Corresponding author S. A. Thayer: Department of Pharmacology, University of Minnesota Medical School, 3-249 Millard Hall, 435 Delaware Street SE, Minneapolis, MN 55455, USA. Email: email@example.com
1Store-operated, voltage-independent Ca2+ channels are activated by depletion of intracellular Ca2+ stores and mediate Ca2+ influx into non-excitable cells at resting membrane potential. We used microfluorimetry, patch-clamp and Mn2+-quench techniques to explore the possibility that a similar mechanism exists in rat dorsal root ganglion (DRG) neurones in primary culture.
2Following caffeine-induced depletion, ryanodine-sensitive Ca2+ stores refilled with Ca2+ at resting membrane potential. The refilling process required extracellular Ca2+, was blocked by 2 mM Ni2+, and was facilitated by membrane hyperpolarization from −55 to −80 mV, indicating a key role for Ca2+ influx. This influx of Ca2+ was not affected by the voltage-operated Ca2+ channel (VOCC) antagonists nicardipine (10 μM), nimodipine (10 μm) or ω-grammotoxin SIA (1 μm).
3When ryanodine-sensitive Ca2+ stores were depleted in Ca2+-free media, a return to 2 mM external Ca2+ resulted in a pronounced [Ca2+]i overshoot, indicating an increased permeability to Ca2+. Depletion of Ca2+ stores also produced a 2-fold increase in the rate of Mn2+ influx. The [Ca2+]i overshoot and Mn2+ entry were both inhibited by Ni2+, but not by VOCC antagonists.
4Caffeine induced periodic Ca2+ release from, and reuptake into, ryanodine-sensitive stores. The [Ca2+]i oscillations were arrested by removal of extracellular Ca2+ or by addition of Ni2+, but they were not affected by VOCC antagonists. Hyperpolarization increased the frequency of this rhythmic activity.
5These data suggest the presence of a Ca2+ entry pathway in mammalian sensory neurones that is distinct from VOCCs and is regulated by ryanodine-sensitive Ca2+ stores. This pathway participates in refilling intracellular Ca2+ stores and maintaining [Ca2+]i oscillations and thus controls the balance between intra- and extracellular Ca2+ reservoirs in resting DRG neurones.
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In non-excitable cells, capacitative Ca2+ influx is the principal route of Ca2+ entry (Berridge, 1995; Clementi & Meldolesi, 1996; Parekh & Penner, 1997). Store-operated Ca2+ channels (SOCCs) that are activated by depletion of intracellular Ca2+ stores mediate this influx. The single channel conductance of SOCCs is estimated to be below 1 pS (Hoth & Penner, 1993). SOCCs are selectively permeable to Ca2+ and blocked by a number of inorganic cations (Hoth & Penner, 1993), but no organic antagonist has proven to be selective for these channels (Lo & Thayer, 1995; Clementi & Meldolesi, 1996). SOCCs are voltage independent; thus, SOCC-mediated Ca2+ influx persists at resting membrane potential. Hyperpolarization increases Ca2+ influx through these channels due to the increased driving force for Ca2+ (Parekh & Penner, 1997). In non-excitable cells that lack voltage-gated Ca2+ channels, SOCCs are the primary pathway for the Ca2+ influx that is needed to replenish intracellular stores, to maintain [Ca2+]i oscillations and to establish long-lasting increases in [Ca2+]i following agonist-induced Ca2+ release (Berridge, 1995; Clementi & Meldolesi, 1996).
In this report, we show that a mechanism similar to capacitative Ca2+ entry maintains Ca2+ influx in mammalian neurones at resting membrane potential. We used indo-1-based microfluorimetry in combination with whole-cell patch-clamp and Mn2+-quench techniques to study Ca2+ entry into cultured rat dorsal root ganglion (DRG) neurones before and after depletion of intracellular Ca2+ stores. We found that Ca2+ influx persisted in resting neurones, was not prevented by selective antagonists of voltage-gated Ca2+ channels and increased with hyperpolarization. Depletion of Ca2+ stores increased Ca2+ influx. Ca2+ influx was needed to replenish ryanodine-sensitive stores and maintain caffeine-induced [Ca2+]i oscillations. This pathway may regulate cellular Ca2+ levels between periods of electrical activity in neurones.
