INTRODUCTION

In many cell types, depletion of intracellular calcium stores either by receptor activation or by inhibition of intracellular Ca²⁺-ATPases results in the activation of a calcium-carrying ``capacitative'' current at the plasma membrane (for review see Refs. 1 and 2)). The calcium permeability underlying this capacitative current (also known as I_CRAC (calcium release-activated current (3)) is of considerable interest, since it may play a role in sustained calcium responses and oscillations (1, 4), T-lymphocyte activation (5, 6), regulation of secretion (7, 8), steroidogenesis in adrenal cells (9), adenylate cyclase regulation (10), predisposition to hypertension in humans (11), human immunodeficiency (12), and perhaps hypothermia-induced organ damage (13).

There exist two major questions regarding the capacitative current: 1) What signal(s) communicates the filling state of the calcium stores to the plasma membrane, and 2) what channel(s) open in response to this signal? No clear candidate for the signal from depleted stores has been identified, although numerous second messengers have been suggested (for review see Refs. 1 and 2)). Similarly, the calcium-specific channel(s) that mediate this pathway are generally unknown and are only observed as whole cell currents (3, 5, 14, 15, 16, 17, 18, 19). However, small conductance plasma membrane channels underlying a capacitative current have been identified using single channel patch methods in an epithelial cell line (20) and in endothelial cells (21). Finally, the gene product underlying the trp Drosophila photoreceptor mutant has been characterized as mediating a calcium-selective leak current that is activated by calcium store depletion (22, 23), but single-channel properties remain to be determined.

Here, we provide evidence that cultured skeletal muscle cells possessed a capacitative current that was mediated by the calcium-specific leak channel, first described in skeletal muscle in studies of Duchenne muscular dystrophy (24, 25, 26, 27, 28). In addition, we describe the effects of two dihydropyridine (DHP)¹ compounds that inhibited the calcium leak channel, capacitative manganese influx, and refilling of intracellular calcium stores. Finally, we examined regulation of the skeletal muscle capacitative pathway by second messenger pathways that have been implicated in other cell types.

EXPERIMENTAL PROCEDURES

Mouse myoblasts and myotubes were prepared from C57/BL normal or mdx mouse hindlimb muscle as described previously (27) and used after 8-16 days. The mdx mouse is an animal model for the human muscle wasting disease Duchenne muscular dystrophy. It possesses similar genetic and biochemical defects as Duchenne humans (29, 30), including elevated activity of the calcium leak channel and elevated resting calcium levels (24, 25, 26, 27, 28, 31, 32, 33). Myoball cultures were prepared from myotube cultures as described (34), using 1-min exposure to 0.05% trypsin, and used after 6-21 days. Manganese quenching assays and calcium measurements were performed as described (27, 28), using the fluorescent, calcium-chelating molecule fura-2/AM ester (35). Changes in intracellular fura-2 fluorescence intensity at the isosbestic wavelength (357 ± 5 nm) were assayed in the presence of extracellular MnCl₂ as a measure of plasma membrane permeability to Mn²⁺, an indirect indicator of Ca²⁺ permeability (36, 37, 38, 39). MnCl₂ was used at 100 µM, unless otherwise indicated. In control experiments without Mn²⁺, the intensity at 357 nm changed no more than 5% during high potassium or CPA-induced calcium transients. The rate of loss of fluorescence intensity was divided by the initial fluorescence intensity in the cell to correct for differences in cell size and expressed as percentage per minute (%/min). Fluorescence experiments were carried out at 32 °C, rather than 37 °C, to reduce compartmentalization of fura-2 (28, 40).

