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Depolarization-induced entry of divalent ions into skeletal muscle has been attributed to a process termed Excitation-Coupled Ca2+ Entry (ECCE), which is hypothesized to require the interaction of the ryanodine receptor (RyR1), the L-type Ca2+ channel (DHPR) and another unidentified cation channel. Thus, ECCE is absent in myotubes lacking either the DHPR (dysgenic) or RyR1 (dyspedic). Furthermore, ECCE, as measured by Mn2+ quench of Fura-2, is reconstituted by expression of a mutant DHPR α1S subunit (SkEIIIK) thought to be impermeable to divalent cations. Previously, we showed that the bulk of depolarization-induced Ca2+ entry could be explained by the skeletal L-type current. Accordingly, one would predict that any Ca2+ current similar to the endogenous current would restore such entry and that this entry would not require coupling to either the DHPR or RyR1. Here, we show that expression of the cardiac α1C subunit in either dysgenic or dyspedic myotubes does result in Ca2+ entry similar to that ascribed to ECCE. We also demonstrate that, when potentiated by strong depolarization and Bay K 8644, SkEIIIK supports entry of Mn2+. These results strongly support the idea that the L-type channel is the major route of Ca2+ entry in response to repetitive or prolonged depolarization of skeletal muscle.
In skeletal muscle, entry of external divalent cations occurs in response to sustained weak depolarizations or repetitive electrical stimulation via a process which has been termed Excitation-Coupled Ca2+ Entry (ECCE).1–7 The entry of divalent ions attributed to ECCE requires expression of both the skeletal muscle L-type Ca2+ channel (or 1,4-dihydropyridine receptor; DHPR) and the type 1 ryanodine receptor (RyR1) since depolarization-induced Ca2+ entry is absent in myotubes lacking either the DHPR α1S subunit (dysgenic) or RyR1 (dyspedic), respectively.1–4, 7 It has been proposed that ECCE is conducted via a cation channel that is distinct from the skeletal L-type channel because: i) ECCE is diminished by agents that block Ca2+ entry via transient receptor potential (TRP) or store-operated Ca2+ entry (SOCE) channels,1–4 ii) ECCE is relatively insensitive to block by the specific L-type channel inhibitor nifedipine,3 and iii) ECCE has been observed as a Mn2+ entry-dependent quench of Fura-2 fluorescence in dysgenic myotubes transfected with an α1S pore mutant (SkEIIIK)8 thought to conduct only monovalent cations.1 Thus, it has been proposed that ECCE involves a still unidentified channel of the plasma membrane whose gating is controlled by conformational coupling to the skeletal DHPR and RyR1.1
Although SOCE channels have been eliminated as the channels that mediate ECCE,7 the identity of the ECCE channel remains unsettled.9 Recently, we proposed that the bulk of Ca2+ entry attributed to ECCE is mediated by the skeletal L-type Ca2+ channel.4 In particular, we showed that agents which block TRP and SOCE channels, as well as nifedipine, have a similar potency for block of the L-type current as previously described for ECCE. Furthermore, we failed to detect Ca2+ entry via SkEIIIK using Fluo-3, a low affinity Ca2+ indicator dye. Although this result indicates that SkEIIIK does not permit a large influx of Ca2+, it does not exclude the possibility that the channel can support small fluxes of divalent cations during the long, weak depolarizations that have previously been employed to evoke ECCE.1–7
If it is correct that the depolarization-induced divalent entry attributed to ECCE arises from the skeletal L-type current, then a similar entry should be supported by the cardiac (α1C) DHPR which produces substantial L-type current when expressed in myotubes.10–18 In the current study, we demonstrate that ECCE-like Ca2+ entry can indeed be reconstituted by expression of the cardiac DHPR α1C subunit in either dysgenic or dyspedic myotubes. Thus, neither expression of the DHPR α1S subunit nor the presence of RyR1 is required for the ECCE-like Ca2+ entry observed after expression of α1C. In addition, we show that SkEIIIK can conduct Mn2+ when potentiated in by the strong depolarization in the presence of the L-type channel agonist ±Bay K 8644, consistent with the possibility that the Mn2+ quench observed previously in SkEIIIK-expressing dysgenic cells may have resulted from Mn2+ entry via the mutant channel itself.
