A capacitative calcium current in cultured skeletal muscle cells is mediated by the calcium-specific leak channel and inhibited by dihydropyridine compounds.

Calcium stores from cultured skeletal muscle cells were depleted using cyclopiazonic acid (CPA), a reversible inhibitor of Ca2+-ATPases at the sarcoplasmic reticulum. Store depletion led to activation of the calcium-specific leak channel, as assayed using single-channel patch clamp analysis and rates of manganese influx and quenching of fura-2 fluorescence. Two novel dihydropyridine compounds inhibited this single-channel leak channel activity, the resting and depletion-induced manganese influx, and refilling of the CPA-depleted intracellular calcium store. These compounds represent the first antagonists for a calcium leak channel and for a channel that mediates a capacitative current. The development of the skeletal muscle capacitative current was inhibited by genistein, a tyrosine kinase inhibitor, but was not affected by okadaic acid, a phosphatase inhibitor, or econazole. Thus, the capacitative current in cultured skeletal muscle cells was mediated by the calcium leak channel and was inhibited by pharmacological antagonists and may provide a model system for uncovering the complete set of signals leading from store depletion to channel activation.

In many cell types, depletion of intracellular calcium stores either by receptor activation or by inhibition of intracellular Ca 2ϩ -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 -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 -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 -28, 31-33). 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 Ϯ 5 nm) were assayed in the presence of extracellular MnCl 2 as a measure of plasma membrane permeability to Mn 2ϩ , an indirect indicator of Ca 2ϩ permeability (36 -39). MnCl 2 was used at 100 M, unless otherwise indicated. In control experiments without Mn 2ϩ , 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 BaCl 2 or MnCl 2 as charge carrier in the pipette. The leak channel is similarly permeable to Mn 2ϩ and Ba 2ϩ (28). Data were recorded at a 3or 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 * This work was funded by National Institutes of Health Grant RO1 AR41129 and Athena Neurosciences (South San Francisco, CA). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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 Ϯ S.E.
Ruling Out Possible Fluorescence Artifacts Associated with the Addition of AN1043-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 M) and AN406 (10 M) did not significantly affect fura-2 fluorescence intensity in cell-free solutions compared with Me 2 SO controls (data not shown). Also, 10 M AN1043 did not affect measured [Ca 2ϩ ] i during calibration of myotubes with ionomycin (1-2 M) in extracellular RRS containing 250 nM calcium (AN1043 present: 245 Ϯ 11 nM calcium, n ϭ 5; AN1043 absent: 250 Ϯ 12 nM calcium, n ϭ 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 [Ca 2ϩ ] i .
A small autofluorescence artifact, detected as slowly increasing fluorescence intensity, was observed after the addition of 10 M 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 Ϯ 11 intensity units/min, n ϭ 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 Ͼ 0.2) between myotubes loaded with fura-2 (219 Ϯ 28 intensity units/min, n ϭ 24 of 34 cells) and unloaded myotubes (154 Ϯ 46 intensity units/min, n ϭ 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.
2) Fluorescence intensity rose upon addition of AN1043 and then reached a plateau after 2.6 Ϯ 0.2 min (n ϭ 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 ϭ 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.