Rat DRG neurones were grown in primary culture as described previously (Thayer & Miller, 1990). In brief, 1- to 3-day-old Sprague-Dawley rats were killed by decapitation with sharp scissors, according to a protocol approved by the University of Minnesota Institutional Animal Care and Use Committee (IACUC). DRG were dissected from the thoracic and lumbar segments of the spinal cord and incubated at 37°C in collagenase-dispase (0.8 and 6.4 U ml−1, respectively) for 45 min. Ganglia were dissociated by trituration through a flame-constricted pipette and then plated onto laminin-coated (50 μg ml−1) glass coverslips (25 mm diameter). Cells were grown in Ham's F12 medium supplemented with 5 % heat-inactivated horse serum and 5 % fetal bovine serum, 50 ng ml−1 nerve growth factor (NGF), 4.4 mM glucose, 2 mM L-glutamine, modified Eagle's medium vitamins, and penicillin- streptomycin (100 U ml−1 and 100 μg ml−1, respectively). All cell culture reagents were purchased from Sigma. Cultures were maintained at 37°C in a humidified atmosphere of 5 % CO2. Cells were used within 2–6 days after plating.
[Ca2+]i was recorded from cultured DRG neurones by using indo-1-based microfluorimetry (Grynkiewicz et al. 1985). The instrumentation has been described in detail previously (Werth et al. 1996). Cells were placed in a flow-through chamber (Thayer & Miller, 1990) (10 s solution exchange) that was mounted on the stage of an inverted epifluorescence microscope equipped with a × 70 objective (Leitz, NA = 1.15). Indo-1 was loaded into the cells by incubation in medium containing 5 μM indo-1 AM and 0.02 % (w/w) Pluronic F-127 for 30 min at room temperature or via the patch pipette when the [Ca2+]i measurements were combined with patch-clamp recordings. The dye was excited at 350 nm (10 nm bandpass) and emission was detected at 405 (20) and 490 (20) nm. Fluorescence was monitored by a pair of photomultiplier tubes (Thorn EMI, Fairfield, NJ, USA) operating in photon-counting mode. The 5 V output signals were then integrated by 8-pole Bessel filters and digitized at 1 or 10 Hz with an analog-to-digital converter (Indec Systems, Sunnyvale, CA, USA). Data were stored and analysed on an IBM-compatible computer.
[Ca2+]i and Mn2+ influx were recorded simultaneously using fura-2-based microfluorimetry. The procedure for dye loading and the microscope configuration were similar to that used for indo-1. Fura-2 fluorescence was alternately excited at 360 (10) nm and 380 (10) nm by using a computer-controlled wheel (Sutter Instruments, Inc., Novato, CA, USA). Fluorescence images (510 nm, 40 nm bandpass) were projected onto a cooled charge-coupled device camera (Photometrics, Inc., Tucson, AZ, USA; 384 pixels × 576 pixels binned to 192 pixels × 288 pixels) controlled by an IBM-compatible computer. Image pairs were collected every 6 s and spatially averaged for further analysis.
Changes in the fluorescence of indo-1 (fura-2) were converted to [Ca2+]i using the formula:
where R is the 405 nm/490 nm (360 nm/380 nm) fluorescence intensity ratio (Grynkiewicz et al. 1985). The dissociation constants used were 250 and 224 nM for indo-1 and fura-2, respectively. β is the ratio of fluorescence emitted at 490 nm for indo-1 or excited at 360 nm for fura-2 measured in the absence and presence of Ca2+. Rmin, Rmax and β were determined in intact cells by applying 10 μM ionomycin in Ca2+-free buffer (1 mM EGTA) and saturating Ca2+ (5 mM Ca2+). Values for Rmin, Rmax and β, respectively, were 0.25, 2.3 and 3.5 for indo-1 and 1.0, 3.5 and 4.2 for fura-2. Mn2+ influx was measured as the change in fura-2 fluorescence excited near the isobestic point (360 nm). Background light levels were determined in an area that did not contain a cell.
Data are presented as means ±s.e.m.; n, number of cells tested. P < 0.05 was considered significant.
