Ca2+ influx is required to refill ryanodine-sensitive Ca2+ stores
We have shown previously that ryanodine-sensitive Ca2+ stores refill spontaneously in resting DRG neurones (Usachev et al. 1993). However, a significant portion of released Ca2+ is extruded immediately from the cell by active transport (Benham et al. 1992; Usachev et al. 1993; Werth et al. 1996) reducing the level of intracellular Ca2+ in the cytosol available for subsequent replenishment of the stores. Therefore, an external source of Ca2+ is probably involved in the refilling process in resting neurones. We tested this hypothesis by studying the role of Ca2+ entry in refilling ryanodine-sensitive stores.
In this series of experiments, [Ca2+]i was monitored in intact DRG neurones loaded with indo-1 AM. Caffeine (5 mM), a modulator of ryanodine receptors, was employed in a paired-pulse protocol in which the first (control) application of caffeine was used to deplete the stores, then after a delay, a second (test) caffeine stimulation was applied to probe the amount of Ca2+ that had reaccumulated in the stores (Fig. 1A). During depolarization, Ca2+ influx through VOCCs supplies Ca2+ to reload intracellular Ca2+ stores (Usachev et al. 1993; Garaschuk et al. 1997). In these experiments, a complete replenishment of the Ca2+ stores was provided before each pair of caffeine applications by a series of action potentials (10 Hz for 5 s) using extracellular field stimulation. Caffeine was added to Ca2+-free media to prevent Ca2+ influx during the release activation. The amplitude of the test [Ca2+]i response was normalized to the amplitude of the control response and used as an index of the efficiency of the refilling process. This paired-pulse protocol renders the peak [Ca2+]i dependent on the refilling state of the stores because basal [Ca2+]i was constant (± 20 nM) for a given pair of stimuli and [Ca2+]i buffering processes were much slower (the time from peak to basal [Ca2+]i was 165 ± 14 s) than the upstroke of the [Ca2+]i transient (the time from basal to peak [Ca2+]i was 24 ± 3 s; n= 16), providing a clear temporal separation of release from recovery. The Ca2+ stores refilled in a time-dependent manner. For example, the amplitude of the test response after 1 min of refilling was only 24 ± 9 % (n= 6) of control, whereas after 20 min the test response recovered to 86 ± 6 % of control (n= 8). The refilling was completely blocked (n= 4, data not shown) by 1 μM cyclopiazonic acid (CPA), a selective antagonist of sarco-endoplasmic reticulum Ca2+-ATPases (SERCAs) (Thomas & Hanley, 1994). This is consistent with the role of SERCAs in actively transporting Ca2+ into intracellular stores (Pozzan et al. 1994).
Figure 1. Ca2+ influx is required to refill ryanodine-sensitive Ca2+ stores
A, Ca2+ release was activated by 5 mM caffeine in Ca2+-free extracellular solution, as indicated by the horizontal bars. The paired [Ca2+]i transients are presented in order of increasing interstimulus interval as shown above the [Ca2+]i traces, although they were intermixed in the actual experiment. In B and C, the effect of extracellular Ca2+ (B) and Ni2+ (C) on the refilling process was studied using a paired-pulse protocol. The interval between two subsequent caffeine applications was 5 min. The horizontal bars indicate the duration of the treatments.
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Next, we tested whether replenishment of ryanodine-sensitive stores depended on extracellular Ca2+. Removal of extracellular Ca2+ during the interval between caffeine applications blocked refilling, as indicated by the failure of the second application of caffeine to evoke a [Ca2+]i response (n= 4; Fig. 1B). The response did not recover if the cell remained in Ca2+-free buffer (data not shown), suggesting that Ca2+ influx is needed to refill the stores. Furthermore, if the cell was treated with 2 mM Ni2+ during the refilling interval, as shown in Fig. 1C, the amplitude of the test [Ca2+]i response decreased from 69 ± 5 to 28 ± 5 % of control (n= 18; P < 0.001, Student's paired t test). The inhibitory effect of Ni2+ was fully reversible.
