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J Neurophysiol 100: 2115-2124, 2008. First published August 13, 2008; doi:10.1152/jn.90473.2008
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Effects of Strontium on the Permeation and Gating Phenotype of Calcium Channels in Hair Cells

Adrian Rodriguez-Contreras1, Ping Lv1, Jun Zhu1,2, Hyo Jeong Kim1 and Ebenezer N. Yamoah1

1Center for Neuroscience, Program in Communication Sciences, University of California, Davis, California; and 2Department of Nephrology, Xijing Hospital, Fourth Military Medical University, Xi'an, China

Submitted 15 April 2008; accepted in final form 30 July 2008


ABSTRACT
 
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To minimize the effects of Ca2+ buffering and signaling, this study sought to examine single Ca2+ channel properties using Sr2+ ions, which substitute well for Ca2+ but bind weakly to intracellular Ca2+ buffers. Two single-channel fluctuations were distinguished by their sensitivity to dihydropyridine agonist (L-type) and insensitivity toward dihydropyridine antagonist (non-L-type). The L- and non-L-type single channels were observed with single-channel conductances of 16 and 19 pS at 70 mM Sr2+ and 11 and 13 pS at 5 mM Sr2+, respectively. We obtained KD estimates of 5.2 and 1.9 mM for Sr2+ for L- and non-L-type channels, respectively. At Ca2+ concentration of ~2 mM, the single-channel conductances of Sr2+ for the L-type channel was ~1.5 and 4.0 pS for the non-L-type channels. Thus the limits of single-channel microdomain at the membrane potential of a hair cell (e.g., –65 mV) for Sr2+ ranges from 800 to 2,000 ion/ms, assuming an ECa of 100 mV. The channels are ≥4-fold more sensitive at the physiological concentration ranges than at concentrations >10 mM. Additionally, the channels have the propensity to dwell in the closed state at high concentrations of Sr2+, which is reflected in the time constant of the first latency distributions. It is concluded that the concentration of the permeant ion modulates the gating of hair cell Ca2+ channels. Finally, the closed state/s that is/are altered by high concentrations of Sr2+ may represent divalent ion-dependent inactivation of the L-type channel.


INTRODUCTION
 
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ca2+ channels are multi-ion pores that allow the passive flow of several divalent, and under restricted conditions, monovalent cations (Hess et al. 1986Go; Hille 2001Go). Among the permeant ions, Sr2+, by virtue of its similar charge and size to Ca2+, produced currents that are akin to Ca2+ currents (Bourinet et al. 1996Go; Byerly et al. 1985Go; McNaughton and Randall 1997Go). The magnitude and time constants of activation and deactivation of whole cell Sr2+ currents are similar to the properties of Ca2+ currents of Cav2.2 channels. The properties of Sr2+ currents are not identical to Ca2+ currents, however, and the differences rely mainly on the magnitude and extend inactivation (McNaughton and Randall 1997Go). Moreover, the effects of Sr2+ on the elementary properties of Ca2+ channels remain uncertain. Instead, much attention has focused on studies of the effects of Ba2+ on the macroscopic and microscopic phenotype of Ca2+ channels, partly because Ba2+ currents are ≥2-fold larger than Ca2+ currents, and Ba2+ currents are moderately stable compared with the fast rundown of Ca2+ currents (Fox et al. 1987Go; Rodriguez-Contreras et al. 2002Go; Wakamori et al. 1998Go; Yue and Marban 1990Go). Ba2+ markedly alters not only the voltage- and time-dependent activation and deactivation kinetics but also abolishes inactivation in Ca2+ channels that rely on Ca2+ and calmodulin in conferring the decay of the current (Imredy and Yue 1994Go; Mori et al. 2004Go). Apparently, whereas Ba2+ is a poor substitute for Ca2+ in various biological processes, Sr2+ can replace Ca2+ in several Ca2+-dependent reactions (Falke et al. 1994Go; Xu-Friedman and Regehr 1999Go).

In addition to traversing Ca2+ channels, Sr2+ mediates neurotransmitter release in the squid giant synapse (Augustine and Eckert 1984Go), the neuromuscular junction (Bain and Quastel 1992Go; Dodge et al. 1969Go), hippocampal synapse (Abdul-Ghani et al. 1996Go), and cerebellar synapses (Falke et al. 1994Go; Xu-Friedman and Regehr 1999Go). Invariably, the effects of Sr2+ on neurotransmitter release are desynchronized and reduced compared with Ca2+, suggesting that Sr2+ is less effective than Ca2+ in triggering exocytotic transmitter release (Falke et al. 1994Go; Xu-Friedman and Regehr 1999Go).

