Orthograde dihydropyridine receptor signal regulates ryanodine receptor passive leak

Abstract

The skeletal muscle dihydropyridine receptor (DHPR) and ryanodine receptor (RyR1) are known to engage a form of conformation coupling essential for muscle contraction in response to depolarization, referred to as excitation–contraction coupling. Here we use WT and Ca_V1.1 null (dysgenic) myotubes to provide evidence for an unexplored RyR1–DHPR interaction that regulates the transition of the RyR1 between gating and leak states. Using double-barreled Ca²⁺-selective microelectrodes, we demonstrate that the lack of Ca_V1.1 expression was associated with an increased myoplasmic resting [Ca²⁺] ([Ca²⁺]_rest), increased resting sarcolemmal Ca²⁺ entry, and decreased sarcoplasmic reticulum (SR) Ca²⁺ loading. Pharmacological control of the RyR1 leak state, using bastadin 5, reverted the three parameters to WT levels. The fact that Ca²⁺ sparks are not more frequent in dysgenic than in WT myotubes adds support to the hypothesis that the leak state is a conformation distinct from gating RyR1s. We conclude from these data that this orthograde DHPR-to-RyR1 signal inhibits the transition of gated RyR1s into the leak state. Further, it suggests that the DHPR-uncoupled RyR1 population in WT muscle has a higher propensity to be in the leak conformation. RyR1 leak functions are to keep [Ca²⁺]_rest and the SR Ca²⁺ content in the physiological range and thus maintain normal intracellular Ca²⁺ homeostasis.

In skeletal muscle the free cytosolic Ca²⁺ concentration at rest ([Ca²⁺]_rest) is ≈120 nM, and the concentration found in the extracellular space and in intracellular stores is in the millimolar range (1). This enormous chemical gradient of Ca²⁺ ions is crucial because all of the Ca²⁺ signaling processes in living cells are based in spatial and temporally controlled Ca²⁺ transients (2). Understanding how cells maintain cytoplasmic Ca²⁺ homeostasis at [Ca²⁺]_rest under normal and pathophysiological conditions can provide essential information about muscle disorders, including muscular dystrophies (3), central core disease/malignant hyperthermia (4, 5), and inclusion body myositis (6, 7).

It is currently thought that the sarco-endoplasmic Ca²⁺ ATPase, the plasma membrane Ca²⁺ ATPase, and the Ca²⁺/Na⁺ exchanger of the plasma membrane regulate the myoplasmic Ca²⁺ concentration in skeletal muscle (2, 8). However, it has also been demonstrated that resting sarcolemmal Ca²⁺ entry and “passive” sarcoplasmic reticulum (SR)-Ca²⁺ leak are working simultaneously, opposing mechanisms that exclude Ca²⁺ from the cytosol. As a result the homeostatic [Ca²⁺]_rest is set by a fine and complex equilibrium among influx and efflux pathways regulating Ca²⁺ ions (9–13).

The physical coupling between the dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR1) is essential for skeletal excitation–contraction coupling, engaging a form of bidirectional signaling. During orthograde signaling, sarcolemmal depolarization elicits conformational changes in Ca_V1.1 that are physically transmitted to RyR1 channels, promoting their opened state. Through retrograde signaling, RyR1 increases the L-type current of the DHPR (14). Freeze-fracture images have shown that DHPRs are clustered in groups of four particles (tetrads), which owing to steric hindrance are associated with alternate RyR1s in each Ca²⁺ release unit. Thus, alternate RyR1 channels are not physically coupled with DHPRs. It is known that the coupled DHPR/RyR1 channels engage retrograde as well as orthograde coupling, but the function of the uncoupled RyR1 population is unknown (15–17).