Ca2+ influx at resting membrane potential is not mediated by voltage-gated Ca2+ channels
Because the refilling of Ca2+ stores required Ca2+ influx, we tried to determine which channels mediated Ca2+ influx in unstimulated neurones. In non-excitable cells this influx is conducted by SOCCs (Berridge, 1995; Parekh & Penner, 1997). In neurones, VOCCs are the principal Ca2+ entry pathway during electrical activity (Lipscombe et al. 1988; Thayer & Miller, 1990). However, the contribution of VOCCs to Ca2+ entry at resting membrane potential is not clear. We investigated whether Ca2+ influx in resting DRG neurones was mediated by VOCCs.
DRG neurones express T-, L-, N- and P/Q-type Ca2+ channels (Fox et al. 1987; Rusin & Moises, 1995). We studied the effects of VOCC antagonists on the refilling of Ca2+ stores by using a paired-pulse protocol in which the caffeine stimuli were separated by 5 min intervals (Figs 2 and 3). Nimodipine and GsTX were used in combination to block L-type channels and N- and P/Q-type channels, respectively (McCarthy & TanPiengco, 1992; Piser et al. 1995). In these experiments, the normalized amplitude of the test [Ca2+]i response was 66 ± 5 % (n= 8) in untreated cells and was dependent on Ca2+ influx, as indicated by sensitivity to Ni2+ (2 mM). However, the combination of 10 μM nimodipine and 1 μM GsTX did not affect refilling of the stores (Fig. 2A) and the normalized amplitude of the test response after this treatment was 71 ± 6 % (n= 8). The same combination of drugs completely blocked the [Ca2+]i response elicited by depolarization with 40 mM K+ (n= 5; Fig. 2B) and inhibited by 94 ± 3 % (n= 6) high-threshold voltage-gated Ca2+ currents elicited by step depolarization from −60 to +10 mV (Fig. 2C).
Figure 2. Refilling of ryanodine-sensitive Ca2+ stores is not dependent on high-threshold voltage-gated Ca2+ channels
A, [Ca2+]i transients were elicited by 5 mM caffeine in Ca2+-free media. The cells were treated with a mixture of 10 μM nimodipine (Nim) and 1 μM ω-grammotoxin SIA (GsTX) during the 5 min period between two subsequent applications of caffeine. The duration of the drug application is indicated by the horizontal bars below the [Ca2+]i traces. B, [Ca2+]i transients were elicited by 40 mM K+. The combination of 10 μM nimodipine and 1 μM GsTX completely blocked depolarization-induced increases in [Ca2+]i. The break in the [Ca2+]i trace corresponds to 20 min. C, whole-cell Ca2+ currents were evoked by depolarization from −60 to +10 mV for 100 ms. Voltage pulses were applied every 30 s. The amplitude of the Ca2+ current is plotted versus time. The traces (right) show currents for the same experiments obtained before treatment (control) and after block with a combination of 10 μM nimodipine and 1 μM GsTX.
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Figure 3. Low-threshold voltage-gated Ca2+ channels do not mediate refilling of ryanodine-sensitive Ca2+ stores
A, [Ca2+]i transients were elicited by 5 mM caffeine in Ca2+-free media. Nicardipine (10 μM; Nic) was applied during the 5 min period between the two caffeine applications as indicated by the filled horizontal bar. B, whole-cell Ca2+ currents were evoked by depolarization from −90 to −30 mV for 200 ms. Voltage pulses were applied every 30 s. The amplitude of the Ca2+ current is plotted versus time. The current traces (right) represent Ca2+ currents before (control) and during treatment of the cell with 10 μM nicardipine.
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We next examined the contribution of T-type Ca2+ channels to the refilling of ryanodine-sensitive Ca2+ stores. We found that amiloride (500 μM), which is often used to block low-threshold voltage-gated Ca2+ channels, strongly interfered with indo-1 fluorescence (n= 4; data not shown) and therefore could not be used for [Ca2+]i measurements. Certain dihydropyridines have been reported to block T-type channels at high concentrations (Akaike et al. 1989). In DRG neurones, nicardipine was shown to be effective (Richard et al. 1991). In our system, 10 μM nicardipine inhibited by 97 ± 3 % (n= 5) low-threshold voltage-gated currents elicited by step depolarization from −90 to −30 mV (Fig. 3B). The effect of nicardipine reversed slowly with time. For comparison, 500 μM amiloride and 100 μM Ni2+ reduced low-threshold Ca2+ currents by 64 ± 3 % (n= 4) and 88 ± 3 % (n= 5), respectively (data not shown). However, 10 μM nicardipine did not slow the refilling process (Fig. 3A). In these experiments, the normalized amplitude of the test [Ca2+]i response was 71 ± 13 % (n= 4) in untreated cells and 77 ± 11 % (n= 4) after treatment with 10 μM nicardipine.