Patch methods were as described previously (27, 28), using 96 mM BaCl₂ or MnCl₂ as charge carrier in the pipette. The leak channel is similarly permeable to Mn²⁺ and Ba²⁺ (28). Data were recorded at a 3- or 10-kHz sampling rate with 2-kHz filtering and filtered at 1 kHz upon playback. Unless otherwise indicated, all experiments were carried out in a low calcium rodent Ringer's solution (RRS, 138 mM NaCl, 2.7 mM KCl, 1.06 mM MgCl₂, 0.18 mM CaCl₂, 5.6 mM glucose, 12.4 mM HEPES, pH 7.2) to suppress spontaneous contractions. Nominally calcium-free Ringer's solution was RRS with no CaCl₂ added.

AN406 (dimethyl 2,6-dimethyl-4-(4-trifluoromethylphenyl)-1,4-dihydropyridine-3,5-dicarboxylate) and AN1043 (dimethyl 2,6-dimethyl-4-(4-bromophenyl)-1,4-dihydropyridine-3,5-dicarboxylate) were generously provided by Athena Neurosciences (South San Francisco, CA), and were prepared in 10 mM stocks in Me₂SO and stored at 20 °C. Cell-free solutions used to test drug autofluorescence contained 130 mM KCl, 10 mM NaCl, 10 mM MOPS, pH 7.02, 0.1% fura-2 buffer (1.1 M D-gluconic acid, K⁺ salt; 10 mM HEPES; 10 mM CaCl₂, pH 7), 10 µM fura-2 free acid, either 0.8 mM EGTA or 0.4 mM calcium, and 0.1% Me₂SO or 0.1% (10 µM) AN compound dissolved in Me₂SO. AN compounds are stable and retain potency for ~4 h at room temperature.

Okadaic acid, econazole, and genistein were from Research Biochemicals International (Natick, MA). Cyclopiazonic acid and all other chemicals were from Sigma. We chose to use CPA instead of thapsigargin, another ATPase inhibitor (37), because effects of CPA are reversible with washout, while those of thapsigargin are not.

All significance values were calculated using a two-tailed Student's t test, unless otherwise indicated. All data are expressed as mean ± S.E.

To ensure that changes in fluorescence intensity after the addition of AN1043 were due to action upon the leak channel, we must ensure that drug interactions with fura-2 or autofluorescence did not significantly contribute to the measured fluorescence intensity changes. AN1043 (10 µM) and AN406 (10 µM) did not significantly affect fura-2 fluorescence intensity in cell-free solutions compared with Me₂SO controls (data not shown). Also, 10 µM AN1043 did not affect measured [Ca²⁺]_i during calibration of myotubes with ionomycin (1-2 µM) in extracellular RRS containing 250 nM calcium (AN1043 present: 245 ± 11 nM calcium, n = 5; AN1043 absent: 250 ± 12 nM calcium, n = 5). Finally, AN1043 did not affect fura-2 detection of ionomycin-induced manganese influx or release of intracellular calcium (data not shown). These data suggest that AN1043 did not affect fura-2 measurement of [Ca²⁺]_i.

A small autofluorescence artifact, detected as slowly increasing fluorescence intensity, was observed after the addition of 10 µM AN1043. This was probably a consequence of drug binding to myotubes, because the rate of fluorescence increase was lower in dishes that had been scraped to remove cells (42 ± 11 intensity units/min, n = 3; intensity values here and below are given for 357 nm and are an arbitrary scale), and the rate of increase was not different (p > 0.2) between myotubes loaded with fura-2 (219 ± 28 intensity units/min, n = 24 of 34 cells) and unloaded myotubes (154 ± 46 intensity units/min, n = 12 of 12 cells). Similar autofluorescence effects were observed with AN406 (data not shown).

As described below, the rate of manganese quenching of fura-2 fluorescence decreased significantly after the addition of AN1043 or AN406 (see Fig. 4 and Table II). Decreased quenching rates could have resulted from AN compounds blocking leak channel activity and preventing manganese entry or could have been due to drug autofluorescence (a rise in fluorescence intensity) superimposed on the manganese-induced fluorescence quenching (a drop in fluorescence intensity). However, the latter possibility is unlikely for the following reasons.