We have previously shown that a significant part of the Ca²⁺ leak pathway from the SR is ryanodine insensitive, arising from a constitutively open (P_O ≈ 1) low conductance conformation of RyR1. This ryanodine-insensitive conformation is distinct from the actively gating ryanodine-sensitive conformation that is involved in excitation-contraction coupling (10, 18). Bastadin 5 (B5), a brominated macrocyclic derivative of dityrosine isolated from the marine sponge Iathella basta (19), interacts with RyR1, modulating RyR1 gating behavior in a FKBP12-dependent manner. B5 can be used as a pharmacological tool to convert RyR1 from its leak conformation into a gating conformation (19).

In the present study, we identify a previously undiscovered orthograde signal from the DHPR to RyR1 that dictates the ratio of actively gating RyR1 channels vs. those in the leak conformation, thus setting resting myoplasmic Ca²⁺ concentration. Using double-barreled Ca²⁺-selective microelectrodes, we demonstrate that dysgenic myotubes, which lack expression of Ca_V1.1, have increased [Ca²⁺]_rest, higher resting Ca²⁺ entry, and decreased SR Ca²⁺ loading compared with WT cells. Pharmacological control of RyR1 leak state, using B5, equalizes all three parameters between WT and dysgenic myotubes. We conclude that a previously unreported orthograde DHPR/RyR1 signaling significantly inhibits the transition of RyR1 into the RyR1 leak state, keeping the [Ca²⁺]_rest and the SR Ca²⁺ content in the physiological range. In addition, our work suggests that the RyR1 population not coupled to the DHPR in WT muscle has a higher propensity to be in the leak conformation and thus may be determinant in maintaining normal intracellular Ca²⁺ homeostasis.

Results

Dysgenic Myotubes Have a Higher [Ca²⁺]_rest than WT Cells.

To determine the influence of the DHPR on the [Ca²⁺]_rest in myotubes, we measured it directly with Ca²⁺-selective microelectrodes in differentiated WT and dysgenic (Ca_V1.1 null) myotubes. In agreement with previous studies (1, 10), [Ca²⁺]_rest in WT myotubes was 121 ± 1.7 nM (n = 18), but it was significantly elevated (163 ± 2.5 nM, n = 17; P < 0.001) in dysgenic myotubes (Fig. 1). To ascertain whether the absence of Ca_V1.1 was solely responsible for this difference, dysgenic cells were permanently transduced with Ca_V1.1 using lentiviral infection, and transduced myoblasts were cloned to ensure homogeneity of expression, as described in Materials and Methods. WT and transduced dysgenic myotubes (dysgenic+Ca_V1.1) express Ca_V1.1 when assessed by Western blotting and have the expected punctate pattern when tested by immunocytochemistry, showing clustering of DHPRs at the cell surface, presumably at sites of peripheral couplings (20). These clusters are absent in untransduced dysgenic myotubes (Fig. 2). This demonstrates that proper targeting of the protein. [Ca²⁺]_rest in dysgenic+Ca_V1.1 myotubes (119 ± 1.1 nM, n = 17) was not different from WT myotubes (Fig. 1), indicating that the difference in [Ca²⁺]_rest between dysgenic and WT cells was due to the absence of Ca_V1.1 in the former.

Fig. 1.

B5 equalizes [Ca²⁺]_rest among Ca_V1.1-expressing and dysgenic (MDG) myotubes. [Ca²⁺]_rest determinations were done in WT (n = 18), dysgenic (MDG, n = 17), and dysgenic myotubes transduced with Ca_V1.1 lentivirus (MDG+Ca_V1.1, n = 16). Where indicated, the measurements were done in the presence of 20 μM B5 (WT n = 18, dysgenic n = 16, and dysgenic+Ca_V1.1 n = 10), FK506 (WT n = 10, dysgenic n = 10, and dysgenic+Ca_V1.1 n = 10), or FK506 plus B5 (FK506+B5, WT n = 10, dysgenic n = 10, and dysgenic+Ca_V1.1 n = 10). Mean ± SEM is shown. *P < 0.001 (ANOVA). NS, not significant.

Fig. 2.

