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CELLULAR |
1 Physiologie Intégrative, Cellulaire et Moléculaire, UMR CNRS 5123, Université C. Bernard Lyon I, 43 bd du 11 Novembre 1918, 69622 Villeurbanne cedex, France
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Abstract |
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1.5 pA at –80 mV was detected in 17 out of 127 and in 21 out of 59 patches in control and mdx dystrophic fibres, respectively. In both populations of fibres, large whole-cell depolarizing pulses did not reproducibly increase this channel activity. This was also true when, repeated application of the whole-cell pulses led to exhaustion of the Ca2+ transient. SR Ca2+ depletion produced by the SR Ca2+ pump inhibitor cyclopiazonic acid (CPA) also failed to induce any increase in the resting whole-cell conductance and in the inward single channel activity. Overall results indicate that voltage-activated SR Ca2+ release and/or SR Ca2+ depletion are not sufficient to activate the opening of channels carrying inward currents at negative voltages and challenge the physiological relevance of a store-operated membrane conductance in adult skeletal muscle.
(Received 27 April 2006;
accepted after revision 15 June 2006;
first published online 15 June 2006)
Corresponding author V. Jacquemond: Physiologie Intégrative, Cellulaire et Moléculaire, UMR CNRS 5123, Université C. Bernard Lyon I, 43 bd du 11 Novembre 1918, 69622 Villeurbanne cedex, France. Email: vincent.jacquemond{at}univ-lyon1.fr
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Introduction |
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Interestingly a calcium entry depending on the calcium content of the intracellular store was described in intact adult and fetal mammalian skeletal muscle fibres using fluorescence measurements from a Ca2+ indicator (Kurebayashi & Ogawa, 2001; Collet & Ma, 2004). However, evidence for the operating of this store-operated calcium entry was obtained under rather drastic non-physiological conditions including the absence of extracellular calcium and the use of SR Ca2+-ATPase inhibitors. Still this calcium influx was suggested to be of functional relevance for the refilling of a diminished intracellular calcium store content, a situation that may occur upon sustained muscle activation. Interestingly Launikonis et al. (2003) observed a store-operated calcium loss in the sealed tubular system of mechanically skinned skeletal muscle fibres, which appeared to be mediated by the inositol trisphosphate receptor. Finally and along the same line, electrical stimulation of skeletal myotubes was also shown to activate a Ca2+ entry pathway with properties similar to the ones of the store-dependent Ca2+ entry, but operating under conditions that did not produce substantial store depletion (Cherednichenko et al. 2004). This whole trend of data thus suggests that SR Ca2+ release and/or SR Ca2+ depletion could control the gating of a plasma membrane calcium conductance.
The physiological relevance of this store-operated calcium entry as well as the identification of a corresponding ion channel activity remain poorly documented. Generally, recording of ion currents through presumed store-operated calcium channels was not available until openings of channels spontaneously active at resting membrane potentials were found to be increased in fibres treated with caffeine or with the SR Ca2+-ATPase inhibitor thapsigargin (Hopf et al. 1996; Vandebrouck et al. 2002). This suggested that these channels described in the early 1990s could correspond to the store-operated calcium channels. Interestingly, in numerous studies, the activity of these channels has been demonstrated to be chronically increased in dystrophin-deficient muscle fibres and it was thus postulated that a dysfunction of store-operated calcium channels might be responsible for the exacerbated calcium influx that characterizes dystrophin-deficient muscle (Vandebrouck et al. 2002).
However, to date, there is a lack of experimental data showing real-time acute changes in membrane conductance at the macroscopic or single channel level in a skeletal muscle fibre, that would correlate with voltage-elicited SR calcium release or depletion, as seen for instance in smooth muscle (Wayman et al. 1996; Albert & Large, 2002). Yet such an approach would provide the most reliable and convincing proof for the existence and physiological relevance of an SR function-dependent transmembrane Ca2+ entry in skeletal muscle. The present study aimed at determining if a store-dependent ion influx develops in response to voltage-activated Ca2+ release in experimental conditions that allowed simultaneous measurement of the macroscopic membrane conductance, the intracellular [Ca2+] and the single channel activity on the same skeletal muscle fibre under control conditions and after depleting the SR. Results seriously question the physiological relevance of a store-operated calcium influx mechanism in skeletal muscle.