When expressed in dysgenic myotubes, cardiac DHPR α1C subunits produce robust L-type Ca2+ current.10–13,15–17 Since our previous results indicated that ECCE can be largely attributed to Ca2+ influx via native skeletal muscle L-type Ca2+ channels,4 we reasoned that α1C would also be capable of supporting ECCE-like Ca2+ entry. In the presence of 2 mM external Ca2+, ECCE can be strongly evoked by prolonged exposure to 60 or 80 mM K+;1–7 these ionic conditions produce membrane potentials in the range of −15 to 0 mV (P.D. Allen and J.R. López, personal communication). To determine whether α1C-CFP-mediated L-type current can be observed this range of potentials, we recorded currents in 2 mM external Ca2+ in response to 200 ms test pulses. As shown Figure 1A and 1B, L-type currents mediated by α1C-CFP began to activate at −20 mV, and produced substantial L-type current at −10 and 0 mV in 2 mM external Ca2+ (n = 6). Since ECCE is normally evoked by exposures to elevated K+ on the order of seconds, we also measured currents in response to 9.8 s depolarizations to −20, −10 and 0 mV in 2 mM external Ca2+. As shown in Figure 1C, steps to −10 and 0 mV yielded L-type current which peaked quickly, inactivated modestly within ~1 s and then was relatively well maintained (Table 1). Taken together, the data in Figure 1A–C indicate that long, weak depolarizations elicit substantial, sustained α1C-CFP-mediated L-type current under conditions similar to those used to evoke ECCE.1–7
In order to assess whether the slowly inactivating L-type current mediated by α1C-CFP could support Ca2+ influx similar to that attributed to ECCE, we applied 60 mM K+ to dysgenic myotubes expressing α1C-CFP after block of SR Ca2+ release by ryanodine pre-treatment (200 μM, >1 hr, 37°C). Upon depolarization with 60 mM K+, dysgenic myotubes expressing α1C-CFPshowed a large Ca2+ entry (n = 9; Fig. 2A). In control experiments, no quantifiable Ca2+ transients were observed in CFP-negative dysgenic cells (n = 18; Fig. 2B). Consistent with the idea that the Ca2+ entry observed in cells expressing α1C-CFP resulted from L-type current, 10 μM nifedipine caused a large reduction in the rate of rise of depolarization-induced Ca2+ transients and a decrease in the peak ΔF/F (n = 7; Figs. 2C, 2E). 50 μM nifedipine completely eliminated the transients (n = 2; Fig. 2D). Thus, the block of Ca2+ entry via α1C channels expressed in dysgenic skeletal myotubes by nifedipine (summarized in Fig. 2E) was comparable to the block of native cardiac L-type channels by nitrendipine in canine ventricular myocytes with a holding potential of −80 mV.19
The presence of ECCE-like Ca2+ entry in dysgenic myotubes expressing α1C-CFP suggests that conformational coupling to RyR1 is not essential for ECCE, because α1C does not engage in interactions with RyR1 that produce skeletal-type EC coupling.10–12,16–18,20,21 To test the requirement for RyR1 more directly, we examined whether Ca2+ transients could be evoked in dyspedic myotubes expressing α1C- CFP. In 7 of 10 dyspedic myotubes displaying cyan fluorescence, we observed a large Ca2+ entry in response to 60 mM K+ (ΔF/F = 1.45 ± 0.21; n = 7; Fig. 3A). It is unlikely that the observed transients in dyspedic myotubes transfected with α1C-CFP arose from RyR3-mediated Ca2+ induced-Ca2+ release from the SR because the myotubes were pretreated with 200 μM ryanodine for 1 hour (37°C) prior to experiments.
Because dyspedic myotubes do express relatively unaltered levels of the native skeletal DHPR,23 it is possible that these endogenous DHPRs contributed to the Ca2+ entry observed in dyspedic myotubes transfected α1C-CFP. However, only small Ca2+ currents are produced by the endogenous DHPRs in dyspedic myotubes.14,23–28 Additionally, in agreement with previous studies,1, 2, 4, 7 no elevated K+-evoked Ca2+ transients were observed in untransfected dyspedic cells (n = 15; Fig. 3B). Furthermore, no measurable Ca2+ entry was observed in dyspedic myotubes treated with 5 μM ±Bay K 8644 (n = 16; Fig. 3C). Thus, the results shown in Figure 3 indicate that expression of α1C can result in ECCE-like Ca2+ entry and that this entry does not require the presence of RyR1.