Voltage dependence can reveal important information about the nature of ionic fluxes. Hyperpolarization would be predicted to decrease Ca2+ influx mediated by VOCCs due to decreased channel activation (Fox et al. 1987; Magee et al. 1996). In contrast, SOCC-mediated Ca2+ influx would be greater at more negative potentials because the driving force for Ca2+ would increase without a decrease in channel opening (Parekh & Penner, 1997). The effects of changes in membrane potential during the refilling process are described in Fig. 4. The paired-pulse protocol was applied to neurones clamped at −55 or −80 mV using the whole-cell configuration of the patch-clamp technique. Because the stability of the electrical recording was impaired in Ca2+-free media (0.1 mM EGTA), extracellular Ca2+ was not removed during caffeine applications in these experiments. When cells were clamped at −55 mV during the interval between two caffeine applications, the normalized amplitude of the test [Ca2+]i response was 65 ± 6 % of the control (n= 8). Hyperpolarization to −80 mV significantly facilitated filling of the Ca2+ stores and the amplitude of the test response increased to 90 ± 7 % (n= 8; Fig. 4B). This facilitation was probably a result of enhanced Ca2+ influx. Indeed, 40 s hyperpolarization from −55 to −80 mV following depletion of the stores with 10 mM caffeine produced a small elevation in [Ca2+]i that could be detected in the presence of 5 mM Ca2+ in the extracellular solution (Fig. 4C). Ni2+ (2 mM) inhibited this elevation, reducing the amplitude of the response from 44 ± 11 to 15 ± 2 nM (n= 5). Similar results were obtained using the amphotericin B-based patch-clamp technique (n= 4; data not shown). These data demonstrate that in DRG neurones significant Ca2+ influx occurs at resting membrane potential that is independent of VOCCs. We next explored the possibility that SOCCs might mediate this Ca2+ influx. If SOCCs are present in these cells then Ca2+ influx might be increased by depletion of intracellular Ca2+ stores.
Figure 4. Hyperpolarization increases Ca2+ influx and facilitates the refilling process
DRG neurones were loaded with 100 μM indo-1 using the patch pipette. A, [Ca2+]i was measured in neurones clamped to −55 or −80 mV following stimulation with 5 mM caffeine, as indicated below the [Ca2+]i traces. For this paired-pulse protocol, the interstimulus time was 5 min. B, histogram displaying the amplitude of the second [Ca2+]i response normalized to the first for a paired-pulse protocol such as that described in A (n= 8). The membrane potential in the interval between the two subsequent caffeine applications is indicated below the bars. **P < 0.01, Student's paired t test. C, hyperpolarization induced an elevation in [Ca2+]i that was inhibited by 2 mM Ni2+. In both instances, hyperpolarization was applied after the stores were depleted with 10 mM caffeine. The extracellular Ca2+ concentration was increased to 5 mM.