Calcium Leak Channel Activity Was Inhibited by Several
Dihydropyridine Compounds-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 [Ca 2ϩ ] i levels, perhaps indicating antagonism of the calcium leak channel. Two analogs ( Fig. 1), called AN406 and AN1043 reduced resting [Ca 2ϩ ] 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 Me 2 SO (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 Me 2 SO include channels from three mdx cell-attached patches, seven mdx excised patches, and one normal excised patch. As excised and cellattached patches showed similar behavior, the data were 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 -27), which makes the channel a possible can-didate 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. Channel activity was measured in the cell-attached patch mode, at a membrane potential of approximately Ϫ140 mV, using 96 mM BaCl 2 as the charge carrier. Data were recorded at a 10-kHz sampling rate with 2-kHz filtering. B, changes in channel P o of the same channel with time. P o was binned in 2.5-s increments. C, sample single-channel records showing decreased channel activity after the addition of 10 M AN406. Channel activity was from an mdx myotube in the excised inside-out patch mode at a holding potential of Ϫ100 mV using 96 mM BaCl 2 as the charge carrier. Data were recorded at a 3-kHz sampling rate with 2-kHz filtering and 1-kHz filtering during play back. D, decrease of leak channel mean P o in single-channel patch measurements by 10 M AN406 but not Me 2 SO. ** indicates p Ͻ 0.001, and * indicates p Ͻ 0.01, for Me 2 SO versus AN406. Due to variability in initial P o , mean P o before drug addition was normalized to 100%, and P o at subsequent times is expressed relative to initial P o . Later time points contained a lower sample size due to rupture of some patches during the course of the experiment. E, all-points amplitude histogram from 2 s of channel activity from trace in C; a shorter segment was chosen due to slight drift in the base line. With the mean amplitude of the closed state normalized to zero, the mean open state amplitude was Ϫ2.24 pA. inhibitor of Ca 2ϩ -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 [Ca 2ϩ ] 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 [Ca 2ϩ ] i elevation and a prolonged plateau phase after the initial [Ca 2ϩ ] i peak (Fig. 3A, C-F).

CPA Increased Cytoplasmic [Ca 2ϩ ] i Levels in Cultured Myotubes, and CPA-induced [Ca 2ϩ ] i Elevations Were Inhibited by
The addition of 10 M AN1043 to the bath resulted in a rapid decrease in the CPA-induced [Ca 2ϩ ] i plateau (Fig. 3C, 64 Ϯ 10% decrease, n ϭ 4). The addition of 3 mM EGTA to the bath (19) also resulted in a rapid decrease in plateau [Ca 2ϩ ] i (Fig. 3, D and E, 51 Ϯ 2% decrease, n ϭ 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 [Ca 2ϩ ] i plateau was not affected by 10 M Gd 3ϩ (Fig. 3D, n ϭ 4), unlike capacitative currents in other cell types (23), but 30 M La 3ϩ did reduce plateau [Ca 2ϩ ] i (Fig. 3F, 56 Ϯ 2% decrease, n ϭ 9), as observed in several cell types (8,10,23,46). Fig. 3D and 3E also show the variability in the [Ca 2ϩ ] i response after CPA addition in 1.8 mM extracellular calcium.
Manganese Influx Rates Were Increased by CPA and Inhibited by Dihydropyridine Compounds-Regulation of resting [Ca 2ϩ ] i is complicated by the number of molecules, such as channels, pumps, and buffers, that can affect calcium homeo-stasis. To assay sarcolemmal divalent cation influx more directly, we measured rates of Mn 2ϩ 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 -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 Mn 2ϩ . Me 2 SO addition did not change Mn 2ϩ quenching rates (Fig. 4B, n ϭ 7). The Mn 2ϩ influx induced by the addition of CPA was significantly inhibited by AN406 and AN1043 (data for 5 M shown in Fig. 4D; data for 10 M shown in Table II). Also, the addition of 10 M AN406 and AN1043 significantly reduced resting Mn 2ϩ quenching rates (e.g. see Fig. 4B). Thus, CPA increased sarcolemmal permeation of Mn 2ϩ , and both DHP analogs prevented Mn 2ϩ entry at the sarcolemma after the addition of CPA and at rest. In Fig. 4B, 100% represented ϳ12,000 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 ϳ13,500 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.
It is possible that stores were partially depleted by incuba- tion 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 Mn 2ϩ flux rates, which would correlate inversely with the amount of calcium released by CPA. However, no correlation was observed (correlation coefficient ϭ 0.06, n ϭ 45, p Ͼ 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").