Ohmori (1985)Go showed that, in vestibular hair cells of the chick, Sr2+ maintained mechanoelectrical transducer currents. In addition, activation of Ca2+-activated K+ channels (BK channels) can be supported by Sr2+ in goldfish saccular hair cells (Sugihara 1998Go). Because hair cells in lower vertebrates rely on the interplay between the activation of voltage-gated Ca2+ channels and the activation of BK channels to confer electrical tuning (Armstrong and Roberts 1998Go; Art et al. 1993Go; Fuchs and Evans 1988Go; Lewis and Hudspeth 1983Go), and Sr2+ can replace Ca2+ to induce membrane electrical resonance, we surmised that understanding the effects of Sr2+ on the elementary properties of Ca2+ channels in hair cells would provide clues to the mechanisms of hair cell functions. Indeed, to prevent cross-talk between different Ca2+-dependent pathways in hair cells, the macro-domain Ca2+ handling is tightly regulated by mobile Ca2+ buffers (Hall et al. 1997Go). Thus incoming Ca2+ at the micro-domains of Ca2+ channels is a major determinant of Ca2+-dependent functions. Additionally, because intracellular Ca2+ buffers bind weakly with Sr2+ (Yawo 1999Go), we rationalized that Sr2+ flux through Ca2+ channels represents an unadulterated means to study the throughput of the channel. Thus we determined the unitary current phenotypes of Ca2+ channels of bullfrog saccular hair cells using Sr2+ as the charge carrier.


METHODS
 
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
American bullfrogs (Rana Castebeina) were purchased from West Jersey Biological Supply Co. (Wenonah, NJ) and Nebraska Scientific (Omaha, NB) and kept in the laboratory in freshwater circulating containers. The methods for dissociating single hair cells from the saccule of bullfrogs and for the set-up of patch-clamp experiments (Hamill et al. 1981Go) have been described previously (Rodriguez-Contreras and Yamoah 2001Go; Rodriguez-Contreras et al. 2002Go; Yamoah and Gillespie 1996Go). All chemicals were obtained from Sigma Chemical (St. Louis, MO) unless otherwise noted. Bullfrogs were killed, and inner ears were removed and placed quickly in oxygenated, low Ca2+ solution (LCS; in mM): 110 NaCl, 2 KCl, 3 D-glucose, 0.1 CaCl2, and 5 HEPES, pH 7.4, with NaOH. To disrupt the macular tight junctions, the preparation was exposed to 4 mM 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) for 20 min. The tissue was washed and transferred to frog saline containing 1 mg/ml bovine serum albumin (Sigma) and 2 mg/ml DNAse I (Worthington, Lakewood, NJ) for 10 min. This procedure has been used previously (Chabbert 1997Go) and differs from more severe enzymatic treatments that can affect ionic conductances (Armstrong and Roberts 1998Go). The preparation was washed and placed on the recording chamber at 4°C before recording at room temperature. The otolithic membrane was removed mechanically, 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 concavalin-A) for 20 min before patch-clamp recording.

Patch pipettes were pulled from borosilicate and quartz glass capillaries (World Precision Instruments Sarasota, FL and Sutter Instruments, San Rafael, CA) on a Flaming-Brown and laser microelectrode pullers (Sutter Instruments). For single-channel recordings, the borosilicate glass electrodes were coated with Sylgard 184 (Dow-Corning, Midland, MI) ≤100 µm from the tip and fire-polished before use. Bath solution contained (in mM) 90 NaCl, 25 tetraethylammonium chloride (TEACl), 5 4-aminopyridine (4-AP), 5 BaCl2/CaCl2/SrCl2, 3 glucose, and 5 HEPES (pH 7.4). Ca2+ currents were recorded using perforated patch electrodes (resistances 2–4 M{Omega}) whose tips contained (in mM) 120 CsCl and 5 HEPES (neutralized to pH 7.3 with CsOH). To gain electrical assess to hair cells and to minimize wash-out of intracellular molecules, patch electrodes were backfilled with solution containing (in mM) 130 CsCl, 1 CaCl2, 5 HEPES, and amphotericin 200 µg/ml (neutralized to pH 7.3 with CsOH) (Korn et al. 1991Go). To ensure that recordings are made 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 to whole cell mode resulted in cell death caused by Ca2+ toxicity. Series resistance (5–10 M{Omega}) was compensated (nominally 50–60%). Liquid-junction potentials were not corrected because they were <2 mV. Voltage-clamped Ba2+/Ca2+/Sr2+ currents were amplified with Axopatch 200B amplifiers (Molecular Devices, Sunnyvale, CA). Cell capacitance was calculated by integrating the area under an uncompensated capacitive-transient elicited by a 20-mV hyperpolarizing pulse from a holding potential of –80 mV. Cell capacitance and series resistance was compensated as much as possible almost to the point of ringing. In general, 60–80% of the series resistance was compensated. Current records were filtered at 2–5 kHz with a low-pass Bessel filter and digitized at 10 kHz with a Digidata interface controlled by custom-made software.