Dysgenic (MDG) myotubes do not express the α1S subunit of the DHPR (Ca_V1.1). The expression of Ca_V1.1 was assessed by immunostaining in WT, dysgenic (MDG), and dysgenic (MDG)+Ca_V1.1 myotubes. Clusters of DHPRs (red foci) are located at the cell surface in WT and in dysgenic+Ca_V1.1 but not in dysgenic myotubes. Western blot confirms lack of Ca_V1.1 expression in dysgenic myotubes and its rescue by stable transduction. (Scale bar, 10 μm.)

[Ca²⁺]_rest of Dysgenic Myotubes Is Normalized by Treatment with B5.

Exposure to B5 resulted in decrease of [Ca²⁺]_rest to comparable values in all three types of myotubes. [Ca²⁺]_rest after treatment was 99 ± 0.7 nM (n = 16) in WT, 103 ± 1.3 nM (n = 16) in dysgenic, and 94 ± 2.2 nM (n = 10) in dysgenic+Ca_V1.1 myotubes (Fig. 1). As previously shown (19), the action of B5 requires an intact RyR1–FKBP12 complex. Pretreatment with FK506 to disrupt the RyR1–FKBP12 interaction caused a significant increase in [Ca²⁺]_rest to 139 ± 2.4 nM, 217 ± 3.0 nM, and 136 ± 1.3 nM (n = 10) in WT, dysgenic, and Ca_V1.1+dysgenic myotubes, respectively (Fig. 1). After this treatment, addition of B5 had no significant effect in lowering [Ca²⁺]_rest in any of the myotubes, the values remaining at 129 ± 1.8 nM, 202 ± 3.5 nM, and 133 ± 0.8 nM, respectively (n = 10; Fig. 1). These experiments clearly show that B5 treatment equalizes the resting [Ca²⁺]_rest in all of the three cell types and that pretreatment with FK506 abolishes this effect.

Resting Ca²⁺ Entry Is Increased in Dysgenic Myotubes.

Resting Ca²⁺ entry was estimated using Mn²⁺ quench technique as described in Materials and Methods. The rate of decay of the Fura-2 fluorescence was −0.485 ± 0.048 arbitrary units (a.u.)/s (n = 32) in WT myotubes but was significantly higher (0.677 ± 0.072 a.u./s, n = 25, P < 0.01) in dysgenic cells (Fig. 3A). To evaluate the contribution of passive Ca²⁺ entry to [Ca²⁺]_rest, we used the Ca²⁺ entry blocker BTP2 (11, 21). After BTP2 (5 μM) pretreatment [Ca²⁺]_rest decreased from 117 ± 1.8 nM (n = 12) to 89 ± 1.8 nM (n = 10) in WT myotubes, and from 158 ± 2.0 nM (n = 11) to 121 ± 1.7 nM (n = 11) in dysgenic myotubes (Fig. 3B). Although BTP2 has a greater effect on dysgenic than WT myotubes, the blocking effect is not sufficient to equalize the difference in [Ca²⁺]_rest between the two genotypes (Fig. 3B). As before, B5 nullified the difference in [Ca²⁺]_rest between WT (99 ± 0.7 nM, n = 17) and dysgenic myotubes (102 ± 1.2 nM, n = 18), and further addition of BTP2 decreased [Ca²⁺]_rest to the same extent in both genotypes [79 ± 1.1 nM (n = 10) and 83 ± 0.9 nM (n = 10) in WT and dysgenic, respectively]. These data suggest that the reduction of RyR1 leak by the action of B5 may have an effect on SR Ca²⁺ content, which in turn signals entry pathways in the sarcolemma.

Fig. 3.