Table II. 10 µM AN406 and AN1043 inhibited manganese quenching of fura-2 fluorescence intensity after the addition of CPA See legend of Table I for methods. Resting quenching rates before CPA addition are not shown. Data from myotubes and myoballs are combined. Significance of quenching rate before versus after addition of AN compound was determined using a paired T-test. Compound tested Fura-2 quenching rates before AN compound Fura-2 quenching rates after AN compound AN1043 (n = 16) 8.9  ± 1.2%/min 0.83  ± 0.4%/mina AN406 (n = 2) 10.6  ± 2.0%/min 0.49  ± 0.35%/minb a  p < 0.001. b  p < 0.04.

1) Fura-2 fluorescence intensity in cells for experiments in which MnCl₂ was added was 9100 ± 1450 intensity units (n = 28). Thus, autofluorescence should have contributed a mean 2.4%/min (219 units/9100 units) increase in fluorescence intensity. This was significantly less than the inhibition in fluorescence quenching observed after the addition of 10 µM AN1043 to CPA- and manganese-treated myotubes (quenching rate before AN1043, 8.9 ± 1.2%/min; after AN1043, 0.83 ± 0.4%/min; see below).

2) Fluorescence intensity rose upon addition of AN1043 and then reached a plateau after 2.6 ± 0.2 min (n = 20 of 24 cells) with no further elevations in intensity. However, in manganese influx experiments, manganese quenching rates remained near 0%/min for more than 10 min after the addition of AN1043 (n = 10, see Fig. 4, B and C). Thus, AN1043 inhibition of manganese-induced quenching persisted long after autofluorescence effects should have contributed to the observed intensity changes. These data also support the idea that autofluorescence was due to drug binding to myotubes at nonspecific sites, which became saturated after several minutes.

RESULTS

Calcium leak channel activity in cardiac and skeletal muscle cells is increased by DHPs such as nifedipine (24, 25, 28, 41), which also acts as an antagonist of the L-type voltage-dependent calcium channel (VDCC) (42). Since DHP compounds could be either antagonists or agonists for the L-type VDCC, we reasoned that DHP analogs that were not good at inhibiting VDCC might be good antagonists for calcium leak channels. Therefore, we screened an assortment of uncommon DHP compounds for ability to lower resting [Ca²⁺]_i levels, perhaps indicating antagonism of the calcium leak channel. Two analogs (Fig. 1), called AN406 and AN1043 reduced resting [Ca²⁺]_i,² and therefore were tested more directly for antagonism of leak channel activity.

AN1043 (Fig. 2, A and B) and AN406 (Fig. 2, C-E), but not Me₂SO (Fig. 2D), significantly decreased the open probability (P_o) of leak channel during single channel patch analysis in myotubes conducted at 22 °C. Due to variability in initial channel activity, P_o before the addition of AN406 in Fig. 2C was normalized to 100%, and P_o at subsequent times was expressed relative to initial P_o. Data for AN406 include channels from eight mdx cell-attached patches, four mdx excised patches, and one normal excised patch. Data for Me₂SO include channels from three mdx cell-attached patches, seven mdx excised patches, and one normal excised patch. As excised and cell-attached patches showed similar behavior, the data were pooled.

Channel open probability was calculated using the following relationship: (mean open time)/(mean open time + mean closed time). Open and closed dwell times of the calcium leak channel are generally very fast (<1 ms) (24, 25, 26, 27), and channel amplitude is small relative to noise. Thus, sampling rate and filtering introduce some error by reducing the apparent duration of shorter open and closed time events and by causing some apparent variability in channel amplitude (see Refs. 43, 44, 45). However, any error in measurement of open and closed time events should have affected the measured P_o before and after drug addition similarly and thus do not detract from our basic observation of inhibition of leak channel activity by AN compounds.