Whole-cell patch-clamp recordings (Hamill et al. 1981) were obtained using a patch-clamp amplifier (PC501; Warner Instrument Corporation, Hamden, CT, USA) and an analog-to-digital converter (Indec Systems). The whole-cell currents were filtered at 1 kHz and sampled every 200 μs, or 100 ms when recorded in combination with measurements of [Ca2+]i. Patch pipettes were pulled from borosilicate glass (Narishige; 2–4 MΩ) on a Sutter Instruments P-87 micropipette puller and filled with the following solution (mM): potassium gluconate, 125; KCl, 10; Mg-ATP, 3; MgCl2, 1; Hepes, 10; indo-1, 0.1; pH 7.25 with KOH, 290 mosmol kg−1 with sucrose. The extracellular recording solution contained (mM): NaCl, 140; KCl, 5; CaCl2, 2; MgCl2, 1; Hepes, 10; glucose, 10; pH 7.35 with NaOH, 310 mosmol kg−1 with sucrose. Ca2+-free solution was obtained by substituting 0.1 mM EGTA for Ca2+, unless otherwise indicated. To isolate Ca2+ currents from other currents, Cs+ was substituted for K+ in the pipette solution and extracellular Na+ and K+ were replaced with TEA+; 10 mM BAPTA replaced indo-1 in the pipette solution when patch-clamp recordings were not accompanied by [Ca2+]i measurements.
For the ‘perforated’ modification (Horn & Marty, 1988) of the patch-clamp technique the following solution was used in the patch pipettes (mM): KCl, 55; K2SO4, 70; MgCl2, 7; Hepes, 10; pH 7.25 with KOH. Amphotericin B was added to filtered pipette solution from a DMSO stock (2 mg amphotericin B in 40 μl DMSO) to a final concentration of 240 μg ml−1. The solution was kept in the dark and used within 2 h. The access resistance became < 20 MΩ within 5–10 min after seal formation and was further compensated by 30–40 %.
To evoke action potentials in intact neurones, extracellular field stimulation was employed (Werth et al. 1996). Field potentials were generated by passing current between two platinum electrodes via a Grass S44 stimulator and a stimulus isolation unit (Quincy, MA, USA). Trains of 1 ms pulses were delivered at a rate 10 Hz. The stimulus voltage sufficient to elicit a detectable increase in [Ca2+]i from a cell was determined before beginning an experiment, and subsequent stimuli were 20 V over this threshold.
Indo-1, indo-1 AM, fura-2 AM and Pluronic F-127 were obtained from Molecular Probes (Eugene, OR, USA). ω-Grammotoxin SIA (GsTX) was a gift from Dr Richard A. Keith at Zeneca Pharmaceuticals. All other reagents were purchased from Sigma.
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).
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).
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.
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.
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).
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).
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).
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.
The state of ryanodine-sensitive stores controls the size and the shape of [Ca2+]i transients elicited by trains of action potentials
We have shown that store-regulated Ca2+ influx is the principal source for refilling ryanodine-sensitive Ca2+ stores at the resting membrane potential in rat DRG neurones. When Ca2+ influx was blocked, intracellular Ca2+ stores remained empty. Ca2+ stores can regulate [Ca2+]i responses evoked by K+ application in neurones (Friel & Tsien, 1992; Usachev et al. 1993). We compared Ca2+ signals in response to physiological stimuli for full and empty stores (Fig. 9A). Trains of action potentials were elicited every 60 or 120 s (indicated by the triangles) by extracellular field stimulation (10 Hz) to produce [Ca2+]i transients in intact cells. For a given cell, the intensity of the stimulus remained constant during the recording. The amplitude (Δ[Ca2+]i) and the time constant for the monoexponential function that described the recovery process (τ) for each [Ca2+]i transient were analysed. Depletion of the stores by 5 mM caffeine significantly reduced the time constant from 12 ± 3 to 5 ± 1 s (n= 5; Fig. 9B) and it decreased the amplitude of the response from 181 ± 20 to 89 ± 12 nM (n= 5; Fig. 9C). This was probably the consequence of the increased capability of the emptied stores to buffer cytosolic Ca2+. Both parameters slowly recovered as the stores recharged during subsequent stimulation. Inhibition of the SERCAs with 5 μM CPA excluded Ca2+ stores from the buffering process and the resulting [Ca2+]i transients increased in amplitude to 167 ± 21 nM (n= 5) and recovery slowed more than 3-fold (τ= 28 ± 5 s; n= 5) relative to the last [Ca2+]i transient before CPA application (Δ[Ca2+]i= 125 ± 11 nM and τ= 9 ± 2 s; n= 5). The effect of CPA reversed completely in 5–8 min (Fig. 9). This is the first demonstration that intracellular stores contribute significantly to the amplitude and recovery kinetics of action potential-elicited [Ca2+]i transients. These findings corroborate previous observations (Friel & Tsien, 1992; Usachev et al. 1993; Toescu, 1998) that depletion of ryanodine-sensitive Ca2+ stores enhances Ca2+ uptake into the stores.