The primary reason that ECCE has been believed to be independent of Ca2+ entry via L-type channels is that Mn2+ quench has been observed in dysgenic myotubes transfected with an α1S pore mutant (SkEIIIK)8 thought to conduct only monovalent cations.1 Previously, we demonstrated that potentiated SkEIIIK channels could pass Ca2+, which suggests that Mn2+ quench observed in SkEIIIK-expressing dysgenic cells arose from Mn2+ passing through the mutant channel.4 In order to test this possibility more directly, we sought to determine whether strong depolarization in the presence of ±Bay K 8644 would produce inward, Mn2+ tail current upon repolarization. Figure 4 shows currents recorded from SkEIIIK-expressing dysgenic myotubes in 10 mM external Mn2+ before (Fig. 4A) and following application of 5 μM ±Bay K 8644 (Fig. 4B). Consistent with the observations of Leuranguer et al.,29 the large outward monovalent current elicited by step depolarizations from −50 mV to 80 mV was reduced by ±Bay K 8644 compared to control (13.6 ± 2.2 vs. 18.5 ± 4.7 pA/pF, respectively; n = 4). More importantly, there was a substantial component of slowly-decaying, inward current in the presence of ±Bay K 8644 upon repolarization from 80 mV to −50 mV (compare Fig. 4A and 4B). This slow rate of decay of this inward current in ±Bay K 8644 relative to control (τdeact = 4.2 ± 0.5 vs. 1.3 ± 0.4 ms, respectively; n = 4; p < 0.006, t-test; Fig. 4C) indicates that it is an inward ionic tail current carried by Mn2+ (see ref. 4).
In the current study, we demonstrated that the cardiac DHPR α1C subunit can produce sustained L-type Ca2+ current in response to weak depolarizations when expressed in dysgenic myotubes (Fig. 1). Furthermore, depolarization of either dysgenic or dyspedic myotubes expressing α1C caused entry of extracellular Ca2+ (Figs. 2 and and3,3, respectively) resembling that previously attributed to ECCE in normal myotubes.1–7 In addition, we also showed that SkEIIIK, a mutant DHPR α1S subunit thought not to be permeable to divalent cations,8 will conduct Mn2+ in a potentiated state induced by ±Bay K 8444 and strong depolarization (Fig. 4). This Mn2+ permeability may account for the depolarization-induced Mn2+ entry previously described in response to depolarization of dysgenic mytoubes expressing SkEIIIK.1 Together, these results provide further support for the view that the L-type Ca2+ current is the molecular basis of the Ca2+ entry which has been attributed to ECCE.
ECCE-like entry of Ca2+ has been described as requiring the presence of both the DHPR α1S subunit and of RyR1. However, we have now shown that Ca2+ entry resembling ECCE occurs after expression of α1C in dysgenic myotubes which lack α1S. Furthermore, we observed similar depolarization-induced Ca2+ entry when we expressed α1C in dyspedic myotubes which lack RyR1. Thus, L-type current is sufficient to produce ECCE-like Ca2+ entry without an absolute requirement for α1S or RyR1. In addition to the L-type channel, skeletal muscle is known to express other cation channels (e.g., TRPs) which could contribute to Ca2+ entry.30 Likewise, divalent cations may also enter the myoplasm via an unidentified non-electrogenic exchanger.31 However, one would expect that ECCE-like depolarization-induced Ca2+ entry in normal myotubes would be largely attributable to the skeletal L-type current since this current is of similar magnitude to the L-type current in dysgenic myotubes expressing α1C.