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Depletion of intracellular Ca2+ stores facilitates Ca2+ influx in resting neurones
In non-excitable cells, store-operated Ca2+ influx is commonly exhibited as a sustained plateau phase following agonist-evoked Ca2+ release from intracellular stores. This plateau phase depends on extracellular Ca2+ and is thought to be mediated by store-operated Ca2+ channels (Jacob, 1990; Parekh & Penner, 1997). We found that in DRG neurones a rapid caffeine-induced [Ca2+]i rise was followed by a sustained [Ca2+]i elevation above the baseline (Fig. 5A). The amplitude of the initial [Ca2+]i increase did not depend on extracellular Ca2+ (172 ± 16 nM, n= 10, with 2 mM Ca2+ in the media, and 186 ± 16 nM, n= 10, in Ca2+-free media) consistent with Ca2+ release from intracellular stores. In contrast, the plateau phase could be abolished by removal of extracellular Ca2+ (n= 10), suggesting that it was mediated by Ca2+ influx. In the absence of extracellular Ca2+, caffeine-induced [Ca2+]i transients recovered completely to the baseline within 3 min, whereas in control (2 mM Ca2+ in the media) [Ca2+]i was elevated 47 ± 13 nM (n= 10) and 9 ± 3 nM (n= 10) above the baseline when measured 3 and 8 min, respectively, after the beginning of the response (Fig. 5B). Caffeine quenches indo-1 fluorescence, although this effect is wavelength independent and does not influence the ratiometric measurements (O'Neill et al. 1990). Thus, the observed plateau phase was unlikely to result from an indo-1-caffeine interaction. Furthermore, a similar sustained [Ca2+]i elevation was observed with another Ca2+-sensitive dye, fura-2 (n= 6, data not shown), the fluorescence of which increases in the presence of caffeine (Nohmi et al. 1992). The plateau phase displayed a graded inactivation. This may result from Ca2+-dependent inhibition of Ca2+ influx (Zweifach & Lewis, 1995; Madge et al. 1997), inactivation of ryanodine receptor-mediated Ca2+ release with subsequent store refilling (Pozzan et al. 1994), or an upregulation of Ca2+ efflux processes (Miller, 1991). Depletion of Ca2+ stores with CPA also produced a sustained elevation in [Ca2+]i; it decreased at a slower rate (plateau amplitude at 8 min = 63 ± 15 nM; n= 7), suggesting that inactivation of Ca2+ release contributed to the decline in [Ca2+]i during the plateau phase.
Figure 5. Caffeine-induced [Ca2+]i response exhibits a Ca2+ influx-dependent plateau phase
A, [Ca2+]i was measured in indo-1 AM-loaded DRG neurones during caffeine (10 mM) application. Representative [Ca2+]i traces obtained in the presence of 2 mM extracellular Ca2+ (2 mM Ca2+) or in Ca2+-free media were superimposed. B, histogram displaying the difference between [Ca2+]i and the baseline measured at the peak of the [Ca2+]i response, and at 3 and 8 min after the beginning of the response (n= 10). The baseline was measured before drug application. Data obtained in the presence or absence of extracellular Ca2+ were compared using Student's paired t test (***P < 0.001). Negative values correspond to [Ca2+]i levels below baseline.
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Store-operated Ca2+ influx in non-excitable cells can also be observed as a pronounced [Ca2+]i increase elicited by switching from Ca2+-free to Ca2+-containing media following depletion of intracellular Ca2+ stores (Clementi et al. 1992; Berridge, 1995; Bennett et al. 1998). Depletion of the stores opens plasmalemmal channels that allow Ca2+ to rush into the cell upon its return to the media. We found that readdition of external Ca2+ to the bath after caffeine-induced discharge of the stores resulted in a [Ca2+]i overshoot in 68 % of DRG neurones tested (n= 47; Fig. 6), which is consistent with the presence of store-operated Ca2+ entry in neurones. During this transient elevation, [Ca2+]i peaked at 37 ± 6 nM above resting [Ca2+]i (n= 28) and the maximal rate of [Ca2+]i rise was 4 ± 1 nM s−1 (n= 28). [Ca2+]i then recovered to the basal level. The recovery could be fitted by a monoexponential function with a time constant of 123 ± 7 s (n= 28). Ni2+ (2 mM), but not the selective VOCC antagonists, inhibited the overshoot by 62 ± 6 % (n= 8; Fig. 6C, see also Figs 1C, 2A and 3A). The overshoot was not observed if the switch from Ca2+-free to Ca2+-containing media (2 mM Ca2+) was not preceded by depletion of the stores (n= 6; Fig. 6A).
Figure 6. Depletion of intracellular Ca2+ stores increases Ca2+ influx
[Ca2+]i was measured in intact neurones loaded with indo-1 AM. A, a [Ca2+]i overshoot was observed after depletion of ryanodine-sensitive stores with caffeine. The breaks between the traces correspond to 15 min during which the cell was stimulated with 3 trains of action potentials (4 s at 10 Hz) elicited by extracellular field stimulation. B, for the recordings shown in A, changes in [Ca2+]i evoked by addition of 2 mM Ca2+ following store depletion with caffeine (empty) or with ‘full’ stores are superimposed. The caffeine-elicited [Ca2+]i transient is indicated (Caff). In C, the [Ca2+]i overshoots elicited by store depletion in the absence (control) or presence (Ni2+) of 2 mM Ni2+ are compared. Application of 5 mM caffeine is indicated on the trace (Caff). D, a [Ca2+]i overshoot was elicited by depletion of the stores with 5 mM caffeine or 5 μM cyclopiazonic acid (CPA), but not by 0.5 mM 3-isobutyl-1-methylxanthine (IBMX). Horizontal bars below the trace indicate the duration of treatment.