Resting Ca²⁺ entry is increased in dysgenic myotubes. Resting Ca²⁺ entry was estimated using the Mn⁺² quench technique. (A) Representative traces for WT and dysgenic are shown. The fluorescence decay induced by Mn²⁺ permeability was fitted to a linear regression for each response: WT (n = 32) and dysgenic (MDG) (n = 25). Mean ± SEM is shown. *P < 0.05 (t test). (B) [Ca²⁺]_rest was measured as described in Materials and Methods. The measurements were done in WT (n = 12) and dysgenic (MDG) myotubes (n = 11) in control conditions, treated with 5 μM BTP2 alone (WT n = 10, dysgenic n = 11), B5 alone (WT n = 17, dysgenic n = 18), or the combination of both drugs (B5+BTP2, WT n = 10, dysgenic n = 10). Results are shown as mean ± SEM. ***P < 0.001 (ANOVA). NS, not significant.

Dysgenic Myotubes Have Decreased SR-Ca²⁺ Content That Can Be Normalized with B5.

To estimate the SR Ca²⁺ content, we loaded the cells with the low-affinity Ca²⁺ probe Fluo-5N and measured the increase in cytosolic fluorescence during a 20-mM caffeine challenge in absence of extracellular Ca²⁺ (1 mM EGTA). Interestingly WT myotubes always gave larger Ca²⁺ responses than dysgenic myotubes (Fig. 4). Analysis of the area under the curve shows that the Ca²⁺ released by caffeine was approximately fourfold smaller in dysgenic (25 ± 2.3 a.u., n = 69) vs. WT myotubes (107 ± 8.5 a.u., n = 66), despite the fact that Western blot analysis showed the same level of RyR1 expression (Fig. 4). As a second approach to estimate the intracellular Ca²⁺ content, we loaded the cells with the ratiometric Ca²⁺ probe Fura-4F and measured the increase of cytosolic Ca²⁺ concentration while emptying the SR using the Ca²⁺ ionophore 4Br-A23187 (Fig. 5) in absence of extracellular Ca²⁺ (1 mM EGTA). Two parameters were calculated for each response: the peak value and the area under the curve. WT myotubes showed greater Ca²⁺ transients than dysgenic myotubes for both parameters: 2.1 ± 0.2 ratio_(340/380) (n = 20) vs. 1.5 ± 0.1 ratio_(340/380) (n = 29) peak values, and 30 ± 5.5 (a.u.) (n = 20) vs. 18 ± 1.9 (a.u.) (n = 29) area values, respectively. B5 treatment had no significant effect on either parameter in WT myotubes but normalized the Ca⁺² content in dysgenic myotubes to WT levels [2.3 ± 0.2 ratio_(340/380) (n = 15) dysgenic vs. 2.3 ± 0.1 ratio_(340/380) (n = 24) WT peak values, and 30 ± 4.4 (a.u.) (n = 15) dysgenic and 30 ± 1.7 (a.u.) (n = 24) WT area] (Fig. 5).

Fig. 4.

Dysgenic myotubes have decreased releasable SR Ca²⁺. WT and dysgenic myotubes loaded with Fluo-5N were exposed to 20 mM caffeine in free Ca²⁺ media (1 mM EGTA) as describes in Materials and Methods. Representative traces are shown for WT and dysgenic (MDG) myotubes; the black rectangle shows when caffeine was applied. The area under the curve of each response was calculated [WT n = 66 and dysgenic (MDG) n = 69], and the data are shown as mean ± SEM. ***P < 0.001 (ANOVA) (Upper). Western blot analysis shows that RyR1 expression is not different between WT and dysgenic myotubes [Lower; WT n = 3 and dysgenic (MDG) n = 3].

Fig. 5.

Intracellular Ca²⁺ content is decreased in dysgenic myotubes and is corrected by B5. WT and dysgenic myotubes were loaded with the ratiometric dye Fura-4F. The total Ca²⁺ released by 4Br-A23187 in Ca²⁺-free solution was measured as an estimation of the Ca²⁺SR loading of the myotubes. Mean traces of the responses [WT black trace and dysgenic (MDG) trace] and the effect of 20 μM B5 are shown [WT+B5 trace and dysgenic (MDG)+B5 trace]. Lower: Graphs show peak and area values plotted as mean ± SEM [WT n = 20, dysgenic (MDG) n = 29, WT+B5 n = 24, dysgenic (MDG)+B5 n = 15]. *P < 0.05; **P < 0.01 (ANOVA).

