable hypothesis is that the single Ca channels activity we recorded involved the CaV1.3 subunit.
On the other hand, we did not find evidence for other Ca channel types. The expression of Ca channels involving different (1) subunits was reported in frog vestibular hair cells (Martini et al. 2000; Rodriguez-Contreras and Yamoah 2001). Moreover, recent works on CaV1.3-(1D)-knock-out mice showed the expression of a persisting Ca channel population in acoustic and vestibular hair cells (Brandt et al. 2003; Dou et al. 2004; Platzer et al. 2000). Because we limited our analysis to the basolateral region of hair cells, and these were mostly from crista zone 2, it is possible that other Ca channel types are expressed in different cell membrane regions, for example close to the apical surface and/or different crista zones.
Ca channel biophysical properties
No data are available in the literature concerning the elementary properties of the CaV1.31-subunit-containing Ca channel. However, present data can be placed in the context of the more general literature on L-type Ca channels. For the following discussion, it is worth recalling that all excitable cells, except skeletal muscle and perhaps retina, express the CaV1.2 and the CaV1.3 gene (see Lipscombe et al. 2004 for a review). The CaV1.3 gene is coexpressed in many cells with CaV1.2; in neurons, CaV1.2 and CaV1.3 are also often found in the same membrane compartments; in the heart, conversely, CaV1.3 is present in atrial tissue, but not in ventricular muscle that expresses CaV1.2.
As far as single-channel gating properties are concerned, the two different mean open and closed times found here resemble brief and long openings of L channels in cardiac ventricular (Hess et al. 1984) and neuronal (Nowycky et al. 1985) cells, called "mode 1" for brief and "mode 2" for long lifetimes, respectively. However, our analysis of the recorded traces did not reveal a clear segregation of mode 1 or mode 2 as in Hess et al. (1984). The frequent occurrence of null sweeps (mode 0) as well is reminiscent of L-type Ca channels described in cardiac (Hess et al. 1984), neuronal (Nowycky et al. 1985), and chromaffin cells (Carabelli et al. 2001). Interestingly, Ca channel opening probability appears to result from voltage dependence of Po between a minimum (intrinsic) and maximum level during active sweeps and voltage dependence of null sweeps frequency.
Finally, the finding that single-channel tail current duration increased with predepolarization is consistent with increased activity of L-type Ca channels following repolarization from depolarized voltages (Jones 1998).
Present experiments revealed that type I and type II hair cells express Ca channels with similar properties (see also Table 1): voltage activation threshold at –80/–60 mV with 5/70 mM Ba2+, negligible inactivation, and a unitary conductance of 11/21 pS with 5/70 mM Ba2+. A similar Ca channel unitary conductance was found in hair cells of the chick cochlea (24 pS in 110 mM Ba2+) (Kimitsuki et al. 1994) and frog saccule (21 pS in 65 mM Ba2+) (Rodriguez-Contreras and Yamoah 2001). The maximum Po in our recordings was 0.54 (with BayK 8644 and excluding null sweeps), analogous to that found in frog sacculus hair cells (0.4 with BayK 8644) (Rodriguez-Contreras and Yamoah 2001), but significantly less than reported in inner hair cells (0.82 without BayK 8644; Brandt et al. 2005). So far dwell times distribution have been characterized only in frog sacculus hair cells (Rodriguez-Contreras and Yamoah 2001), where L channels showed three mean open times (o = 0.3, 9.7, and 22.7 ms), and three mean closed times (c = 1.5, 10.3, and 56.6 ms) rather similar to those found here (o = 2.4, 6.8, and 24.2 ms; c = 0.5, 8.8, and 76.6 ms).
An original finding of the present paper concerns the hyperpolarized voltages at which single L-type Ca channel activity could be detected. With 5 mM Ba2+ in the patch pipette, brief unitary Ca channel openings were detected at voltages as negative as –80 mV. Single events were rather short and infrequent at –80 and –70 mV; therefore they should produce a small total inward current, possibly hard to detect in whole cell configuration. Indeed, in this same preparation we reported whole cell inward currents through Ca channels activating close to –60 mV (Masetto et al. 2005). However, in rat crista type I hair cells, Bao et al. (2003), by interpolating the I-V relationship, estimated that 1% of the peak total Ca current recorded with 1.3 mM Ca2+ was available at –72 mV, thus providing a first hint that L-type Ca channels in hair cells might be active at voltages more negative than previously thought.
The 20-mV more-negative activation range found here with Ba2+ 5 mM, relative to Ba2+ 70 mM, accompanied with a –23 mV shift in Ca channel Po. These phenomena should reflect the local increase in negative surface charge screening effect exerted by divalent cations, which shifts the activation curve of voltage-dependent Ca channels toward more depolarized voltages (Frankenhaeuser and Hodgkin 1957; Hille 2001; Kostyuk et al. 1982). As a result, stronger depolarization would be required to open the same number of channels following increase of divalent cations concentration.
We speculate that in physiological conditions (i.e., with 2 mM Ca2+ in the perilymph) Ca channels might activate at such negative voltages as found here in 5 mM Ba2+ (i.e., at –80 mV). The rationale for this hypothesis is that we found that the activation kinetics of the inward current at several potentials was similar in 5 mM Ba2+ relative to 2 mM Ca2+ (Masetto et al. 2005), and it has been reported that the position of the CaV1.31 current-voltage curve obtained with 2 mM Ca2+ overlaps with that recorded in 5 mM Ba2+ (Xu and Lipscombe 2001), as also reported for the activation curve of L-type Ca channels in rat neocortical neurons (Lorenzon and Foehring 1995). These results are consistent with previous studies showing that Ca2+ is more effective than Ba2+ at shifting the gating of voltage-dependent Na (Hille et al. 1975) and Ca (Smith et al. 1993) channels; that is, the surface charge associated with channel gating has a much higher affinity for Ca2+ than for Ba2+.