Previously, no role has been discerned for the calcium leak channel. However, this channel exhibits many properties, such as small size and conductance (7-14 pS) and voltage-independent activity (24, 25, 26, 27), which makes the channel a possible candidate for a conductance underlying a capacitative current (see Ref. 21). We examined whether cultured myotubes exhibited a capacitative current and used AN1043 and AN406 to investigate the potential role of the calcium leak channel in this process.

Fig. 3A shows changes in intracellular free calcium ([Ca²⁺]_i) in fura-2-loaded skeletal myotubes after the addition of 50 µM cyclopiazonic acid (CPA), an inhibitor of Ca²⁺-ATPases from the endoplasmic and sarcoplasmic reticulum (37). As observed in other cell types (19, 37), the addition of CPA in calcium-free RRS led to a rapid increase in [Ca²⁺]_i, which returned rapidly to base line (Fig. 3A), while the addition of CPA in RRS containing normal (1.8 mM) extracellular calcium concentrations resulted in a much greater [Ca²⁺]_i elevation and a prolonged plateau phase after the initial [Ca²⁺]_i peak (Fig. 3A, C-F).

The addition of 10 µM AN1043 to the bath resulted in a rapid decrease in the CPA-induced [Ca²⁺]_i plateau (Fig. 3C, 64 ± 10% decrease, n = 4). The addition of 3 mM EGTA to the bath (19) also resulted in a rapid decrease in plateau [Ca²⁺]_i (Fig. 3, D and E, 51 ± 2% decrease, n = 8), indicating that part of the plateau calcium corresponded to the influx of calcium across the sarcolemma, presumably in response to store depletion. The CPA-induced [Ca²⁺]_i plateau was not affected by 10 µM Gd³⁺ (Fig. 3D, n = 4), unlike capacitative currents in other cell types (23), but 30 µM La³⁺ did reduce plateau [Ca²⁺]_i (Fig. 3F, 56 ± 2% decrease, n = 9), as observed in several cell types (8, 10, 23, 46). Fig. 3D and 3E also show the variability in the [Ca²⁺]_i response after CPA addition in 1.8 mM extracellular calcium.

Regulation of resting [Ca²⁺]_i is complicated by the number of molecules, such as channels, pumps, and buffers, that can affect calcium homeostasis. To assay sarcolemmal divalent cation influx more directly, we measured rates of Mn²⁺ entry as evidenced by quenching of fura-2 fluorescence at 357 nm. This method is used as an indirect indicator of calcium permeation in many studies (e.g. Refs. 36, 37, 38, 39), since many capacitative currents also carry manganese.

The addition of CPA significantly increased the rate of manganese quenching of fura-2 fluorescence intensity in cultured myotubes (Fig. 4, A and D, and Table I) and myoballs (Fig. 4C, Table I), indicating increased sarcolemmal permeation of Mn²⁺. Me₂SO addition did not change Mn²⁺ quenching rates (Fig. 4B, n = 7). The Mn²⁺ influx induced by the addition of CPA was significantly inhibited by AN406 and AN1043 (data for 5 µM shown in Fig. 4D; data for 10 µM shown in Table II). Also, the addition of 10 µM AN406 and AN1043 significantly reduced resting Mn²⁺ quenching rates (e.g. see Fig. 4B). Thus, CPA increased sarcolemmal permeation of Mn²⁺, and both DHP analogs prevented Mn²⁺ entry at the sarcolemma after the addition of CPA and at rest. In Fig. 4B, 100% represented ~12,000 intensity units in the AN406 trace; the rate of fluorescence quenching by manganese was 9.7%/min before AN406 and near 0%/min after AN406. In Fig. 4C, 100% represented ~13,500 intensity units; the rate of fluorescence quenching after the addition of CPA was 6.7%/min and near 0%/min after the addition of AN1043.