Mn 2ϩ Influx Was Mediated by the Leak Channel and Not the Voltage-dependent Calcium Channel-Several lines of evidence suggest that Mn 2ϩ influx rates in skeletal myotubes were an accurate measure of leak channel activity, and not that of some other channel, such as the VDCC. Mn 2ϩ 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 -28), and were not inhibited by antagonists of VDCC, including nifedipine, nimodipine (28), diltiazem, and verapamil (5-50 M, n ϭ 9; Fig. 4A) (24). Finally, depolarization of GH3 or adrenal chromaffin cells with elevated K ϩ leads to a rapid increase in Mn 2ϩ influx, which is blocked by DHP antagonists of the VDCC (47), but depolarization of myotubes with 25 mM K ϩ decreased manganese quenching rates (before K ϩ , 4.85 Ϯ 1.03%/min; after K ϩ , 1.68 Ϯ 0.51%/min; n ϭ 4, p Ͻ 0.02, paired t test). These data from myotubes are consistent with observations (48) suggesting that depolarization decreases Mn 2ϩ influx by reducing the driving force for divalent cations but are inconsistent with the hypothesis that depolarization activates a Mn 2ϩ -permeable VDCC.
Increased Calcium Permeability Was Not Due to the Increased [Ca 2ϩ ] i after CPA Addition-It is possible that the increased permeability to manganese and calcium after CPA addition was triggered directly by the elevated [Ca 2ϩ ] i (e.g. see Fig. 4C), and was not necessarily related to store depletion. To test this possibility, calcium stores were depleted by the addi-tion of CPA in calcium-free RRS, and permeability to calcium was determined 10 -12 min after CPA addition. By this time, resting [Ca 2ϩ ] 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 [Ca 2ϩ ] 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 [Ca 2ϩ ] i and rate of [Ca 2ϩ ] i rise were determined. Both peak [Ca 2ϩ ] i and rate of [Ca 2ϩ ] i rise were significantly greater after CPA-related store depletion compared with Me 2 SO controls (p Ͻ 0.001, Fig.  5, A and C-E), indicating that store depletion led to increased sarcolemmal calcium permeability even when resting [Ca 2ϩ ] i levels had been at base line for greater than 5 min. Also, peak [Ca 2ϩ ] i and rate of [Ca 2ϩ ] i rise were significantly reduced by the addition of 10 M AN1043 2-4 min before the addition of 1.8 mM calcium (p Ͻ 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 [Ca 2ϩ ] 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.
Calcium Leak Channel Activity Was Increased by CPA and Reduced by Dihydropyridine Compounds-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 ϭ 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.
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 (ϳϩ50 mV, Fig. 6C) were consistent with previously described properties of the calcium leak channel (24 -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 Ϫ50 mV, as previously reported (24,49). Although this estimate of resting potential is not exact, the resting potential would have to be greater than Ϫ120 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 Me 2 SO 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 [Ca 2ϩ ] i levels after CPA addition, but not peak [Ca 2ϩ ] i levels, were depressed at 22°C (Fig. 7, B and C (Fig. 6C,  p Ͻ 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 [Ca 2ϩ ] i elevation; this prevented analysis of the effects of AN compounds upon channel activity after the addition of CPA. AN1043 Prevented Refilling of Depleted Calcium Stores-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 [Ca 2ϩ ] i rise after the addition of CPA in calcium-free RRS) was significantly decreased when 10 M 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 M AN1043 (added 2-4 min before CPA; Fig. 8B), nor did AN1043 affect the rate of CPA-related [Ca 2ϩ ] 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.
Regulation of the Capacitative Current-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 -53). In myotubes, 100 M genistein (added 10 -15 min before CPA) significantly reduced CPA-induced increases in sarcolemmal Mn 2ϩ permeability (Fig. 9A). The addition of CPA normally increased the Mn 2ϩ influx rate by at least 4%/min (see above). In contrast, in the presence of genistein the CPA-induced Mn 2ϩ influx rate was reduced to 0.29 Ϯ 0.4%/min (before CPA, 0.33 Ϯ 0.48%/min; after CPA, 0.62 Ϯ 0.36%/min; p Ͼ 0.6; no difference before and after CPA; n ϭ 7 of 8 cells tested, 3 myoballs, 4 myotubes). The lack of effect in the one genisteinloaded cell may have been due to incomplete penetration of genistein during the loading period (see Ref. 50).