Increased [Ca²⁺]_rest in Dysgenic Myotubes Is Not Caused by the Occurrence of Spontaneous Local Ca²⁺ Release Events.

Myotubes from WT and dysgenic mice exhibited similar very low rates of spontaneous local Ca²⁺ release events (LCRE) behavior (Fig. 6, Left). The occurrence of LCREs comprising Ca²⁺ sparks and macrospark release events (22) was similar despite the dysgenic myotubes having a significantly higher level of [Ca²⁺]_rest as estimated by the resting myofiber Fluo-4 fluorescence (Fig. 6, Right). After a 2-h incubation with 20 μM FK506, both WT and dysgenic myotubes showed a minor, nonsignificant increase in Ca²⁺ spark activity. On the other hand, in both groups, FK506 treatment resulted in a relatively large and significant increase in resting fluorescence, consistent with the results obtained when Ca²⁺-selective microelectrodes were used (compare Figs. 1 and 6).

Fig. 6.

Increased [Ca²⁺]_rest in dysgenic myotubes does not correlate with frequency of spontaneous Ca²⁺sparks. Spontaneous LCREs were measured as described in Materials and Methods on WT and dysgenic (MDG) myotubes. The frequency of spontaneous LCRE was calculated for each individual cell, and the data are shown as mean ± SEM (Left; WT n = 10, dysgenic n = 10). In addition, the global fluorescence of each myotube was calculated and plotted (Right; WT n = 10, dysgenic n = 10) as mean ± SEM. ^#P < 0.05 (ANOVA). The effect of FK506 treatment on both LCRE frequency and global fluorescence is shown.

Discussion

It has been previously reported that the rate of passive Ca²⁺ release from SR vesicles is 10³- to 10⁴-fold smaller than the rate of active Ca²⁺ release in vivo and is likely to reflect passive Ca²⁺ flux from the SR in relaxed muscle. The comparison of the passive Ca²⁺ release rate in light and heavy SR fraction vesicles (LSR and HSR, respectively) shows that the rate in HSR is at least fourfold faster than in LSR membranes (23). This observation is in good agreement with the preferential location of RyR1 in the terminal cisternae of the SR and HSR vesicles, suggesting that RyR1 could be a significant contributor to the passive Ca²⁺ release from the SR. B5, an agent that directly interacts with RyR1, is able to reduce passive leak (18) and increase SR filling capacity (19). More direct evidence that supports the participation of RyR1 in the passive Ca²⁺ release from the SR and the control of [Ca²⁺]_rest in muscle cells is that when RyR1s bearing mutations that cause malignant hyperthermia are expressed in RyR1-null myotubes, the amount of Ca²⁺ release in response to caffeine is decreased (24, 25), and [Ca²⁺]_rest is increased compared with WT. In addition, B5 had a greater effect in decreasing [Ca²⁺]_rest in cells expressing malignant hyperthermia RyR1s than WT RyR1 (4), and the combined action of B5 and ryanodine on WT myotubes, which controls the RyR1 leak and blocks the channels at the same time, decreased [Ca²⁺]_rest to the same level as RyR1-null myotubes, which do not express any RyR isoform and are unresponsive to B5 and ryanodine treatment (10). In addition to RyR1, Ca_V1.1 is a locus for mutations that cause the malignant hyperthermia syndrome (26–28). The mechanism by which DHPR structural alterations may affect RyR1 function to cause this syndrome is currently unknown. It is possible that the disruption of the unique orthograde communication described in this work may be a plausible explanation of how RyR1 leak is enhanced by these mutations, leading to the increase in [Ca²⁺]_rest that is a common feature of malignant hyperthermia susceptibility.