Table I. CPA increased the rate of manganese quenching of fura-2 fluorescence intensity 100 µM MnCl2 was added 2-4 min before CPA. The rate of loss of fluorescence intensity at 357 nm was divided by the initial fluorescence intensity in the cell to correct for differences in cell size and expressed as percentage per min (%/min). Significance of quenching rate before versus after CPA addition was determined using a paired T-test. Cell type Fura-2 quenching rates before CPA Fura-2 quenching rates after CPA Myotube (n = 45) 1.79  ± 0.37%/min 6.64  ± 0.67%/mina Myoball (n = 7) 0.67  ± 0.41%/min 4.67  ± 0.72%/minb a  p < 0.0001. b  p < 0.001.

It is possible that stores were partially depleted by incubation in RRS containing 0.18 mM calcium (which is used to suppress spontaneous contractions as well as delay store refilling). If so, cells with depleted stores should have exhibited higher resting Mn²⁺ flux rates, which would correlate inversely with the amount of calcium released by CPA. However, no correlation was observed (correlation coefficient = 0.06, n = 45, p > 0.6), suggesting that myotubes maintained their store integrity in the presence of 0.18 mM external calcium.

Changes in fluorescence intensity after the addition of AN1043 were not due simply to drug interactions with fura-2 or autofluorescence effects, although a small fluorescence artifact was observed after the addition of AN1043 to some cells (see ``Experimental Procedures'').

Several lines of evidence suggest that Mn²⁺ influx rates in skeletal myotubes were an accurate measure of leak channel activity, and not that of some other channel, such as the VDCC. Mn²⁺ influx, calcium leak channel P_o, and resting calcium levels were all increased by nifedipine (25, 28) and CPA (this study), decreased by AN406 and AN1043 (this study), are greater in mdx than normal myotubes (24, 25, 26, 27, 28), and were not inhibited by antagonists of VDCC, including nifedipine, nimodipine (28), diltiazem, and verapamil (5-50 µM, n = 9; Fig. 4A) (24). Finally, depolarization of GH3 or adrenal chromaffin cells with elevated K⁺ leads to a rapid increase in Mn²⁺ influx, which is blocked by DHP antagonists of the VDCC (47), but depolarization of myotubes with 25 mM K⁺ decreased manganese quenching rates (before K⁺, 4.85 ± 1.03%/min; after K⁺, 1.68 ± 0.51%/min; n = 4, p < 0.02, paired t test). These data from myotubes are consistent with observations (48) suggesting that depolarization decreases Mn²⁺ influx by reducing the driving force for divalent cations but are inconsistent with the hypothesis that depolarization activates a Mn²⁺-permeable VDCC.

It is possible that the increased permeability to manganese and calcium after CPA addition was triggered directly by the elevated [Ca²⁺]_i (e.g. see Fig. 4C), and was not necessarily related to store depletion. To test this possibility, calcium stores were depleted by the addition of CPA in calcium-free RRS, and permeability to calcium was determined 10-12 min after CPA addition. By this time, resting [Ca²⁺]_i had returned to base line (see Fig. 5, A and B). Therefore, any increased sarcolemmal permeability to calcium or manganese would be related directly to calcium store depletion, and not to elevated [Ca²⁺]_i, and would thus represent a ``capacitative'' current (see Refs. 1 and 2).

To test permeability to calcium, 1.8 mM calcium was added to the bath 10-12 min after the addition of CPA, and peak [Ca²⁺]_i and rate of [Ca²⁺]_i rise were determined. Both peak [Ca²⁺]_i and rate of [Ca²⁺]_i rise were significantly greater after CPA-related store depletion compared with Me₂SO controls (p < 0.001, Fig. 5, A and C-E), indicating that store depletion led to increased sarcolemmal calcium permeability even when resting [Ca²⁺]_i levels had been at base line for greater than 5 min. Also, peak [Ca²⁺]_i and rate of [Ca²⁺]_i rise were significantly reduced by the addition of 10 µM AN1043 2-4 min before the addition of 1.8 mM calcium (p < 0.001, Fig. 5, B, D, and E).