For single-channel recordings, patch electrodes were filled with a Sr2+ solution (5–70 mM) containing (in mM) 20 TEACl, 5 4-AP, and 5 HEPES at pH 7.4 (adjusted with TEAOH). N-methyl-D-glucamine (NMG) was used to substitute for divalent Sr2+ and to maintain an osmolarity of ~280 mmosmol. Quartz glass electrodes were used to record single-channel fluctuations when the Sr2+ concentration was <20 mM. To identify L-type channels, experiments were conducted in the presence of the channel's agonist Bay K 8644 (Calbiochem, La Jolla, CA). 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, 1 CaCl2, 5 4AP, and 5 HEPES, pH 7.4, with TEAOH, to shift the resting potential to ~0 mV (Rodriguez-Contreras and Yamoah 2003Go). Here, liquid junction potentials were measured and corrected as described previously (Rodriguez-Contreras et al. 2002Go). Experiments were carried out at room temperature (~21°C).

Data analysis

Whole cell Ba2+/Ca2+/Sr2+ current amplitudes at varying test potentials were measured at the peak and steady-state levels using a peak and steady-state detection routine. For single channel records, leakage and capacitative transient currents were subtracted by fitting a smooth template to null traces. Leak-subtracted current recordings were idealized using a half-height criterion. Transitions between closed and open levels were determined using a threshold detection algorithm, which required that two data points exist above the half mean amplitude of the single-unit opening. The computer-detected openings were confirmed by visual inspection, and sweeps with excessive noise were discarded. Amplitude histograms at a given test potential were generated and fitted to a single Gaussian distribution using a Levenberg-Marquardt algorithm to obtain the mean and SD. At least five voltage steps and their corresponding single-channel currents were used to determine the unitary conductance. Single-channel current-voltage relations were fitted by linear least-square regression lines and single-channel conductances were obtained from the slope of the regression lines. Idealized records were used to construct ensemble-averaged currents and open probability (Po), as well as 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, Northampton, MA). To ensure that the recordings were made on single L-type channels, only patches containing single channel events that were blocked by nimodipine (5 µM) were analyzed. Additionally, kinetic analyses were performed on patches, which contained only one channel. The criteria consisted of quantitative determination followed by visual inspection of the data. The patches contained one channel, as there was no stacking of events. Furthermore, direct transition from fast to slow kinetics with similar current amplitude, and vice versa, were observed in the selected recordings, indicating that the gating modes were derived from a single channel. The cumulative first latency histograms were determined as described by (Zei and Aldrich 1998Go). Curve fits and data analyses were performed using Origin software (MicroCal). All averaged and normalized data are presented as means ± SE. Frogs were housed and killed using a protocol approved by the University of California, Davis, IACUC Committee.


RESULTS
 
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
We determined the similarities and differences in the magnitude and profile of whole cell Ba2+, Ca2+, and Sr2+ currents in hair cells. Whereas replacing Ca2+ with Ba2+ in the recording bath solution strongly affected the amplitude and decay of the whole cell inward currents in saccular hair cells, the effects of Sr2+ were relatively modest (Fig. 1). Inward currents were elicited from a holding potential of –60 mV and a step potential to –10 mV. Outward K+ currents were blocked with external 4-AP and TEA as well as internal Cs+. On switching the bath solution, which contained 5 mM Ca2+, to one that contained the same concentrations of Ba2+, the inward current increased by ~1.4-fold. The mean amplitudes of the current (in pA) for Ca2+ and Ba2+ were 778 + 56 and 992 ± 101 (n = 7; P < 0.01), respectively. Additionally, Ba2+ abolished the slow decay of the current profile. In contrast, in 5 mM Sr2+, the inward current mirrored roughly the Ca2+ current in magnitude, activation, and deactivation. The mean amplitudes of the current (in pA) for Sr2+ and Ca2+ were 755 ± 62 and 811 ± 45 (n = 7; P = 0.08), respectively. Inactivation kinetics differed slightly, which is shown in the inset in Fig. 1. Although some of the macroscopic features of Sr2+ and Ca2+ currents appeared similar, single-channel currents should confirm these parallels and reveal the distinct properties of Sr2+ currents.


Figure 1
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  		FIG. 1. Whole cell Sr2+ current compared with Ca2+ and Ba2+ currents from hair cells. Examples of inward current traces recorded using a depolarizing voltage step from a holding potential of –60 mV to a step potential of –10 mV. The current traces were obtained from the same cell after changing the external solution in the order from Sr2+ to Ca2+ to Ba2+ (5 mM). Shown in the inset are normalized current traces to show the effects of the charge carrier on the activation and inactivation of the current traces. For comparison, the Sr2+ current trace is shown in solid, and the Ca2+ and Ba2+ currents are depicted with dotted lines. The mean amplitude of the current (in pA) for Sr2+ and Ca2+ were 755 ± 62 and 811 ± 45 (n = 7; P = 0.08) and Ca2+ and Ba2+ were 778 + 56 and 992 ± 101 (n = 7; P < 0.01), respectively.