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Methods |
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Experiments were performed on single skeletal fibres enzymatically isolated from the flexor digitorum brevis muscles from adult Swiss OF1 and C57BL/10ScSn-mdx/J male mice. All experiments were performed in accordance with the guidelines of the French Ministry of Agriculture (87/848) and of the European Community (86/609/EEC).
Mice were killed by cervical dislocation before removal of the muscles. Procedures for enzymatic isolation of single muscle fibres, partial insulation of the fibres with silicone grease and intracellular microinjection were as previously described (Jacquemond, 1997; Collet et al. 1999, 2004; Collet & Jacquemond, 2002). In brief, the major part of a single fibre was electrically insulated with silicone grease so that whole-cell voltage-clamp could be achieved on a short portion of the fibre extremity. Prior to voltage clamp, fibres were pressure microinjected with a solution containing millimolar concentrations of EGTA and CaCl2 in a 10: 4 ratio. The pH of this solution was buffered at 7.20 and the corresponding calculated free calcium concentration was 130 nM, within the range of commonly measured levels for resting free calcium under our conditions (see for instance Pouvreau et al. 2004). Under these calcium buffering conditions, the fibre integrity was well preserved during the experiments even when large amounts of calcium remained outside of the SR (as for instance in the presence of SR Ca2+ uptake inhibitors); this also allowed the tip of a cell-attached patch pipette to remain sealed on the surface membrane when whole-cell depolarizing pulses were applied. The concentration of EGTA in the microinjection pipette was set to 10 mM for the experiments described in relation to Fig. 5 and to 90 mM for all other experiments.
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Unless otherwise specified the silicone-clamp technique was combined with simultaneous measurements of the single channel activity of the surface membrane using the cell-attached configuration of the patch-clamp technique, as previously described (Jacquemond & Allard, 1998). For the silicone clamp an RK-400 patch-clamp amplifier (Bio-Logic, Claix, France) was used in whole-cell configuration. Command voltage pulse generation and data acquisition were done using the WinWCP software (John Dempster, University of Strathclyde, Glasgow, UK) driving an A/D, D/A converter (BNC-2090 or BNC-2120, National Instruments, Austin, TX, USA). Analog compensation was systematically used to decrease the effective series resistance. Voltage clamp was performed with a microelectrode filled with the whole-cell silicone-clamp intrapipette solution (see Solutions) and having a typical resistance of 2 M
. The tip of the microelectrode was inserted through the silicone, within the insulated part of the fibre. Analog compensation was systematically used to decrease the effective series resistance. Values for the capacitance of the whole-cell voltage-clamped cell portion ranged between 400 and 900 pF. The tip of the patch pipette was sealed on the silicone-free end portion of the fibre in order to record single channel activity in the cell-attached configuration at a pipette potential of 0 mV, with an additional RK400 patch-clamp amplifier. The resistance of the cell-attached pipettes were between 2 and 3 M. Currents flowing into the pipette were considered to be positive. Acquisition of the whole-cell and unitary currents was synchronized with the command voltage pulse generation and was done using pCLAMP 9 software (Axon Instruments Inc.) driving an A/D converter (Digidata 1322A, Axon Instruments Inc.). The average single channel current was measured after filtering at 300 Hz and sampling at 1 kHz. Holding whole-cell voltage was always set to –80 mV. In the experiments described in Fig. 8A, fibres were voltage clamped using the whole-cell configuration of the patch-clamp technique using pipettes of low resistance (ranging from 0.4 to 0.6 M) filled with the whole-cell patch-clamp intrapipette solution (see Solutions). Analog compensation was systematically used to decrease the effective series resistance. In Fig. 8B, the single channel activity was recorded in the cell-attached configuration with the pipette potential held at 0 mV.