These data show that the addition of CPA increased sarcolemmal permeation of calcium and that increased permeation could occur even at base-line resting [Ca²⁺]_i levels. Thus, the calcium-permeant pathway was activated by store depletion and could be considered a ``capacitative'' pathway. Also, sensitivity to AN1043 suggests that at least part of this increased permeability was mediated by the calcium leak channel.

CPA increased the P_o of a calcium leak channel during single-channel patch clamp analysis in cell-attached patches from myoballs measured at 32 °C (Figs. 6 and 7A, n = 16). In five other patches, no channel activity was observed before or after the addition of CPA. The myoball configuration was used for patch analysis of CPA effects instead of myotubes because myotubes contracted more vigorously upon addition of CPA, destroying seal integrity. Nonetheless, in 19 myoballs, seal damage occurred very soon after CPA addition, preventing analysis of CPA effects in these cells.

Store depletion with 50 µM CPA increases activity of the calcium leak channel in cultured skeletal myoballs. A, sample traces of episodic recording of a calcium leak channel before and after exposure of a myoball to 50 µM CPA, measured at 32 °C. Channel activity was measured in the cell-attached patch mode, at a membrane potential of approximately 140 mV, using 96 mM BaCl₂ (n = 10, this trace) or MnCl₂ (n = 6) as the charge carrier. Data were recorded at a 10-kHz sampling rate, with 2-kHz filtering and filtered at 1 kHz upon playback. B, changes in channel P_o of the same channel with time. P_o was binned in 2.5-s increments. C, current-voltage relationship from the same channel. Voltages indicated were determined from the applied holding potential and the estimated resting potential (50 mV).

Leak channel activity, which was very low at rest, appeared in intermittent bursts after CPA addition, often after a delay of a few minutes (see Fig. 6B). Single channel conductance (14 pS, Fig. 6C) and reversal potential (~+50 mV, Fig. 6C) were consistent with previously described properties of the calcium leak channel (24, 25, 26, 27, 28). When estimating the membrane potential at a given holding potential in cell-attached patches, we assumed the resting potential of the cell to be 50 mV, as previously reported (24, 49). Although this estimate of resting potential is not exact, the resting potential would have to be greater than 120 mV for the reversal potential of the leak channel to approach 0 mV. Thus, the apparent channel selectivity for calcium, based on reversal potential, could not be an artifact of uncertainty in measurements of membrane potential. Ion substitution experiments in excised patches also confirm selectivity of the leak channel for calcium (25).

Channel activity was not altered after the addition of Me₂SO to cell-attached patches (Fig. 7A) or the addition of CPA to excised patches (Fig. 7A). Also, interestingly, channel activity in cell-attached patches was not increased by CPA when experiments were performed at room temperature (22 °C, Fig. 7A). Similarly, plateau [Ca²⁺]_i levels after CPA addition, but not peak [Ca²⁺]_i levels, were depressed at 22 °C (Fig. 7, B and C) compared with 32 °C. The rate of [Ca²⁺]_i rise at the two temperatures was not different (37 °C, 28.8 ± 5.3 nM/s; 22 °C, 38.2 ± 11.3 nM/s; p > 0.4). Resting [Ca²⁺]_i at the different temperatures was slightly but significantly different (Fig. 6C, p < 0.05). Seal damage, indicated by increased root mean square noise and drift in the base line, often appeared several minutes after CPA addition, presumably due to cell motion related to CPA-induced [Ca²⁺]_i elevation; this prevented analysis of the effects of AN compounds upon channel activity after the addition of CPA.

If the capacitative current mediated by the leak channel is involved in store refilling, then AN1043 should prevent refilling of depleted calcium stores. To test this possibility, calcium stores were depleted with CPA in calcium-free RRS for 8-10 min and then placed for 5 min in 1.8 mM calcium RRS to allow refilling. The amount of store calcium present after the refilling period (assayed by the peak [Ca²⁺]_i rise after the addition of CPA in calcium-free RRS) was significantly decreased when 10 µM AN1043 was present during the refilling period (Fig. 8A). The effects of AN1043 were not due to direct action upon store release or content, because the magnitude of CPA-induced calcium release in calcium-free RRS was not altered by the presence of 10 µM AN1043 (added 2-4 min before CPA; Fig. 8B), nor did AN1043 affect the rate of CPA-related [Ca²⁺]_i rise or decline (data not shown). Thus, AN1043 acts by blocking the entry of calcium, which was required to refill the calcium stores after depletion.