Caffeine-induced [Ca2+]i oscillations at resting membrane potential are controlled by store-regulated Ca2+ influx
We found that in 37 % (n= 134) of large (28–34 μm) DRG neurones 5 mM caffeine initiated [Ca2+]i oscillations (Fig. 10). These oscillations resulted from periodic Ca2+ release from and reuptake into ryanodine-sensitive stores (Lipscombe et al. 1988; Nohmi et al. 1992). The oscillations were maintained in neurones voltage clamped at −55 mV. However, they required a persistent Ca2+ influx, because addition of 2 mM Ni2+ to the buffer (Fig. 10A) or removal of extracellular Ca2+ (Fig. 10B) abolished the oscillations. The influx was not mediated by VOCCs because nimodipine (10 μM) and GsTX (1 μM) were without effect (n= 4). As shown in Fig. 10B-D, hyperpolarization from −55 to −80 mV increased significantly the frequency of oscillations from 0.6 ± 0.3 to 0.8 ± 0.3 min−1 (n= 5). This probably resulted from an increase in Ca2+ influx upon hyperpolarization and faster recharge of the Ca2+ stores, which subsequently accelerated the onset of the next [Ca2+]i spike.
In this report, we describe a Ca2+ influx pathway present in quiescent DRG neurones. The influx was not mediated by VOCCs, but rather shared certain features with capacitative Ca2+ influx. Hyperpolarization and depletion of ryanodine-sensitive Ca2+ stores enhanced the influx. This modest but persistent Ca2+ influx appeared to regulate the rate at which Ca2+ stores refilled with Ca2+, and produced marked effects on store-regulated processes such as Ca2+ buffering and [Ca2+]i oscillations.
Routes of Ca2+ entry in resting neurones
Neurones express multiple types of VOCCs (Bertolino & Llinás, 1992; Tsien et al. 1995). Upon excitation, Ca2+ enters the cell through these channels to initiate a number of processes including neurotransmitter release and transcription (Ghosh & Greenberg, 1995). This depolarization-induced Ca2+ influx also potentiates replenishment of intracellular Ca2+ stores, and thus depolarization is commonly used to refill Ca2+ stores in neurones (Usachev et al. 1993; Garaschuk et al. 1997). However, at the resting membrane potential, we found that VOCCs contributed little to Ca2+ influx or to refilling of Ca2+ stores in rat DRG neurones. Ca2+ influx was the rate-limiting step in the refilling of Ca2+ stores as indicated by the requirement for extracellular Ca2+, its block by Ni2+ and the enhanced refilling produced by hyperpolarization. In spite of this exquisite sensitivity to factors that influence Ca2+ influx, pharmacological block of VOCCs with dihydropyridine drugs and Ca2+ channel toxins had no effect on the refilling of the stores, resting Ca2+ influx, or Mn2+ influx. These drugs were clearly effective under our recording conditions (Figs 2B and C and 3B). The action of dihydropyridines is voltage dependent; depolarization enhances their inhibitory effect on L-type channels. However, at concentrations above 100 nM these drugs are effective even at hyperpolarized membrane potentials (McCarthy & TanPiengco, 1992). Therefore, it is unlikely that a lack of effect of 10 μM nimodipine on the refilling process could be explained by its voltage dependence. In fact, short pre-treatment of DRG neurones with 10 μM nimodipine at the resting membrane potential completely abolished high-K+-induced Ca2+ flux through L-type channels (Fig. 2B). Our observations are in agreement with the observation that dihydropyridines did not affect Ca2+ influx at rest in a neuronal cell line (Takemura et al. 1991), sympathetic (Lampe et al. 1995) or hippocampal neurones (Garaschuk et al. 1997) and are in contrast to another study that described a steady-state current mediated by dihydropyridine-sensitive channels in central neurones (Magee et al. 1996). This discrepancy might result from differences in dihydropyridine-sensitive (L-type) channels expressed in brain relative to peripheral tissues (Tsien et al. 1995) or the different methods used in these studies. The fact that VOCCs do not contribute