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For these, the solution that was microinjected in the fibres also contained 1 mM of either indo-1 or fluo-3. The optical set-up and the procedures used for the indo-1 fluorescence measurements were as previously described (Jacquemond, 1997; Collet et al. 1999; Collet & Jacquemond, 2002; Pouvreau et al. 2004). In brief, a Nikon Diaphot epifluorescence microscope was used in diafluorescence mode. The beam of light from a high-pressure mercury bulb set on the top of the microscope was passed through a 335 nm interference filter and focused onto the preparation. The emitted indo-1 fluorescence light was collected by a 40 x objective and simultaneously detected at 405 ± 5 nm (F405) and 470 ± 5 nm (F470) by two photomultipliers. The fluorescence measurement field was 40 µm in diameter and the silicone-free extremity of each tested fibre was placed in the middle of the field. Background fluorescence at both emission wavelengths was measured next to each tested fibre and was then subtracted from all measurements. The standard ratio method was used with the parameters: R = F405/F470, and Rmin, Rmax, KD and ß having their usual definition. Results were either expressed in terms of indo-1 percentage saturation or in actual free calcium concentration (for details of calculation, see Jacquemond, 1997). In vivo values for Rmin, Rmax and ß were measured using procedures previously described (Collet et al. 1999; Collet & Jacquemond, 2002). For the results described in relation to Fig. 5D the concentration of the Ca–EGTA complex was calculated assuming the intracellular EGTA concentration to be one fifth of the concentration present in the injected solution (for details concerning microinjections see Csernoch et al. 1998) and a KD value for calcium binding to EGTA of 0.2 µM.
The optical set-up and procedures used for the fluo-3 fluorescence measurements were as previously described (Jospin et al. 2002). The set-up was used for all experiments combining whole-cell and cell-attached voltage clamp with intracellular Ca2+ detection. In brief, we used an inverted microscope (Olympus IMT2) equipped for epifluorescence. Cells were imaged using a 20 x objective. Fluo-3 fluorescence was produced and collected by excitation from a 100 W mercury-vapor lamp, using an appropriate filter set combination (excitation, 450–480 nm; emission, above 515 nm; dichroic mirror, 500 nm). Images from a circular region of interest centred on the silicone-free extremity of the cell under study and close to the tip of the cell-attached patch pipette, were captured with a Coolsnapfx charge-coupled device camera (Roper Scientific, Evry, France) at a frequency of 20 Hz. Although this sampling rate precluded accurate detection of the actual peak and time course of the voltage-activated transients, these measurements allowed us to assess the voltage-induced calcium release activity and how stable it was upon successive membrane depolarizations in a given fibre. For assessing the effect of 4-chloro-m-cresol on the fluo-3 fluorescence (see Fig. 7) a sampling rate of 1 Hz was used. Image acquisition and processing were performed using the MetaVue imaging workbench (Universal Imaging Corporation, Downingtown, PA, USA). Fluorescence values were expressed as F/F0, F being the background corrected intensity of fluorescence from the portion of cell under study and F0 the corresponding baseline fluorescence. No attempt was done to calibrate the fluo-3 signal in terms of actual [Ca2+].
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The whole-cell silicone-clamp intrapipette solution contained (mM): 120 potassium glutamate, 5 Na2-ATP, 5 Na2-phosphocreatine, 5.5 MgCl2, 5 glucose, 5 Hepes adjusted to pH 7.20 with KOH. The whole-cell patch-clamp intrapipette solution contained (mM): 145 caesium aspartate, 5 MgCl2, 20 EGTA, 10 Hepes adjusted to pH 7.20 with CsOH. Except for the experiments described in Fig. 8B, the standard extracellular solution contained (mM): 140 TEA-methanesulphonate, 2.5 CaCl2, 2 MgCl2, 10 TEA-Hepes and 0.002 tetrodotoxin, pH 7.20. For the experiments described in Fig. 8B, the extracellular solution was Tyrode solution, containing (mM): 140 NaCl, 5 KCl, 2.5 CaCl2, 2 MgCl2, 10 Hepes, pH 7.2; 250 µM EGTA-AM (acetoxymethyl ester) was also added to this solution. In the experiments depicted in Fig. 8B, the cell-attached pipette was filled with Tyrode solution. Cyclopiazonic acid and 4-chloro-m-cresol were dissolved in DMSO at 50 mM and 1 M, respectively, and diluted to the required concentration in the extracellular solution. Caffeine was dissolved at 25 mM in the extracellular solution. Cells were exposed to different solutions by placing them in the mouth of a perfusion tube from which flowed by gravity the rapidly exchanged solutions. Experiments were carried out at room temperature, ranging from 20 to 23°C.