Some second messenger pathways have been implicated in signaling the filling state of the stores to the plasma membrane (for review see Ref. 1). In several cell types, tyrosine kinase inhibitors, such as genistein, inhibit development of the capacitative current (50, 51, 52, 53). In myotubes, 100 µM genistein (added 10-15 min before CPA) significantly reduced CPA-induced increases in sarcolemmal Mn²⁺ permeability (Fig. 9A). The addition of CPA normally increased the Mn²⁺ influx rate by at least 4%/min (see above). In contrast, in the presence of genistein the CPA-induced Mn²⁺ influx rate was reduced to 0.29 ± 0.4%/min (before CPA, 0.33 ± 0.48%/min; after CPA, 0.62 ± 0.36%/min; p > 0.6; no difference before and after CPA; n = 7 of 8 cells tested, 3 myoballs, 4 myotubes). The lack of effect in the one genistein-loaded cell may have been due to incomplete penetration of genistein during the loading period (see Ref. 50).

Okadaic acid, a protein phosphatase inhibitor (54), inhibits the capacitative current in some cell types (55, 56) but potentiates it in others (17). In four of nine myotubes, okadaic acid (30 nM) increased resting Mn²⁺ influx rates (Fig. 9B; before okadaic acid, 2.82 ± 0.83%/min; after okadaic acid, 6.65 ± 0.88%/min; p < 0.03). No effect was observed in the other five cells. This increased Mn²⁺ influx was inhibited by AN1043 (n = 2) but not diltiazem or verapamil (n = 2), suggesting activation of the leak channel. However, in the presence of okadaic acid, CPA addition still significantly increased the Mn²⁺ influx rate (Mn²⁺ influx rate in presence of okadaic acid; before CPA, 3.76 ± 1.0%/min; after CPA, 10.1 ± 1.4%/min; n = 9, p < 0.01). Thus, okadaic acid can increase resting Mn²⁺ influx but does not inhibit CPA-induced changes in Mn²⁺ influx in skeletal myotubes.

Finally, econazole (30 µM), a cytochrome P450 inhibitor, blocks the capacitative current in many systems (1, 38). However, these inhibitions may have been the result a lower driving force for calcium entry due to nonspecific effects on potassium or chloride channels (73). In myotubes, econazole did not significantly affect resting Mn²⁺ influx rates (before econazole, 1.21 ± 0.64%/min; after econazole, 2.42 ± 0.49%/min; n = 5, p > 0.15) and did not inhibit CPA-induced changes in Mn²⁺ quenching rates (in the presence of econazole; before CPA, 2.42 ± 0.49%/min; after CPA, 20.8 ± 7.6%/min; n = 5, p < 0.01). Thus, based on Mn²⁺ quenching assays, regulation of leak channel activation during store refilling involves some but not all signaling pathways that have been implicated in other cell types.

DISCUSSION

Evidence from changes in [Ca²⁺]_i, rates of Mn²⁺ quenching of fura-2 fluorescence, and single-channel cell-attached patch measurements indicate that cultured skeletal myotubes possessed a capacitative current that was mediated by the calcium-specific leak channel and inhibited by the DHP compounds AN406 and AN1043. To our knowledge, this is the first description of a capacitative refilling current mediated by a well characterized channel and possessing a DHP antagonist, although small-conductance channels mediating a capacitative current have been previously identified by single channel patch methods in an epithelial cell line (20) and in endothelial cells (21). This is also the first report of a capacitative current from skeletal muscle tissue.