To demonstrate that the effect of B5 treatment was specific to reducing RyR1 leak and not due to a primary reduction of Ca²⁺ sarcolemmal entry, we showed that in WT and dysgenic myotubes that have had their SR Ca²⁺ stores depleted with thapsigargin, that application of B5 has no effect on Ca²⁺ entry (Fig. S1). Our results confirm the previous observations that B5 requires an intact RyR1–FKBP12 complex to stimulate the transition of RyR1 from the leak to a gating state because FK506 treatment almost completely blocked the action of B5 in all of the cell types tested. Thus, all of these data strongly suggest that the RyR1 leak is a normal component that is required for the maintenance of normal Ca²⁺ homeostasis in muscle. Interestingly, profound differences between WT and dysgenic myotubes in relation to leak channel activity at rest suggest an important role of DHPR in controlling RyR1s transitions between the gating and leak states. The pharmacological control of leak-RyR1s using B5 clearly shows that dysgenic cells have a higher proportion of leak-RyR1s than WT cells.

Under physiologic culture conditions cultured WT and dysgenic myotubes show Ca²⁺ sparks at very low frequency that are predominantly located in the central portion of the myotubes away from the peripheral regions where sarcolemma–SR couplings and partially developed t-tubules are found. However, spark frequency was differentially increased in dysgenic myotubes when the culture medium contains 1 mM caffeine and high Ca²⁺. This led to the suggestion that the DHPR negatively regulates the spontaneous Ca²⁺ release through RyR1 (29). Support for this negative regulation comes from our discovery that when RyR3, which is not regulated by the DHPR, is expressed in dyspedic (RyR null) cells, spark frequency is high and [Ca²⁺]_rest is elevated (1, 22, 30). Our present results agree with the previous observations because we see no difference in the frequency or characteristics of spontaneous sparks between dysgenic and WT myotubes in standard resting conditions measured at 25 °C. Because sparks are not more frequent in dysgenic than WT, we believe that the increased [Ca²⁺]_rest in dysgenic myotubes can be accounted for by a greater proportion of RyR1s existing in a low-conductance leak state with high open probability, rather than to an increase in release events.

The constant repressive action by the presence of the DHPR on the transition of RyR1 into the leak state is very important not only to maintain normal [Ca²⁺]_rest in skeletal myotubes but to prevent depletion of the SR–Ca²⁺ content, as demonstrated by the fact that 20-min treatment with B5 is enough to restore normal SR Ca²⁺ loading in dysgenic myotubes. In this new equilibrium, because the SR–Ca²⁺ content is normal, the plasmalemmal Ca²⁺ permeability is low. Thus, these data suggest that the RyR1 leak controls the fine-tuning of the SR–Ca²⁺ content as well as maintaining [Ca⁺²]_rest constant within a tight homeostatic range (Fig. 7, Left). The absence of expression of Ca_V1.1 in dysgenic myotubes releases this orthograde repression of RyR1 transitions to the leak state and results in an increased passive Ca²⁺ efflux from the SR through RyR1. It has been suggested that, in resting condition, plasmalemmal Ca²⁺ permeability is constantly controlled by the Ca²⁺ content level in the SR, through a feedback mechanism that involves coordination between Stim1 and/or STIM2 in the SR and the highly Ca²⁺-selective Orai1 channel (31). In muscle cells we recently have shown that Orai1-mediated resting Ca²⁺ entry accounts for approximately half of the basal entry and is sensitive to BTP2 (11). Thus, supporting the idea of an inverse relationship between SR Ca²⁺ load and plasmalemmal Ca²⁺ permeability at rest, dysgenic myotubes, which have decreased SR Ca²⁺ loading, showed 40% higher resting Ca²⁺ entry than WT myotubes (Fig. 7, Right), and BTP2 decreased [Ca²⁺]_rest more in dysgenic myotubes (37 nM decrement) than in WT (27 nM decrement). In contrast, in myotubes that have RyR1 leaks converted to gating channels with B5, where SR loading is equalized, the effect of BTP2 on [Ca²⁺]_rest is not different in dysgenic (19 nM decrement) and WT myotubes (20 nM decrement).

Fig. 7.