 
Inward Ca2+ currents from hair cells in the saccule are generated by two distinct channels: nimodipine-sensitive (L-type) and -insensitive (non-L-type) channels (Rodriguez-Contreras and Yamoah 2001Go). Figure 2, A–D, shows examples of single-channel Sr2+ currents recorded from hair cells isolated from the frog saccule. Data in Fig. 2, A and B, represent Bay K 8644–modified currents. In contrast, the non-L-type channel currents were recorded with patch-pipettes containing 10 µM nimodipine (Fig. 2, C and D). We examined the permeation phenotype of the two-channel subtypes using different concentrations of patch-pipette Sr2+. For the traces shown, the amplitude histograms at different step potentials were generated to determine the mean single-channel amplitude as shown in Fig. 2E, and the resultant current-voltage relations yielded single-channel conductances ({gamma}, in pS) of 17, and 8 for 70 and 5 mM, respectively for the L-type channel, whereas 18 and 13 pS were obtained for the same concentrations of Sr2+ for the non-L-type channel. The mean values for the single-channel conductances for Sr2+ followed in descending sequence from 70 to 5 mM for the L-type and non-L-type channels (Rodriguez-Contreras and Yamoah 2001Go). Respectively, they are 16.2 ± 1.9 and 18.7 ± 1.7 for 70 mM (n = 6) and 11.2 ± 1.8 and 13.3 ± 2.6 for 5 mM (n = 5).


Figure 2
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  		FIG. 2. Permeation of single L-type and non-L-type channels by Sr2+. Under outside-out cell-attached configuration, hair cell membrane patches were held at –70 mV and stepped to the depolarizing potentials, indicated beside the traces. The potentials have been corrected for liquid junction potential errors. A: typical consecutive inward single-channel current traces using 70 mM Sr2+ and 5 mM (B) as charge carriers are shown for the L-type channel. The L-type channel activity was recorded in the presence of 5 µM BayK 8644 to promote long openings. The solid lines indicate the closed levels. C and D: non-L-type current traces recorded using patch pipettes containing 10 µM nimodipine using 70 and 5 mM Sr2+, respectively, as the charge carrier. E: examples of the amplitude histograms from current traces at the step potentials indicated, which were used to generate the I-V plots. F: the scattered points of the I-V relation were fitted with a linear regression to determine to conductance of the channels. For the examples shown, the single channel conductance values for L-type and non-L-type channels in 70 and 5 mM Sr2+ are (in pS; legend = L) as follows: L70, {circ} = 17; L5, bullet = 5; non-L70, {blacksquare} = 18; non-L5, {square} = 13.

 
To determine the sensitivity range of Ca2+ channels in hair cells for Sr2+, we examined the relation between the single-channel conductances versus Sr2+ concentrations. Using a Langmuir isotherm, a fit to the data accrued from recordings from the L-type channel (Fig. 3A) and the non-L-type channel (Fig. 3B) showed two important features of the channels: 1) the apparent dissociation constants of L- and non-L-type channels for Sr2+ ions were (in mM) 5.2 ± 1.9 and 1.9 ± 1.5 (n = 7), respectively; 2) meanwhile, the maximum conductances for the two channels were (in pS) 16.6 ± 1.4 and 19.2 ± 1.9 (n = 7) for the L- and non-L-type channels, respectively, and were reached at external Sr2+ concentrations above ~20 mM. Similar ranges of results have been reported for Ca2+ and Ba2+ currents (Rodriguez-Contreras et al. 2002Go). The ascending limbs of the conductance versus concentration curves for the two channel subtypes suggest that the sensitivity range of the channel for the permeant ion lies between 1 and 5 mM. Thus optimum assessment of Ca2+ channel functions should be evaluated within these narrow ranges of concentrations.


Figure 3
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  		FIG. 3. Conductance vs. concentration curves. The conductance of the L- and non-L-type channels were determined using different concentrations of Sr2+ (2.5–70 mM). The affinity of Sr2+ ({circ}) for L- (A) and non-L-type ({square}). (B) Ca2+ channels in hair cells in the frog saccule was estimated from these plots. The solid lines represent fits with a Langmuir isotherm of the form {gamma} = {gamma}max/(1 + KD/[Sr2+]), where {gamma} and {gamma}max represent the single channel conductance and maximum conductance, respectively. The dotted lines show the conductance-concentration curves for Ca2+ (Rodriguez-Contreras and Yamoah 2003Go). The estimated KDs and {gamma}maxs of the 2-channel subtypes for Sr2+ were as follows: L-type, 5.2 ± 1.9 and 16.6 ± 1.4 (n = 7); non-L-type, 1.9 ± 1.5 and 19.2 ± 1.9 (n = 7), respectively.