The Ca2+ entry in dysgenic myotubes expressing α1C clearly does not depend on the conformational state of RyR1, as previously proposed for ECCE,1 since α1C cannot engage in either orthograde10–12,14,16–18,20 or retrograde14, 21 conformational coupling with RyR1. The robust Ca2+ entry observed in dyspedic myotubes expressing α1C was not attributable to endogenous α1S since we never saw such entry in naïve dyspedic myotubes. The absence of ECCE-like Ca2+ entry in dyspedic myotubes can be explained by the loss of retrograde coupling between RyR1 and the skeletal muscle DHPR, without which the L-type current has very small amplitude.14,23–28 Moreover, we did not detect depolarization induced Ca2+ entry in dyspedic myotubes even after treatment with the dihydropyridine agonist ±Bay K 8644 (Fig. 3C). The inability of ±Bay K 8644 to potentiate the natively-expressed DHPRs of dyspedic myotubes to the point of producing ECCE is not surprising in this regard because Bay K 8644 only weakly potentiates L-type current in dyspedic myotubes.25 In particular, Bay K 8644 only targets the fast, transient activation phase of the L-type current leaving the slow, sustained component of the current unaffected.25
If ECCE actually represents divalent cation influx via L-type channels, then the question arises as to why ECCE was observed in dysgenic myotubes expressing SkEIIIK, as assayed by Mn2+ quench of Fura-2 dye.1 In Figure 4, the slowly decaying tail currents produced by SkEIIIK indicate that potentiated SkEIIIK channels can conduct Mn2+. In addition to producing slowly decaying tail currents after potentiation by ±Bay K 8644 and strong depolarization (Fig. 4B,C), SkEIIIK channels which entered into the potentiated state during prolonged step depolarizations would also be expected to produce inward Mn2+ current and this might account for the Mn2+ quench observed by Cherednichenko et al. in dysgenic myotubes expressing SkEIIIK.1 It should be noted that we have not been able to detect such inward Mn2+ currents during step depolarizations (data not shown). However, even during prolonged depolarization, only a fraction of all channels would be converted to the potentiated state.32 Thus, any inward current through the potentiated SkEIIIK channels would be obscured by outward current through non-potentiated channels. Whether or not this explanation is correct, our present results indicate that skeletal L-type current alone is sufficient to account for the bulk of the voltage-dependent Ca2+ entry observed in ryanodine-treated normal myotubes4 and that this large entry would make it very difficult to detect any additional entry that might occur via a pathway other than the L-type channel.
In the present study, we have provided additional support for our hypothesis that the skeletal muscle L-type Ca2+ current is the molecular basis of ECCE.4 Since ECCE has been shown to help maintain myoplasmic Ca2+ levels during repetitive electrical stimuli,1, 6 it now appears that the L-type Ca2+ current contributes to this essential function and may also be partially responsible for SR Ca2+ store refilling during activity. Furthermore, previous work has shown that mutations of RyR1 that result in malignant hyperthermia appear to accentuate ECCE and that this altered behaviour of ECCE could contribute to triggering episodes of this pharmacogenetic muscle disease.3,5,33 Thus, an important goal for future research will be to define the involvement of the skeletal L-type Ca2+ current in malignant hyperthermia.
All procedures involving mice were approved by the University of Colorado-Denver Institutional Animal Care and Use Committee. Primary cultures of dysgenic (mdg/mdg) or dyspedic (RyR1−/RyR1−) myotubes were prepared as described previously.34 Cultures were grown for 6–7 days in a humidified 37°C incubator with 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM; #15-017-CM, Mediatech, Herndon, VA), supplemented with 10% fetal bovine serum/10% horse serum (Hyclone Laboratories, Logan, UT). This medium was then replaced with differentiation medium (DMEM supplemented with 2% horse serum). For electrophysiology, dysgenic myotubes were microinjected with a solution of either α1C-CFP (200 ng/μl)35 and pEYFP-C1 (10 ng/μl; Clontech, Palo Alto, CA), or SkEIIIK (400 ng/μl)8 and pEYFP-C1 (10 ng/μl). For Ca2+ imaging experiments, single nuclei of either dysgenic or dyspedic myotubes were microinjected with a solution of cDNA encoding α1C-CFP(200 ng/μl). Fluorescent myotubes were used in experiments two days following microinjection.