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Results |
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Figure 1 shows the recordings of these three parameters in response to four consecutive 2 s-long voltage-clamp pulses to +20 mV. The cell-attached pipette contained Tyrode solution while the bath contained a 140 mM TEA-MeSO3 and TTX-containing solution to optimize voltage control of the fibre portion under study. At the single channel level, cell depolarization elicited opening of delayed rectifier K+ channels which inactivated with time during pulses as already described under similar experimental conditions in a previous study (Jacquemond & Allard, 1998). At the macroscopic level an inward current through L-type voltage-dependent calcium channels was detected, somewhat contaminated by residual voltage-activated K+ conductances. The peak amplitude of the calcium current progressively decreased as the successive voltage pulses were applied, probably due to voltage-dependent inactivation and/or t-tubule calcium depletion (see for instance Collet et al. 2003; Friedrich et al. 2001). In the same fibre, the simultaneously recorded fluo-3 fluorescence showed that calcium was released by the SR in response to the depolarizing pulses. The 3 s interval between the pulses was insufficient to allow a complete return of the signal to its basal level, indicating that some of the released calcium remained outside the SR in between the pulses. We focused on the single channel activity between voltage pulses because, should that arise, currents through putative store-operated calcium channels would be expected to develop in response to, and as a consequence of, calcium release. To ascertain that the patch pipette remained sealed on the surface membrane all along the experiments, all patches that did not display delayed rectifier K+ channel activity upon whole-cell depolarization were discarded. In 110 cell-attached patches exhibiting delayed rectifier K+ channel activity during whole-cell pulses, activity of single channels carrying inward currents at –80 mV was never observed. In 17 other cell-attached patches, the unitary activity of a channel carrying an inward current of about 1.5 pA amplitude at –80 mV was detected before depolarizing pulses were given (Fig. 2A). The conductance properties of this channel indicated that it corresponds to the channel open at rest described by Haws & Lansman (1991), Hopf et al. (1996) and Mallouk & Allard (2002) under similar ionic conditions. As shown in Fig. 2B, while channels carrying inward currents at –80 mV were present in the patch, the single channel activity was not increased in response to the repetitive whole-cell depolarizing pulses given to 0 mV.
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In another set of experiments, we tested the possibility that single channels carrying inward currents would activate at negative potentials during calcium release as described by Cherednichenko et al. (2004). For this purpose, we constrained the cell-attached patch potential to remain at –80 mV during the whole-cell depolarizing pulses by simultaneously applying in the patch pipette a hyperpolarizing pulse of the same amplitude as the whole-cell depolarization. Whereas delayed rectifier K+ channels activated when the cell-attached patch potential was driven by the whole-cell depolarizing pulse (as shown in Fig. 1), channel opening was not observed during whole-cell voltage pulses with the patch potential held at –80 mV (Fig. 3). In four patches tested under these conditions, activation of channels carrying inward currents was never detected during the whole-cell depolarizations.
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The effect of CPA was then tested on single channel activity recorded on fibres repetitively depolarized by voltage pulses. Figure 6A shows the activity of a channel carrying inward currents at –80 mV, present in a cell-attached patch on a whole-cell voltage-clamped portion of fibre. As observed previously, four consecutive depolarizing pulses to 0 mV did not lead to any increase in the single channel activity in between pulses in the presence of the control bath solution. A subsequent 5 min treatment of the cell with CPA, during which 16 depolarizing pulses were given, also did not lead to opening of the inward current-carrying channels. Similar results were obtained in three other fibres. It should be mentioned that, also under these conditions of long-lasting depolarizations, CPA had no effect on the macroscopic holding current measured in between the pulses (not illustrated).