Altered resting Ca²⁺ homeostasis in dysgenic (MDG) myotubes (a model). We propose a model in which association with the DHPR does not allow RyRs to enter into the leak state. This limits the availability of leaky channels and preserves a restricted passive Ca⁺² efflux from the SR. At equilibrium the SR Ca²⁺ content is not depleted, and negative feedback controls the plasmalemmal Ca⁺² permeability (Left). In the absence of DHPRs, in dysgenic (MDG) myotubes, there is an increased passive Ca²⁺ efflux from the SR through RyR1, resulting in an increase of [Ca²⁺]_rest and a decrease of Ca²⁺content in the SR, thus activating store operated calcium entry. The overall effect is an increase of the [Ca²⁺]_rest (Right). SERCA, sarco-endoplasmic Ca²⁺ ATPase; PMCA, plasma membrane Ca²⁺ ATPase; NCX, Ca²⁺/Na⁺ exchanger of the plasma membrane.

On the basis of the mechanism of orthograde coupling between RyR1 and DHPR reported here, the RyR1 population not physically associated with DHPRs should have an increased probability to be in the leak conformation. This observation suggests that the function of RyR1s that are not under direct control by a DHPR is to maintain a certain amount of SR leak to preserve physiologic [Ca²⁺]_rest and overall Ca²⁺ homeostasis at rest.

Materials and Methods

Determination of [Ca²⁺]_rest.

Ca²⁺-selective microelectrodes.

Double-barreled Ca²⁺-selective microelectrodes were prepared and calibrated as previously described (32). Only those electrodes with a linear relationship between pCa3 and pCa8 (Nernstian response, 28.5 mV per pCa unit at 24 °C) were used experimentally. To better mimic the intracellular ionic conditions, all calibration solutions were supplemented with 1 mM Mg²⁺. All electrodes were then recalibrated after making measurements of [Ca²⁺]_rest, and if the two calibration curves did not agree within 3 mV from pCa7 to pCa8, the data from that microelectrode were discarded.

Recording of Vm and [Ca²⁺]_rest.

Myotubes were impaled with the double-barreled microelectrode, and potentials were recorded via high impedance amplifier (WPI FD-223). The potential from the 3M KCl barrel (Vm) was subtracted electronically from VCa_E, to produce a differential Ca²⁺-specific potential (V_Ca) that represents the [Ca²⁺]_rest. Vm and V_Ca were filtered (30–50 KHz) to improve the signal-to-noise ratio and stored in a computer for further analysis.

Lentiviral Transduction of Dysgenic Myoblasts.

The Ca_V1.1 cDNA kindly provided by Manfred Grabner (33) was subcloned between the LTRs of a lentiviral vector driven by an EF1α human promoter. The lentiviral particles were packaged in HEK 293T cells according to a standard method (34). Dysgenic myoblasts were transduced with lentiviral particles and 24 h later were plated in one cell per well in a 96-well plate. After 2 to 3 wk, myoblasts from 20–30 clones were plated in 96-well imaging plates and differentiated to myotubes. After loading with Fluo-4 AM the myotubes were challenged to KCl depolarization. Cells expressing Ca_V1.1 showed strong Ca²⁺ transient in response to depolarization, whereas negative and uninfected dysgenic cells showed no signal. Ca_V1.1 expression and targeting in each clone was evaluated using Western blot and immunofluorescence. After 5 to 6 d of differentiation the cultures showed multinucleated myotubes, with clustering of DHPRs at the cell surface (evaluated by immunofluorescence).

Resting Ca²⁺ Entry.