Finally, econazole (30 M), 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 Mn 2ϩ influx rates (before econazole, 1.21 Ϯ 0.64%/min; after econazole, 2.42 Ϯ 0.49%/min; n ϭ 5, p Ͼ 0.15) and did not inhibit CPA-induced changes in Mn 2ϩ quenching rates (in the presence of econazole; before CPA, 2.42 Ϯ 0.49%/min; after CPA, 20.8 Ϯ 7.6%/min; n ϭ 5, p Ͻ 0.01). Thus, based on Mn 2ϩ 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 [Ca 2ϩ ] i , rates of Mn 2ϩ 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 [Ca 2ϩ ] i elevation. Channel activation solely by elevated [Ca 2ϩ ] i was unlikely, since the calcium-permeable pathway that was inhibited by AN1043 was activated by CPA even when resting [Ca 2ϩ ] 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 [Ca 2ϩ ] 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 [Ca 2ϩ ] 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 M Gd 3ϩ , an inhibitor of stretch-gated channels (57), did not affect the CPA-induced calcium plateau (Fig. 3D) or Mn 2ϩ influx (n ϭ 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 Mn 2ϩ after store depletion, which presumably indicates increased plasma membrane perthe same channel. Voltages indicated were determined from the applied holding potential and the estimated resting potential (Ϫ50 mV).
FIG. 6. Store depletion with 50 M 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 M CPA, measured at 32°C. Channel activity was measured in the cell-attached patch mode, at a membrane potential of approximately Ϫ140 mV, using 96 mM BaCl 2 (n ϭ 10, this trace) or MnCl 2 (n ϭ 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 meability to Mn 2ϩ (36 -39). Our studies regarding Mn 2ϩ 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 Ba 2ϩ , Ca 2ϩ , and Mn 2ϩ (24,25,28), as is the depletionactivated channel from endothelial cells (58). However, deple-tion-activated single channels from epithelial cells (20) and depletion-activated whole cell currents from many cell types (3, 5, 14 -19) are impermeable to or are inhibited by Mn 2ϩ . In contrast, in our preliminary studies of whole-cell currents from FIG. 7. Addition of CPA increased leak channel activity and activated the capacitative calcium current at 32°C but not room temperature (22°C). A, CPA increased leak channel open probability in the cell-attached mode at 32°C but not 22°C. Also, CPA failed to activate the leak channel in the excised mode, and Me 2 SO (DMSO) had no effect on leak channel activity (p Ͼ 0.15). Channel activity was measured for each channel for 3-4 min before the addition of CPA and for 1-4 min after CPA. * indicates p Ͻ 0.025 before versus after the addition of CPA at 32°C. B and C, maintaining cells at 22°C significantly reduced the plateau phase (**, p Ͻ 0.002) but not the peak of the [Ca 2ϩ ] i transient (p Ͼ 0.3) stimulated by the addition of CPA. Resting levels were slightly but significantly different (*, p Ͻ 0.05). In B, the peak [Ca 2ϩ ] i of the 22°C has been scaled to ϳ1000 nM to emphasize changes in [Ca 2ϩ ] i after the peak. Peak [Ca 2ϩ ] i from this trace was originally 450 nM.
FIG. 8. AN1043 prevented refilling of depleted intracellular calcium stores. A, we depleted the calcium store by the addition of CPA in a nominally calcium-free RRS for 8 -10 min, washed out the CPA, and then switched to 1.8 mM calcium RRS Ϯ 10 M AN1043 for 5 min to allow store refilling. After washout of calcium and AN1043, the magnitude of the [Ca 2ϩ ] i increase in response to a second application of CPA in calcium-free RRS was measured. The presence of AN1043 during the refilling period resulted in significantly smaller calcium release from stores, indicating inhibition of refilling (*, p Ͻ 0.002). B, 10 M AN1043 (added 2-4 min before CPA) did not directly affect the magnitude of CPA-induced calcium release in calcium-free RRS, indicating that the effects of AN1043 in the refilling experiment were not due to direct action upon store release or content. skeletal myoballs, the addition of CPA increased the leak current by 37 Ϯ 8.9 pA with 20 mM extracellular Mn 2ϩ (four of six cells tested) and by 40 and 510 pA with 100 mM extracellular Mn 2ϩ (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 Mn 2ϩ -impermeable whole-cell currents and depletion-induced Mn 2ϩ 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 Mn 2ϩ 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 -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 -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.