 
To ascertain whether the concentrations of Sr2+ ions have noticeable effects on the voltage dependence and kinetics of the channels, we examined the properties of the L-type channel at nonsaturating (5 mM) and saturating (70 mM) concentrations. To ensure that the recordings were made on single L-type channels, only patches containing single channel events that were blocked by nimodipine (5 µM) were analyzed. Additionally, kinetic analyses were performed on patches, which contained only one channel. The criteria consisted of quantitative determination followed by visual inspection of the data. The patches contained one channel, because there was no stacking of events. Furthermore, direct transition from fast to slow kinetics with similar current amplitude, and vice versa, were observed in the selected recordings, indicating that the gating modes were derived from a single channel. Figure 4 is a layout of cell-attached recordings of a family of consecutive single channel current traces, recorded using 5 (A) and 70 mM (B) Sr2+ as the charge carrier. With 5 mM Sr2+, the dwell times of the openings were frequent and long compared with recordings in 70 mM. Another visible difference was that, at low concentrations of the permeant ion, the openings were sustained, which was reflected in the persistent ensemble-averaged currents shown below the corresponding family of traces. In contrast, in 70 mM Sr2+, the channel had frequent openings predominantly only at the early phase of the step voltage, which is readily visible in the well-resolved decay of the ensemble-averaged currents. Notwithstanding the presence of 5 µM Bay K, an agonist of L-type channels, in the bath and pipette solutions, the probability of channel opening (Po) was invariably <1 (Fig. 5). The rightward shift in the voltage dependence of activation as the pipette Sr2+ concentrations were raised from 5 to 70 mM is in keeping with surface charge screening effect of divalent cations (Rodriguez-Contreras and Yamoah 2003Go; Rodriguez-Contreras et al. 2002Go; Zhou and Jones 1995Go). The inference from the finding that the Po was less than ~0.5 is that the channel has multiple nonconducting states. Indeed, by increasing the Sr2+ concentrations (70 mM), fluctuations of the open probability (Po) plummeted in comparison to reduced concentrations (5 mM; Figs. 5 and 6, A and B), suggesting that the dwell times in the closed states may be current or ion concentration dependent. For graphic illustrations, we depict the voltage and time dependence of the normalized Po of recordings performed at Sr2+ concentrations of 70 (Fig. 6A) and 5 mM (Fig. 6B). The gating of the channel was noticeably altered in several ways: 1) at saturating concentrations of Sr2+ (70 mM), nonconducting states are more favored than open states; 2) although channel openings are stochastic in nature, the Po of the channel remains infinitesimally low at negative step potentials (–60 to –40 mV), and even at step potentials greater than –40 mV, the increase in Po is relatively modest compared with recordings done at Sr2+ concentrations (5 mM) within the sensitivity range of the channel; 3) finally, at the steeper phase on the sensitivity curve of the channel, the Po of the channel was high at negative voltages and remained so at more positive voltages, indicating that, at low concentrations of the permeant ion, the channel has increased propensity to exit from the nonconducting states. The shift in the activation of the channel using 5 and 70 mM Sr2+ is shown in the normalized Po versus voltage plot (Fig. 6C). The midpoints of activation (in mV) were –24.1 ± 1.2 and –13.7 ± 1.5 (n = 7), and the slope factor of the curves were virtually unchanged (in mV) at 4.3 ± 0.7 and 4.4 ± 0.5 (n = 7) for 5 and 70 mM Sr2+, respectively. Moreover, the slope of the relation between the midpoint of activation and Sr2+ concentrations (0.20 VM–1; Fig. 6D) was similar to the value obtained for Ca2+ as the permeant ion (Rodriguez-Contreras and Yamoah 2003Go). Although the ~10-mV rightward shift in the activation curves as the external divalent cation concentration was increased from 5 to 70 mM was consistent with surface charge screening effects, the value fell short of the predicted shift assuming the absence of permeant ion binding interaction with surface charges (~25 mV) (Rodriguez-Contreras and Yamoah 2003Go; Zhou and Jones 1995Go). Thus Sr2+ binding to the surface charge may dictate the extent of the shift in the activation curve (Rodriguez-Contreras and Yamoah 2003Go).


Figure 4
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  		FIG. 4. Family of single channel [dihydropyridine-sensitive (L-type)] traces recorded in 5 and 70 mM Sr2+. A: exemplary consecutive single channel traces were generated at different test potentials (indicated on top of each column of traces). The holding potential was –70 mV and the concentration of Sr2+ in the patch pipette was 5 mM. The ensemble averaged current traces shown below the single channel current traces were derived from ~150 consecutive sweeps. At 5 mM Sr2+, the ensuing ensemble averaged currents were sustained in nature. The scale bars (horizontal = 100 ms and vertical = 1 pA). B: similar consecutive sweeps of single channel current traces obtained when the patch pipette contained 70 mM Sr2+. The vertical scale bar = 2 pA; the horizontal bar = 100 ms. The holding potential was –70 mV and the step potentials are indicated. Note that the ensemble averaged currents derived from ~150 traces showed a slow decay.