It is possible that activation of the leak channel after the addition of CPA was not due to depletion of the intracellular stores, and instead was a result of [Ca²⁺]_i elevation. Channel activation solely by elevated [Ca²⁺]_i was unlikely, since the calcium-permeable pathway that was inhibited by AN1043 was activated by CPA even when resting [Ca²⁺]_i levels had returned to base line (see Fig. 5). This suggests that activation of the capacitative pathway and the calcium leak channel was not dependent on elevated [Ca²⁺]_i. However, since it is not possible to patch clamp in calcium-free RRS, we cannot use patch clamp methods to test directly whether the leak channel is activated under such conditions. Our data do not exclude the possibility that the leak channel may also be directly activated by elevated [Ca²⁺]_i, although previous studies suggest that the channel is not directly activated by calcium (24, 25).

Channel activation after the addition of CPA could also be due to contraction of the muscle cells or some other mechanically related phenomenon. This is unlikely, since 10 µM Gd³⁺, an inhibitor of stretch-gated channels (57), did not affect the CPA-induced calcium plateau (Fig. 3D) or Mn²⁺ influx (n = 6, data not shown). Also, similar contractile activity was observed at 22 and 32 °C (data not shown), but at 22 °C capacitative calcium influx was greatly reduced (Fig. 7, B and C).

Many studies identify capacitative currents using increased quenching of fura-2 fluorescence by Mn²⁺ after store depletion, which presumably indicates increased plasma membrane permeability to Mn²⁺ (36, 37, 38, 39). Our studies regarding Mn²⁺ permeability suggest a relationship between the calcium leak channel from cultured skeletal muscle cells and conductances underlying capacitative currents in other cell types. The calcium-selective leak channel studied here is similarly permeable to Ba²⁺, Ca²⁺, and Mn²⁺ (24, 25, 28), as is the depletion-activated channel from endothelial cells (58). However, depletion-activated single channels from epithelial cells (20) and depletion-activated whole cell currents from many cell types (3, 5, 14, 15, 16, 17, 18, 19) are impermeable to or are inhibited by Mn²⁺. In contrast, in our preliminary studies of whole-cell currents from skeletal myoballs, the addition of CPA increased the leak current by 37 ± 8.9 pA with 20 mM extracellular Mn²⁺ (four of six cells tested) and by 40 and 510 pA with 100 mM extracellular Mn²⁺ (two of three cells tested).³ Thus, this ion selectivity of the leak channel-mediated capacitative conductance differs from those demonstrated for depletion-activated currents in some other cell types. The possibility that there may be multiple depletion-activated pathways within the same cell must be considered (59), since some cell types exhibit both Mn²⁺-impermeable whole-cell currents and depletion-induced Mn²⁺ quenching of fura-2 fluorescence (e.g. Jurkat lymphocytes (5, 19, 39) and pancreatic acinar cells (14, 36)). The leak channel or a related channel may underlie the observed depletion-induced Mn²⁺ influx in these cell types.

Our results imply a functional role for the calcium leak channel in the refilling of depleted intracellular calcium stores. Calcium leak channels have been previously observed in several muscle (24, 25, 26, 27, 41, 60, 61) and neural (62, 63) cell types, but no clear function has previously been determined. The importance of store refilling in muscle is underscored by observations that calcium release decreases during continuous activation of skeletal muscle in calcium-free media, suggesting store depletion (64), and that the ability to develop sustained force requires extracellular calcium influx in some studies (65, 66, 67). Although calcium entry through VDCC may also mediate store refilling under some conditions (68, 69, 70), evidence presented here suggests that the leak channel represents an important refilling mechanism in skeletal myotubes (see also Ref. 68). Further, concentrations of nifedipine that block VDCC can increase leak channel activity (24, 25, 28) and also potentiate force generation during repetitive contractions in the diaphragm (71). By filling the intracellular calcium stores, the calcium leak channel may modulate calcium influx and force development during normal contractile activity in skeletal muscle.