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In our voltage-clamp experiments, the SR compartment within the silicone-covered main portion of fibre did not release Ca2+ in response to depolarization and it is also likely to be poorly accessible to CMC applied externally. It could then be argued that this part of the SR, which is not Ca2+ depleted, may influence the membrane conductance in the silicone-free portion of the fibre under voltage control so as to prevent the occurrence of store-operated channel activity. We thus tested the effect of the SR Ca2+ releasing agent caffeine on the whole-cell membrane current recorded from entire fibres with the conventional whole-cell configuration of the patch-clamp technique following the procedure described by Wang et al. (1999). The intrapipette solution contained a high concentration of EGTA (20 mM) in order to prevent caffeine-induced contracture. The fibres were held at –80 mV and a 20 mV hyperpolarizing pulse of 50 ms duration was applied every 20 s. The superposition of the corresponding last membrane current record obtained in control conditions and of the one taken 8 min 40 s after exposition of the whole fibre to 25 mM caffeine shows that caffeine only induced a small reduction in the amplitude of the inward background current associated with a somewhat decrease in membrane conductance (Fig. 8A, upper panel). The middle and lower panels of Fig. 8A show that in 12 cells tested under these conditions, the holding inward background current as well as the current change induced by a 20 mV hyperpolarizing step were only slightly reduced in response to caffeine exposition. Furthermore, we also checked whether exposition of the whole cell to caffeine could induce opening of channels recorded at the unitary level. For that series of experiments, fibres were bathed 30 min prior to recording in a Tyrode solution containing 250 µM of the membrane-permeant Ca2+ chelator EGTA-AM. Single channel activity was recorded in the cell-attached configuration with Tyrode solution in the bath and in the pipette, at a holding pipette potential of 0 mV. All patches that did not display delayed rectifier K+ channel activity upon depolarization of the patch membrane were discarded. In Fig. 8B the upper panel shows that channels carrying inward currents were present in the patch (see inset) in control conditions, but that the single channel activity was not increased in response to the exposure of the whole fibre to caffeine. On average, in eight cells tested under these experimental conditions and in which opening of channels carrying inward currents was detected in control conditions, exposition of the whole fibre to caffeine failed to induce any change in single channel activity. These data thus exclude the possibility that non-uniform depletion of the SR in the entire fibre may be the reason for the absence of depletion-induced changes in membrane conductance in the silicone-clamped fibres.
Since a possible dysregulation of store-operated calcium channels in dystrophin-deficient muscle cells has been put forward, we performed a series of experiments on mdx mouse skeletal muscle fibres. Out of 59 patches tested on mdx muscle fibres, 21 exhibited spontaneous activity of the channels carrying inward currents at negative voltages. Figure 9 shows that in an mdx muscle patch where the channels opened before whole-cell depolarizations were applied, the activity of the channel was clearly not altered by repetitive whole-cell depolarizations of the fibre. On average, as observed in control fibres, there was no tendency for an increase in inward single channel activity as the depolarizing pulses were given (see Fig. 2C).
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Discussion |
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In the majority of studies investigating store-operated Ca2+ influx, depletion of internal calcium stores was induced by the use of SR Ca2+-ATPase inhibitor. Exhaustion of the voltage-activated Ca2+ transients and of the CMC-induced Ca2+ rise confirmed that, under our experimental conditions, depolarizing pulses applied in the presence of CPA induced a strong SR Ca2+ depletion. However, even under such drastic conditions, we did not detect any activation of channels carrying inward currents at negative voltages. Moreover, whenever ion channel activity might have been missed at the single channel level, analysis of the macroscopic current at negative membrane potentials revealed that CPA-induced SR depletion did not lead to any change in the whole-cell conductance indicating that no net ion influx occurred.