However, a caveat concerns the use of Bay K 8644, which has been reported to exert part of its agonistic effect on L-type calcium channels by shifting the voltage-dependence of channel gating toward more negative membrane potentials. However, this would be obtained by Bay K 8644 increasing the chances that the channel stays open longer (Cena et al. 1989; Hess et al. 1984; Markwardt and Nilius 1988; Nowycky et al. 1985; Sanguinetti et al. 1986). Also, in pancreatic b-cells, which express the CaV1.3 subunit of the L-type Ca channel (Ertel et al. 2000; Scholze et al. 2001), no effects of Bay K 8644, tested 1 mM, were found on the Ca channel voltage dependence (Smith et al. 1993).
Functional implications of single Ca channel properties
Depending on the cell system of expression, L-type Ca channels may have quite different roles. In neurons, L-type (CaV1.2 and CaV1.3) Ca channels are particularly important in translating synaptic activity into alterations in gene expression and cellular function (e.g., Bito et al. 1996; Deisseroth et al. 2003; Dolmetsch et al. 2001; Graef et al. 1999; Weick et al. 2003). On the other hand, L-type Ca channels do not appear involved in synaptic transmission between neurons, which conversely depends on presynaptic Ca2+ influx through N-type (CaV2.2) and P/Q-type (CaV2.1) Ca channels (Dunlap et al. 1995). L-type Ca channels are expressed in vertebrate cardiac, skeletal, and smooth muscle and are prominent in several endocrine cells, in rod photoreceptors, and in certain nonspiking synaptic terminals that secrete continuously. Their most obvious function is to mediate Ca2+ entry in cells that contract or secrete in response to long or steady depolarizations (Hille 2001).
A similar role appears convenient for L-type calcium channels in hair cells, which sustain a tonic release of afferent transmitter. Consonant with this, several pieces of evidence exist that hair cell afferent transmission involves L-type Ca channels (Brandt et al. 2003; Perin et al. 2000; Spassova et al. 2001). Furthermore, the hyperpolarized threshold of the CaV1.3 subunit would suit the voltage operating range of hair cells, the resting membrane potential of which has generally been reported to be close to, or more negative than, –60 mV.
The present results, together with previous whole cell data showing that Ca channel properties are very similar in 5 mM Ba2+ or 2 mM Ca2+ (Masetto et al. 2005), suggest that vestibular hair cells Ca channels might be already active at –80 and –70 mV in physiological conditions (i.e., in 2 mM Ca2+). If this hypothesis was true, hair cell bidirectional operating range would result extended toward the negative voltage region. Moreover, it might at least in part account for the type I hair cell "paradox." In vitro in fact, type I hair cells show a resting membrane potential more negative than –70 mV, which, together with a low membrane input resistance (Masetto et al. 2000; Rennie and Correia 1994; Rüsch and Eatock 1996), would preclude Ca-dependent neurotransmitter release if the Ca current activated at, or less negative than, –60 mV. This would not be consistent with in vivo recordings from rodent calyx afferents, showing both background and evoked afferent discharge (Baird et al. 1988; Goldberg et al. 1990; Rennie and Streeter 2006) and spontaneous excitatory postsynaptic currents (Rennie and Streeter 2006). To reconcile these results, it has been proposed that type I hair cells are depolarized by K+ accumulation in the synaptic cleft (Goldberg 1996; Soto et al. 2002) or by IK,L inhibition by cGMP (Behrend et al. 1997) or NO (Chen and Eatock 2000). Here we add the possibility that Ca2+ inflow resulting from the short and infrequent openings at voltages between –80 and –60 mV be sufficient to trigger afferent transmitter release in these cells. Noteworthy, the driving force for Ca2+ inflow is high at such negative voltages; moreover, Brandt et al. (2005) have recently suggested that transmitter release in inner hair cells is under the control of an intracellular Ca2+ nanodomain shaped by the gating of one or few Ca channels.
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Address for reprint requests and other correspondence: S. Masetto, Dept. di Scienze Fisiologiche-Farmacologiche Cellulari-Molecolari, Sez. di Fisiologia Generale e Biofisica Cellulare, Università di Pavia, Via Forlanini 6, 27100 Pavia, Italy (E-mail: smasetto{at}unipv.it)
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, mean percentage of null sweeps at each voltage. Error bars are shown in only 1 direction for better clarity.
). - - -, sub-conductance levels; —, closed level. B: mean-variance relationship of currents shown in A. Window width = 50 sample points. The 3-dimensional histogram illustrate number of observations for any single combination of mean and variance (see METHODS). The large peak on the right represents number of events in the closed state (mean current = 0 pA). The variance at the base of the peak spans the background noise. The other peaks in the same low-variance region (i.e., variance <0.1) reflected conductive states (main open and sublevels). Events in the arch region (variance >0.1) reflect the transitions among the different current levels. These points were excluded from the analysis. Note that, consistent with visual inspection of the traces in A, transitions into and out the sublevels appear to arch from the open level.