 

Figure 5
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  		FIG. 5. Probability (Po) of L-type channel openings. The Po was determined for Sr2+ currents using records from ~200 400-ms consecutive sweeps at different step potentials using 5 (bullet) and 70 mM ({circ}) in the patch pipettes. Note that the maximum Po was <0.5. The solid curves are fits to a Boltzmann equation (also see Fig. 6).

 

Figure 6
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  		FIG. 6. Changes from high to low Sr2+ concentrations A: average Po of individual single channel fluctuations recorded using 70 mM Sr2+ in the patch pipette plotted as a function of time and voltage. B: similar data were collected in 5 mM Sr2+. The surface plots show distinct modal gating in the L-type channel at different Sr2+ concentrations. C: shift in activation curves with changes in concentrations of Sr2+. Normalized nPo values (n = number of channels in a given patch) at different test potentials and Sr2+ concentrations [5 (bullet) and 70 mM ({circ})]. The solid lines represent fits to the Boltzmann equation Po = 1/[1+(e(V – V1/2))/k], where V1/2 is the half-activation potential and k is the slope of the e-fold change. The V1/2s (in mV) were –24 ± 1.2 and –13.7 ± 1.5 (n = 7) and ks (in mV) were 4.3 ± 0.7 and 4.4 ± 0.5 for 5 and 70 mM, respectively. D: the shift in V1/2 as function of Sr2+ concentration ({square}). The solid line represents a linear regression through the experimental data, and the dotted line is data from Ca2+ currents (Rodriguez-Contreras and Yamoah 2003Go).

 
If indeed the gating of the channel is altered by the permeant ion concentrations, and that at saturating levels the transition rate constants are altered, we would expect the waiting time to first opening or the first latency distribution to change in recordings performed at the two different spectrums of the sensitivity range of the channel. Figure 7 shows the normalized cumulative first latency distribution at 70 (A) and 5 mM Sr2+ (B) in the patch pipette solution. In accord with a voltage-dependent channel, the time constant of the first latency ({tau}f) was faster at depolarized than at hyperpolarized voltage steps. However, in keeping with the notion that the transition between closed states may be ion concentration dependent, {tau}fs were starkly faster in recordings performed in pipette Sr2+ concentrations that lay within the most sensitive phase of the channel's conductance–concentration relations (Fig. 3) than at saturating concentrations. Figure 7C compares the {tau}fs of Ba2+, Ca2+, and Sr2+ to assess the divalent cations that may promote multiple closed states or reluctance to transition from the closed states to first openings.


Figure 7
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  		FIG. 7. Changes in first latency distribution at different concentrations of Sr2+ A: cummulative 1st latency distribution (solid lines) at different step potentials indicated. The data were collected using 70 mM Sr2+ as the permeant ion. The dotted lines represent exponential fits to the distribution. As expected for a voltage-dependent current, the time constants of the 1st latency (F) were brief at depolarized potentials. B: similar data collected at low concentrations of the permeant ion (Sr2+ at 5 mM). The inset compares the time constants at 5 and 70 mM after liquid junction potential and surface charge screening effects have been considered. C: comparisons of the data obtained in Sr2+ (solid line) to 2 other divalent cations Ba2+ and Ca2+ (dotted lines) (Rodriguez-Contreras and Yamoah 2003Go).

 
Another direct way to determine the effects of charge carrier concentration on the kinetics of Sr2+ currents is to obtain the open and closed time distributions from the single-channel records. Figure 8 shows open and closed time distributions at Sr2+ concentrations of 70 (Fig. 8, A and B) and 5 mM (Fig. 8, C and D). At 70 mM Sr2+, channel gating could be described by two open and two closed states, whereas at 5 mM Sr2+ (Fig. 8, C and D), an additional open state was included in the fit of open time distributions. As shown, patch depolarization increased the duration of the open states, whereas it had the opposite effect on the duration of the closed states. In addition, the time constant of longer duration in both closed and open time distributions was more frequent than the time constant of brief duration as judged from the relative areas of each component. We also show the relation between open and closed state duration and the membrane potential at 70 (Fig. 9, A and B) and 5 mM Sr2+ (Fig. 9, C and D). Comparison of open and closed times suggests that, at 5 mM, when the channel is at its sensitivity range, not only does the dwell time of the open state increase, but also the dwell time of the closed state plummets. It is the opposite case for recordings at saturating concentrations of Sr2+ (70 mM).