Channels spontaneously active at negative membrane voltages in freshly dissociated mouse skeletal muscle fibres have been considered as possible candidates for store-dependent channels (Hopf et al. 1996; Vandebrouck et al. 2002). Indeed, Vandebrouck et al. (2002) observed that store depletion induced by thapsigargin or caffeine in the absence of external calcium significantly increased the open probability of these channels in control mouse muscle. We thus paid particular attention to membrane patches in which spontaneous activity of these channels was revealed before depolarizing pulses were applied. As previously demonstrated by others, in control muscle, a low fraction of patches (13%) displayed such an activity which, in addition, was very weak. We observed that the activity of these channels open at rest was not significantly modified by either a single or by repetitive bursts of voltage-activated calcium release. Moreover, in contrast to what Vandebrouck et al. (2002) described, we also found that repetitive series of depolarizations in the presence of CPA did not affect the activity of these channels.
As reported many times by others (Franco-Obregon & Lansman, 1994; Hopf et al. 1996; Vandebrouck et al. 2002), the occurrence as well as the open probability of the spontaneously active channels is significantly higher in adult skeletal muscle from the dystrophic mdx mouse. Vandebrouck et al. (2002) postulated that the lack of dystrophin in mdx muscle might induce an up-regulation of the channels which were shown to be activated by store depletion to a similar extent as control muscle. While we also found that the occurrence of these channels was higher in mdx than in control muscle although from a different mouse strain (36% of mdx patches), we did not observe any modification of their activity in response to repeated voltage-induced calcium release.
Taken together, our results show that neither a transient nor a chronic SR Ca2+ depletion elicits or increases opening of single channels carrying inward currents at negative voltages in control as well as in dystrophic mdx skeletal muscle. These results thus contrast with the data from Vandebrouck et al. (2002) and the discrepancy may be due to differences in the experimental conditions between the two studies. In the present work, we elicited SR Ca2+ release and depletion under whole-cell voltage clamp, a condition that ensures control of the Ca2+-release activating parameter (i.e. the whole-cell membrane voltage) as well as control of the plasma membrane integrity by monitoring the membrane current. Our experimental conditions further differ in that Vandebrouck et al. (2002) compared different subsets of patches from control and treated fibres whereas we could compare the membrane activity on the same fibre under different conditions. Finally, Vandebrouck et al. (2002) induced SR Ca2+ depletion by a pharmacological treatment associated with the presence of an external solution devoid of Ca2+. To our knowledge, in all the studies describing a store-operated Ca2+ influx in mammalian skeletal muscle, store depletion was induced in the presence of a free-Ca2+ external solution. It may be that the consecutive removal and re-addition of external Ca2+ together with SR depletion promotes a sarcolemmal Ca2+ permeability while SR depletion alone does not, as demonstrated here. In this respect the situation in skeletal muscle seems to differ from the one observed in smooth muscle; in smooth muscle a store-operated inward current characterized at the whole-cell as well as the single channel level was indeed found to develop in response to the emptying of the internal stores by treatment with SR Ca2+-ATPase inhibitors without the need to remove calcium in the external solution (Wayman et al. 1996; Albert & Large, 2002).
Our results do not exclude the possibility that the store-operated Ca2+ influx detected by others in skeletal muscle through either the Mn2+ quenching technique or by intracellular [Ca2+] measurements may flow across the sarcolemma via channels whose conductance is too low for the unitary currents to be resolved or via channels exclusively located in the t-tubule membrane. However, in that latter case, one would still expect changes in the whole-cell membrane conductance of the fibre upon Ca2+ store depletion, unless the amplitude of the corresponding current is too small to be detected. An alternative possibility is that Ca2+ may enter the cell through electrically silent transport or leak processes which, if true, would question the existence of store-operated channels in skeletal muscle.
In conclusion, this study shows that voltage-activated SR Ca2+ release and/or SR Ca2+ depletion are not sufficient to activate the opening of channels carrying inward currents at negative voltages. These results thus seriously challenge the physiological relevance of a store-operated membrane conductance in skeletal muscle, although it cannot be excluded that other conditions also need to be fulfilled for substantial activation of this calcium entry pathway.
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) and in 5 fibres first challenged with several series of depolarizing pulses in the presence of CPA until there was exhaustion of the voltage-activated Ca2+ transients (•). Each fluo-3 fluorescence data point presented for CMC experiments corresponds to the mean change in fluorescence measured in 5 consecutive images captured every second. In all fibres the intracellular EGTA concentration was estimated to be 