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In addition to its action on ryanodine receptors, caffeine is also known to inhibit phosphodiesterases and will thus increase the intracellular concentrations of cyclic AMP and cyclic GMP (Daly, 1993). These cyclic nucleotides may affect the Ca2+ permeability of the plasma membrane (Clementi & Meldolesi, 1996). To address the question of whether the action of caffeine resulted from an increase in cyclic nucleotides, we treated cells with 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), which is an approximately 30-fold more potent inhibitor of phosphodiesterases than caffeine (Daly, 1993), but will not release Ca2+ from stores at this concentration (Usachev & Verkhratsky, 1995). As shown in Fig. 6D, treatment with 0.5 mM IBMX did not produce a [Ca2+]i overshoot (n= 3). Furthermore, the induction of the overshoot did not depend on the delay between removal of caffeine and addition of Ca2+ to the external media. Thus, the overshoot resulted from depletion of Ca2+ stores and subsequent activation of Ca2+ influx. An alternative means of depleting Ca2+ stores is to inhibit the SERCAs with agents such as CPA (Thomas & Hanley, 1994). [Ca2+]i transients elicited by CPA (5 μM) developed more slowly and had a smaller amplitude than those induced by caffeine (Figs 6D and 9A). To ensure an effective discharge of the stores we applied CPA for 4–8 min (compare with 2–3 min for caffeine). After treatment with 5 μM CPA in Ca2+-free media, addition of 2 mM Ca2+ elicited a pronounced overshoot in [Ca2+]i in five of six neurones tested (Fig. 6D). The amplitude of the overshoot relative to the [Ca2+]i baseline was 75 ± 23 nM (n= 5).
Figure 9. Action potential-induced [Ca2+]i transients are modulated by the level of Ca2+ in ryanodine-sensitive Ca2+ stores
A, [Ca2+]i transients were elicited by extracellular field stimulation (10 Hz, 4 s) in intact neurones loaded with indo-1 AM, as indicated by triangles above the [Ca2+]i trace. Horizontal bars indicate application of 5 mM caffeine or 5 μM CPA. A comparison of corresponding [Ca2+]i transients before (1) and after (2) caffeine application, or in the presence of CPA (3), is shown in the inset. The traces were offset along the [Ca2+]i axes. In B and C, the changes in time constant that characterize the [Ca2+]i recovery process (B) and the amplitude of the [Ca2+]i elevation (C) for the experimental protocol described in A are analysed for 5 cells. Each point represents the mean ±s.e.m. of the corresponding parameter for the action potential-induced [Ca2+]i transients. A monoexponential function was fitted to the [Ca2+]i recovery process and the corresponding time constant was calculated by using a non-linear, least-squares curve fitting algorithm (Origin software). The data are plotted versus time, which was set to zero during the caffeine treatment (vertical dotted line).
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These data suggest that the overshoot reflects facilitation of Ca2+ influx after depletion of Ca2+ stores that, in turn, should accelerate replenishment of the stores. We compared the rate of refilling of the Ca2+ stores in cells that displayed an overshoot (Fig. 7A) with that in cells in which the overshoot could not be detected (Fig. 7B). As summarized in Fig. 7C and D, the replenishment occurred significantly faster in cells for which Ca2+ influx was facilitated by store depletion. Using a paired-pulse protocol (Fig. 1A), we found that the refilling process could be described by a monoexponential function with a time constant of 137 s for cells with the overshoot and 414 s for those without it (Fig. 7C). Furthermore, the amplitudes of the caffeine-induced [Ca2+]i response after 5 min of refilling recovered to 85 ± 4 % (n= 21) and 36 ± 8 % (n= 12) for the cells with and without overshoot, respectively (Fig. 7D).