Pipettes were fabricated from borosilicate glass and had resistances of ~1.5 MΩ when filled with internal solution, which consisted of (mM): 140 Cs-aspartate, 10 Cs2-EGTA, 5 MgCl2, and 10 HEPES, pH 7.4 with CsOH. The external solution contained (mM): 145 tetraethylammonium-Cl,2 CaCl2 or 10 mM MnCl2, 0.003 tetrodotoxin, and 10 HEPES, pH 7.4 with tetraethylammonium-OH. For generation of current-voltage (I–V) relationships, linear capacitative and leakage currents were determined by averaging the currents elicited by eleven, 30-mV hyperpolarizing pulses from the holding potential of −80 mV. Test currents were corrected for linear components of leak and capacitive current by digital scaling and subtraction of this average control current. In all other experiments, −P/4 subtraction was employed. Electronic compensation was used to reduce the effective series resistance (usually to < 1 MΩ) and the time constant for charging the linear cell capacitance (usually to < 0.5 ms). L-type currents were filtered at 2 kHz and digitized at 5–10 kHz. In most cases, a 1-s prepulse to −20 mV followed by a 50-ms repolarization to −50 mV was administered before the test pulse (prepulse protocol; see ref. 36) to inactivate T-typeCa2+ channels. Cell capacitance was determined by integration of a transient from −80 mV to −70 mV using Clampex 8.0 and was used to normalize current amplitudes (pA/pF). I–V curves were fitted using the following equation:
where I is the peak current for the test potential V, Vrev is the current reversal potential, Gmax is the maximum Ca2+ channel conductance, V1/2 is the half-maximal activation potential and kG is the slope factor. The deactivation phase of SkEIIIK tail current was fitted as:
where I(t) is the current at time t after the repolarization, A is the peak tail current amplitude, τ is the deactivation time constant, and C represents the steady current (see ref. 37). All electrophysiological experiments were performed at room temperature (~25°C).
In order to block SR Ca2+ release, all cells used in Ca2+ imaging experiments were pretreated with 200 μM ryanodine for > 1 hr at 37°C. Myotubes were washed with Ca2+/Mg2+-free Ringer’s solution (in mM: 146 NaCl, 5 KCl, 10 HEPES, 11 glucose, pH 7.4 with NaOH) twice and subsequently loaded with 5 μM Fluo-3-AM (Molecular Probes, Eugene, OR, #F-1242) dissolved in Rodent Ringer’s solution (in mM: 146 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, pH 7.4 with NaOH) for 20 minutes. Myotubes were then washed 3X in Rodent Ringer’s solution. Fluo-3-AM-loaded myotubes bathed in Rodent Ringer’s solution (~25°C) were then placed on the stage of an LSM META scanning laser confocal microscope (Zeiss, Thornwood, NY) and viewed with 10X magnification. N-Benzyl-P-toluensulfonamide (BTS; 10–100 μM; #S949760, Sigma, St. Louis, MO) was continuously present in the bath. Fluo-3 was excited with the 488-nm line of an argon laser (30-milliwatt maximum output, operated at 50% or 6.3A, attenuated to 1–2%). The emitted fluorescence was directed to a photomultiplier equipped with a 505-nm long-pass filter. Confocal fluorescence intensity data were digitized at 8-bits, with the photomultiplier gain and offset adjusted such that maximum pixel intensities were no more than~70% saturated and cell-free areas had close to zero intensity. Ca2+ transients were elicited by application of 60 mM K+ Ringer’s solution (in mM: 91 NaCl, 60 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, pH 7.4 with NaOH) via a manually-operated, gravity-driven global perfusion system which produced complete exchange within ~5 s. Fluorescence data are expressed as ΔF/F, where F represents the baseline fluorescence prior to application of high K+ Ringer’s solution and ΔF represents the change in peak fluorescence during the application of high K+ Ringer’s solution. tpeak/2 was calculated as the time required for the transient to reach half-peak amplitude measured from the onset of the initial upswing of the transient.
Ryanodine (#Asc-083, Ascent Biosciences, Princeton, NJ) was reconstituted at 20 mM in 40% EtOH and diluted to 200 μM in differentiation medium. Nifedipine (#481981, Calbiochem, La Jolla, CA) was dissolved in 50% EtOH at 10 mM and diluted to 10 or 50 μM in Rodent Ringer’s solution just prior to experiments. In some experiments, Ca2+ currents were recorded following application of racemic Bay K 8644 (kindly supplied by Dr. A. Scriabine, Miles Laboratories Inc., New Haven, CT). Racemic Bay K 8644 was stored as a 20 mM stock in 50% EtOH. Dihydropyridines were stored and used in the dark.
Figures were made using the software program SigmaPlot (version 7.0, SSPS Inc., Chicago, IL). All data are presented as mean ± SEM. Statistical comparisons were by unpaired, two-tailed t-test, with p < 0.05considered significant.
We thank Dr. D.C. Sheridan, Mr. J.D. Ohrtman and Ms. Ong Moua for insightful discussion. We are grateful to Drs. W.A. Sather and S.F. Oliveria (University of Colorado-Denver) for the gift of α1C-CFP. This work was supported in part by National Institutes of Health Grant NS24444 (to K.G.B.). R.A.B. was supported by a Developmental Grant from Muscular Dystrophy Association (MDA4155).