Fura-2–loaded myotubes were perfused with imaging solution [140 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 5.5 mM glucose, and 10 mM Hepes (pH 7.4)] for 1 min and then the perfusion system switched to Mn²⁺-containing solution [140 mM NaCl, 5 mM KCl, 0.5 mM MnCl₂, 5.5 mM glucose, 10 mM Hepes (pH 7.4), ≈7 μM Ca²⁺ (unbuffered nominally Ca²⁺ free buffer)] for 3 min. Some recordings showed motion artifact due to perfusion switching, and to minimize errors in calculating the rate of decrease in Fura-2 fluorescence after Mn²⁺ exposure, the rate was measured when the signal was linear and stable (30 s after solution twitching). To calculate the fluorescence quench rate, the stable part of the signal was fitted to a linear regression (y = a + bx). The slope derived is expressed as fluorescence arbitrary units per second (f.a.u/s). The excitation wavelength used to measure Mn²⁺ quench of Fura-2 was monitored using a 357/7-nm excitation and 510/80-nm emission filter.

Sarcoplasmic Reticulum Ca²⁺ Content.

Myotubes preparations were loaded with 5 μM Fluo-5N AM for 20 min at 37 °C. The myotubes were placed on the stage of an epifluorescence microscope (Nikon TE2000) coupled to a digital acquisition system (Stanford Photonics). The filter set used was excitation 480/30 nm and emission 535/40 nm. The emission signal was acquired at a frequency of 30 frames per second. The amount of SR Ca²⁺ was estimated by taking the area under the curve of the signal induced by 20 mM caffeine in nominally Ca²⁺-free media [140 mM NaCl, 5 mM KCl, 2 mM MgCl₂, 5.5 mM glucose, 1 mM EGTA, 10 mM Hepes (pH 7.4), ≈5 nM free Ca²⁺] to minimize Ca²⁺ entry. To estimate the total amount of Ca²⁺ stored in the intracellular compartments, ratiometric Ca²⁺ sensor Fura-4F AM loaded myotubes were exposed to the Ca²⁺ ionophore 4Br-A23187 in Ca²⁺-free media buffered with 1 mM EGTA. The area under the curve and the peak of Ca²⁺ transient were computed for each myotube genotype studied. Fluorescent emission at 510 nm was captured from regions of interest within each myotube at two frames per second and expressed as ratio between 340-nm and 380-nm excitation wavelengths.

Western Blot and Immunofluorescence.

Membrane vesicles were prepared from differentiated myotubes by homogenizing them in a Polytron cell disrupter in buffer containing 5 mM imidazole (pH 7.4) and 300 mM sucrose supplemented with protease inhibitor (Complete, Roche Applied Science) and collected as previously described (1). Proteins were separated using SDS/PAGE and transferred to polyvinylidene difluoride membranes. Expression of specific proteins was assessed by incubation of the membranes with monoclonal antibodies against RyR (34C, Developmental Studies Hybridoma Bank) or Ca_V1.1 (MA3-920, ABR-Affinity BioReagents). For immunofluorescence localization of Ca_V1.1, differentiated myotubes were fixed in cold methanol for 15 min at −20 °C. Cells were rinsed with PBS, then were blocked for nonspecific interactions with PSB-1% BSA for 1 h at room temperature and incubated overnight with anti-Ca_V1.1 monoclonal antibody at a 1:400 dilution at 4 °C. Cells were washed and then incubated for 1 h with Alexa Fluor-514 goat anti-mouse antibody (Invitrogen). DAPI was used for nuclear visualization.

LCRE Activity (Ca²⁺ Sparks).

Myotubes grown in 96-well special optics plates (Corning) were loaded with the Ca²⁺ indicator dye Fluo-4 AM (Teflabs) as previously described (22). Plates were mounted over an inverted confocal fluorescence microscope system (Zeiss LIVE5; 25×, 0.8 NA H₂0 objective) and fields of myotubes (193.9 μm in x and y) were imaged (30 Hz; 6–8 s) using x/y scanning. Image series were analyzed with custom routines (IDL; Kodak) for spontaneous LCRE activity as previously described (22, 35, 36).

Isolation of B5.

B5 was extracted from lyophilized Ianthella basta sponge collected from Guam using previously described methods (19).

Statistics.

All values are expressed as mean ± SEM. Statistical analysis was performed using one-way analysis of variance and Tukey's posttest for multiple measurements to determine significance (P < 0.05).