to Ca2+ influx in DRG neurones at rest is in agreement with the voltage dependence of activation and inactivation for VOCCs in these cells (Fox et al. 1987). Despite the narrow ‘window current’ for T-type channels near the resting membrane potential (between −70 and −60 mV), their involvement is unlikely because hyperpolarization to −80 mV did not reduce, but instead enhanced, Ca2+ influx in resting DRG neurones (Figs 4 and 10). Similarly, the Na+-Ca2+ exchanger, which could potentially carry Ca2+ into the cell in reverse mode (Miller, 1991), can also be excluded from consideration because hyperpolarization would favour Ca2+ extrusion from the cell. Thus, in this study we describe a novel route of Ca2+ influx into resting DRG neurones.
The Ca2+ influx described here shares a number of features with SOCCs found in non-excitable cells (Berridge, 1995; Parekh & Penner, 1997). Ca2+ influx was: (1) enhanced by Ca2+ store depletion, (2) enhanced by hyperpolarization, (3) blocked by Ni2+, and (4) not sensitive to selective VOCC antagonists. The idea that SOCCs might participate in neuronal function is consistent with the high level of expression of mammalian homologues of the Drosophila trp gene in brain; TRP channels are believed to mediate capacitative Ca2+ entry (Mori et al. 1998; Philipp et al. 1998). Ca2+ influx induced by store depletion was most easily detected as a sustained [Ca2+]i plateau phase during the caffeine-induced response (Fig. 5), and as a [Ca2+]i overshoot following the switch from Ca2+-free to 2 mM Ca2+-containing media (Fig. 6). In the latter case, a similar result might occur if Ca2+ buffering mechanisms were downregulated during the Ca2+-free treatment, but this was not the case because the overshoot was dependent on depletion of the store by caffeine (Fig. 6), a treatment that actually enhanced buffering of electrically induced [Ca2+]i increases (Fig. 9). Furthermore, the influx of Mn2+, a cation that permeates SOCCs (Jacob, 1990), was enhanced by approximately 2-fold after depletion of the stores. The recovery of the overshoot was approximately 10-fold slower than that of electrically induced [Ca2+]i transients suggesting that termination of the overshoot was determined by inactivation of Ca2+ influx rather than by Ca2+ buffering processes. Once activation of SOCCs is initiated by depletion of the stores, these channels inactivate as the consequence of refilling of the store (Jacob, 1990; Zweifach & Lewis, 1995). In DRG neurones, the time course for refilling the stores with Ca2+ was characterized by virtually the same kinetics (τ= 137 s) as the recovery from the overshoot (τ= 123 s), suggesting that inactivation of Ca2+ influx in these cells was also controlled by replenishment of intracellular Ca2+ stores.
Store-operated Ca2+ influx in non-excitable cells has been mainly attributed to depletion of inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores (Parekh & Penner, 1997). Here we demonstrate a similar link between depletion of ryanodine-sensitive Ca2+ stores and Ca2+ influx. Both types of store are associated with the endoplasmic reticulum and there is a high level of structural and functional similarity between IP3 and ryanodine receptors (Pozzan et al. 1994). The fact that Ca2+ influx can be activated by depletion of Ca2+ stores with SERCA inhibitors (Thomas & Hanley, 1994) indicates that formation of IP3 is not required for this activation. Therefore, it is plausible that depletion of ryanodine-sensitive stores might initiate mechanisms similar to those proposed for the IP3-sensitive stores (Berridge, 1995; Parekh & Penner, 1997). Indeed, activation of Ca2+ influx by depletion of ryanodine-sensitive Ca2+ stores has been reported recently in PC12 cells (Clementi et al. 1992; Bennett et al. 1998). This influx was similar to that evoked by IP3 or the SERCA inhibitor thapsigargin in the same cells, suggesting that different intracellular Ca2+ stores may utilize common pathways to induce capacitative Ca2+ entry.