Figure 7. Ryanodine-sensitive stores refill faster in neurones that exhibit a [Ca2+]i overshoot
A and B, [Ca2+]i transients were elicited by 5 mM caffeine in DRG neurones with (A) or without (B) an overshoot, to test the rate of refilling of the stores using a paired-pulse protocol. The interstimulus time was 5 min. In C, the time dependence of the refilling process was studied using a paired-pulse protocol in neurones with (•; n= 4) or without (▴; n= 5) a [Ca2+]i overshoot. An overshoot was defined as a net [Ca2+]i increase greater than 10 nM that recovered to the basal level within 5–10 min. Ryanodine-sensitive Ca2+ stores were depleted by the first (control) application of 5 mM caffeine in Ca2+-free media. The level of replenishment of the Ca2+ stores at a given time was evaluated by applying a second (test) stimulus with 5 mM caffeine in Ca2+-free media. Each point represents the mean ±s.e.m. of the test response normalized to the control for various interstimulus time intervals. Data points were fitted with a single exponential function (smooth curves) using a non-linear, least-squares curve fitting algorithm (Origin software, Microcal). In D, the normalized amplitudes of the test response for neurones with (n= 21; overshoot) and without (n= 12; not detected) a [Ca2+]i overshoot are compared for experiments such as those described in A and B. ***P < 0.001; Student's unpaired t test.
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Mn2+ permeates many Ca2+ pathways and, because it quenches the fluorescence of the Ca2+ indicator fura-2, it can be used as a probe for divalent cation entry (Jacob, 1990). We measured Mn2+ influx in intact neurones as a decrease in fura-2 fluorescence excited near the isobestic point for Ca2+ (360 nm). [Ca2+]i was monitored simultaneously by taking the ratio of the fluorescence signals excited at 360 and 380 nm (F360/F380). Figure 8A shows an example of such a recording. In untreated cells, F360 decreased slowly with time (0.07 ± 0.01 arbitrary units per second (a.u. s−1); n= 15) presumably as a result of fura-2 bleaching. To maximize Ca2+ influx, Ca2+ stores were depleted by application of 5 mM caffeine in Ca2+-free buffer. Addition of 300 μM Mn2+ increased significantly the rate of decay of F360 (0.39 ± 0.04 a.u. s−1; n= 11). A mixture of the VOCC antagonists GsTX (1 μM) and nimodipine (10 μM) did not change the Mn2+ influx, whereas it was inhibited by 2 mM Ni2+ (Fig. 8A); the F360 decay rate was slowed to 0.12 ± 0.02 a.u. s−1 (n= 11). Nicardipine (10 μM) was also without effect (n= 4; data not shown). These data are consistent with our previous observations (Figs 2A, 3A and 6C) and suggest that Mn2+ can be used as a probe for Ca2+ influx in neurones. Next we compared the rate of Mn2+ influx before and after depletion of Ca2+ stores by 5 mM caffeine (Fig. 8B and C) or 10 μM CPA (Fig. 8D and E). Both treatments significantly increased the F360 decay rate (P < 0.01 and < 0.05, respectively; Student's paired t test). After correcting for background decay of the F360 signal caused by fura-2 bleaching, we found that depletion of the stores with 5 mM caffeine or 10 μM CPA increased the rate of Mn2+ influx by 2.3 ± 0.5-fold (n= 6) and 2.0 ± 0.4-fold (n= 5), respectively.
Figure 8. Depletion of Ca2+ stores increases the rate of Mn2+ influx in DRG neurones
[Ca2+]i and Mn2+ (300 μM extracellular concentration) influx were measured simultaneously in fura-2 AM-loaded DRG neurones. Mn2+ influx was detected as a quench of 360 nm fluorescence (F360) shown in arbitrary units (a.u.). A, to maximize Mn2+ influx, Ca2+ stores were depleted with 5 mM caffeine (Caff) in Ca2+-free media. Mn2+ influx was slowed significantly by 2 mM Ni2+ but not by combined application of the VOCC antagonists GsTX (1 μM) and nimodipine (10 μM; Nim). Horizontal bars above the traces indicate the duration of the drug treatments. B-E, depletion of Ca2+ stores with 5 mM caffeine (B and C) or 10 μM CPA (D and E) increased the influx of Mn2+. The corresponding traces before (control) and after (C, Caff; E, CPA) depletion of the stores were offset along the fluorescence intensity axis and compared in C and E.
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