Figure 8
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  		FIG. 8. Open and closed time distributions. Exponential distributions of open and closed time intervals were observed at all membrane potentials. Dwell time histograms were binned logarithmically (time in log scale) using 10 bins per decade and plotted with a square root transformation of the number of events. Data at 70 mM Sr2+ (A and B) were described by 2 open and 2 closed states. As shown in the insets of each panel, the duration of open states increased, whereas the durations of closed states decreased with membrane depolarization. The areas of each component, which represent approximately the time spent in the respective states, are shown in parentheses. At 5 mM (C and D), open and closed times were best described by 2 states at most potentials but at some potentials the open time distributions were best fit with 3 time constants.

 

Figure 9
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  		FIG. 9. Open and closed times are voltage dependent. Time constants of open and closed states were plotted at different membrane potentials for Sr2+ current recordings at 70 (A and B) and 5 mM (C and D). At 70 mM, brief open states and long closed states dominated. However, at 5 mM, long open times and brief closed times were the norm. A 3rd time constant is shown for open times (C) to show another component that was detected at more depolarized potentials (> –20 mV).

 

DISCUSSION
 
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Here, we report on the first extensive single-channel analysis of Sr2+ currents in native hair cells. These recordings showed elementary properties that are usually impossible to identify from reports that use saturating concentrations of divalent cations to study Ca2+ channels. By determining the sensitivity ranges of the channels and by using saturating and nonsaturating concentrations of Sr2+, nuances of the permeation and gating characteristics of the channels that are prone to both permeant ion concentration and surface charge screening effects, are shown. First, we showed that macroscopic Sr2+ currents essentially mirror the profile of the current of the physiological charge carrier Ca2+ in magnitude and activation. The inactivation of whole cell Sr2+ currents is much more similar to Ca2+, although subtle differences are apparent, which is in keeping with reports that Sr2+ can substitute for Ca2+ in several biological processes (Abdul-Ghani et al. 1996Go; Xu-Friedman and Regehr 1999Go). Second, these findings show that, for Ca2+ channel subtypes, such as the Cav1.3 channels in hair cells that are subjected to Ca2+-dependent inactivation, mediated by increase in intracellular Ca2+ within a few angstroms of the channel via calmodulin binding (Peterson et al. 2000Go; Rodriguez-Contreras and Yamoah 2003Go; Shen et al. 2006Go; Yang et al. 2006Go), outcomes of using high concentrations of the permeant ion should be interpreted with the following caveats. Specifically, analyses of the open probability, time constants of openings and closures, and the kinetics of first latency of openings can be influenced considerably by the concentration of the permeant ion. Additionally, these findings should be substantiated with the underpinnings of the key features of the handling of the divalent cation used in the study (Carter et al. 2002Go; Xu-Friedman and Regehr 1999Go). Third, increased Po, number of open states, and reduced dwell times of closed states of the channels in the physiologically important 1- to 5-mM range promotes the increase in whole cell current amplitude and duration. Thus in the functional context of the channels as biological Ca2+ transporters, the cellular physiological implications of these findings could be vast.

By virtue of its similar charge and size, Sr2+ substitutes well for Ca2+ in mediating synaptic transmission at the squid giant synapse (Augustine and Eckert 1984Go), neuromuscular junction (Bain and Quastel 1992Go; Dodge et al. 1969Go), and synapses in the brain (Tokunaga et al. 2004Go; Xu-Friedman and Regehr 1999Go), albeit at different kinetics. Indeed, Sr2+ can maintain mechanoelectrical transduction (Ohmori 1985Go) and activate Ca2+-activated K+ channels in hair cells (Sugihara 1998Go) and in other systems (Oberhauser et al. 1988Go). However, because intracellular Ca2+ buffers are less efficient in chelating Sr2+ than Ca2+ (Yawo 1999Go), replacing pipette Ca2+ with Sr2+ was predicted to reduce the influence of Ca2+-dependent modifications and intracellular Ca2+ buffers. Thus we surmised that Sr2+ entry through Ca2+ channels would be more suitable to examine the elementary properties of voltage-gated Ca2+ channels in hair cells than Ca2+ flux itself, which is confounded by Ca2+-dependent buffering and signaling.