Physiological role of the store-regulated Ca2+ influx in neurones
While voltage-gated Ca2+ channels are the major pathway for Ca2+ influx during excitation in neurones, the novel mechanism of Ca2+ influx described in this study may be important between periods of electrical activity. We estimate that the electrical current corresponding to this influx, based on the maximal rate of [Ca2+]i rise during the [Ca2+]i overshoot (Fig. 6), is approximately 3 orders of magnitude smaller than the Ca2+ current activated during an action potential (Fox et al. 1987; Thayer & Miller, 1990). However, because this influx persists at resting membrane potential, in 1–2 s it carries as much Ca2+ as that carried during an action potential. The sustained Ca2+ influx elicited by store depletion in sympathetic neurones protected these cells from apoptosis induced by nerve growth factor deprivation (Lampe et al. 1995).
The Ca2+ influx described here served to replenish intracellular Ca2+ stores. The Ca2+ level within the stores is important for several reasons. The stores participate in removal of Ca2+ from the cytosol during cell excitation. We found that the efficiency of this transport was significantly increased when the stores were depleted. Depletion of the stores with 5 mM caffeine resulted in a more than 2-fold decrease in the amplitude and duration of electrically induced [Ca2+]i responses (Fig. 9), both of which recovered as the stores refilled. Caffeine (10 mM) reduced Ca2+ currents by approximately 25 % in sympathetic neurones, although the current recovered within a few seconds after removal of the drug (Lipscombe et al. 1988). An effect of caffeine on VOCCs was unlikely to influence the experiments described here because the first post-caffeine electrical stimulation was applied 60 s after the drug was completely washed from the bath (Fig. 9A), as confirmed by recovery from the caffeine-induced quench of indo-1 fluorescence (O'Neill et al. 1990). Thus, our observations indicate that the Ca2+ level within the store determines the size and duration of the [Ca2+]i transients elicited by a given pattern of electrical activity, which in turn would control triggering of various Ca2+-dependent cell functions (Ghosh & Greenberg, 1995). Chronic depletion of Ca2+ stores has been shown to disturb gene expression by inhibiting molecular transport across the nuclear envelope (Perez-Terzik et al. 1997) and to block protein synthesis (Thomas & Hanley, 1994).
Ca2+ influx was required to maintain caffeine-induced [Ca2+]i oscillations at resting membrane potential in neurones (Fig. 10). Store-regulated Ca2+ entry enabled changes in membrane potential to tune the frequency of [Ca2+]i oscillations. The exact role of [Ca2+]i oscillations in neurones is not clear, although some observations suggest that certain patterns of periodic [Ca2+]i spikes are important for regulation of gene expression (Itoh et al. 1995) and cell growth and differentiation (Gu & Spitzer, 1995; Gomez et al. 1995).
We have shown that mammalian sensory neurones possess a distinct Ca2+ entry pathway that is active at rest and enhanced by depletion of Ca2+ stores. This observation is consistent with studies in non-excitable cells that describe specialized channels that are somehow activated by store depletion and play an important role in refilling the store with Ca2+ and maintaining sustained Ca2+ influx. Why neurones, with their extensive complement of voltage- and receptor-operated Ca2+ channels, need yet another Ca2+ entry pathway is not entirely clear. However, the pathway is present and appears to maintain the Ca2+ balance between intracellular and extracellular pools in DRG neurones at resting membrane potential.
The National Science Foundation (IBN9723796) and the National Institutes of Health (DA07304, DA09293) supported this work. Y. M. U. is a research fellow of the American Heart Association, Minnesota Affiliate. We thank Dr Richard A. Keith at Zeneca Pharmaceuticals for providing ω-grammotoxin SIA, and Kyle T. Baron for excellent technical assistance.