Previous analyses of the concentration dependence of unitary current and conductance to determine the KD of Ca2+ channels have shown that the concentration versus conductance curves provide a more reliable measure since they are independent of surface charge screening effects and voltage errors introduced by liquid junction potentials (Church and Stanley 1996Go; Rodriguez-Contreras and Yamoah 2003Go; Rodriguez-Contreras et al. 2002Go). From the calculated maximum conductances and the KDs of the two Ca2+ channel subtypes, at the equivalent physiological Ca2+ concentration of ~2 mM, the conductance (in pS) of Sr2+ for the L- and non-L-type channels were 1.5 and 4.0, respectively. These conductance values dovetail well with extrapolated data from concentration dependence curves of Ca2+, which were ~1.0 and 2.7 pS for the L- and non-L-type channels (Rodriguez-Contreras et al. 2002Go). Indeed, direct measurement of the physiological Ca2+ conductance of the L-type channel yielded ~1.2 pS (Rodriguez-Contreras et al. 2002Go), which is within the range of these results. Thus at a membrane potential of hair cells (e.g., –65 mV), and a reversal potential for Sr2+/Ca2+ of ~100 mV, the limits of the flux rate of Ca2+ ions ranges from 500 to 1,400 ions/ms and that of Sr2+ ions ranges from 800 to 2,000 ion/ms. For clustered channels in hair cells, these unitary Ca2+/Sr2+ domains are expected to increase by ≥100-fold (Roberts et al. 1990Go; Rodriguez-Contreras and Yamoah 2001Go). Another essential feature of the concentration dependence curves is the difference in the steepness at 1–5 mM (Sr2+: 0.36 pS/mM; Ca2+; 0.34 pS/mM) and 10–70 mM (Sr2+: 0.08 pS/mM; Ca2+; 0.04 pS/mM), suggesting that the channels are ~8-fold and 4-fold more sensitive at physiological concentration ranges of Ca2+ and Sr2+, respectively. By retaining high selectivity and sensitivity, an optimal throughput of divalent cations is ensured at physiological concentration ranges.

A question that arises from this study is how can one evaluate the respective roles of subsaturating and saturating concentrations of permeant ions on 1) permeation, as it relates to means by which the permeating ions interact with binding sites within the pore of the channel (Kuo and Bean 1993Go); 2) allosteric effects of binding of divalent and/or monovalent ions to sites of the channel that mediate changes in protein conformational changes that produce alterations in gating mechanism (Zamponi and Snutch 1996Go); and 3) last, electrostatic actions of different concentrations of divalent ions on the membrane electric field (Hille 2001Go; Zhou and Jones 1995Go). Our Sr2+ current amplitudes and conductances ({gamma}) at saturating concentrations are consistent with those reported previously for Cav1.2, 1.3, 2.2, and 2.3 channels, which follow the sequence ({gamma}Ba > {gamma}Sr > {gamma}Ca) (Fox et al. 1987Go; Rodriguez-Contreras and Yamoah 2003Go). Moreover, comparison of the conductance obtained at subsaturating concentrations of divalent ions was within the range of prior observations (Rodriguez-Contreras and Yamoah 2003Go). In particular, the overall sequence in unitary conductances at subsaturating concentrations (5 mM) was distinct from data obtained at saturating concentrations (70 mM) for both channels (at 5 mM L-type: {gamma}Ba = {gamma}Sr > {gamma}Ca; non-L-type: {gamma}Sr > {gamma}Ba = {gamma}Ca). Of note, the results cannot be accounted for by the shifts in the activation curves from high to low concentrations of the permeant ions.

Although it is difficult to disentangle the effects of surface charge screening from allosteric effects, it has been shown that the binding of Ca2+ to Cav2.1 channels produces functional allosteric mechanisms that alter channel gating (Zamponi and Snutch 1996Go). In this study, we showed that changing the concentration of Sr2+ from saturating to subsaturating levels profoundly alters the gating of the Cav1.3 channels. In particular, at subsaturating levels, the Ca2+ channel exhibits increased long openings and a reduced closed time. Because both charge screening and selective binding of Sr2+ (Hille 2001Go) to the channel cannot account for these findings, we suggest that an allosteric interaction of Sr2+ with the L-type channel in hair cells may mediate a state-dependent alterations of a closed state (Gilly and Armstrong 1982aGo,bGo), which is in keeping with the ensuing alterations of the first latency distributions (Fig. 7). Specifically, the time constant of the first latency distribution was shorter at subsaturating concentrations of Sr2+ than at saturating levels.

The physiological interest of this study lies in the increased realization that Ca2+ channels in hair cells may exhibit strong Ca2+-dependent inactivation (Rodriguez-Contreras and Yamoah 2003Go; Shen et al. 2006Go; Song et al. 2003Go; Yang et al. 2006Go) that may vary during different stages of development and regeneration (Levic et al. 2007Go; Yang et al. 2006Go) against the backdrop of previous reports that hair cells use an efficient Ca2+-buffering mechanism (Roberts 1993Go) that may mask the genuine phenotype of Ca2+ currents. It is also clear that the Ca2+ channels are extremely sensitive at subsaturating concentrations of the permeant divalent ions, and we can infer that Ca2+-dependent inactivation might actually have dramatic effects on Ca2+ currents at physiological Ca2+ concentrations at the resting potential because the channels have a sizable Po at baseline. Analysis of the Ca2+ handling at the subnanometer Ca2+ domains of the Ca2+ channel in hair cells will show further details of the basic properties of the channel as a physiological Ca2+ transporter.


FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Address for reprint requests and other correspondence: E. N. Yamoah, Ctr. for Neuroscience, Program in Communication Science, Univ. of California, Davis, 1544 Newton Ct., Davis, CA 95618 (E-mail: enyamoah{at}ucdavis.edu)


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TOP
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 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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