The voltage-activated fluxes of Ca2+ from the sarcoplasmic reticulum (SR) and from the extracellular space were studied in skeletal muscle fibres of adult mice. Single fibres of the interosseus muscle were enzymatically isolated and voltage clamped using a two-electrode technique. The fibres were perfused from the current-passing micropipette with a solution containing 15 mM EGTA and 0.2 mM of either fura-2 or the faster, lower affinity indicator fura-FF. Electrical recordings in parallel with the fluorescence measurements allowed the estimation of intramembrane gating charge movements and transmembrane Ca 2+ inward current exhibiting half-maximal activation at −7.60 ± 1.29 and 3.0 ± 1.44 mV, respectively. The rate of Ca 2+ release from the SR was calculated after fitting the relaxation phases of fluorescence ratio signals with a kinetic model to quantify overall Ca 2+ removal. Results obtained with the two indicators were similar. Ca 2+ release was 2-3 orders of magnitude larger than the flux carried by the L-type Ca 2+ current. At maximal depolarization (+50 mV), release flux peaked at about 3 ms after the onset of the voltage pulse and then decayed in two distinct phases. The slower phase, most likely resulting from SR depletion, indicated a decrease in lumenal Ca 2+ content by about 80% within 100 ms. Unlike in frog fibres, the kinetics of the rapid phase of decay showed no dependence on the filling state of the SR and the results provide little evidence for a substantial increase of SR permeability on depletion. The approach described here promises insight into excitation-contraction coupling in future studies of genetically altered mice. Mechanical force in muscle is controlled by rapid changes in the myoplasmic calcium concentration that result from Ca 2+ release and uptake mechanisms of the sarcoplasmic reticulum (SR) (for overviews see for instance Rüegg, 1988;Melzer et al. 1995;Bers, 2001). The release of Ca 2+ ions stored in the SR is activated by a depolarization of the transverse tubular membrane (Ríos & Brum, 2002). The fundamentals of Ca 2+ release and its voltage control have been elucidated in voltage clamp experiments mainly performed on isolated frog muscle fibres (see summaries by Ríos & Pizarro, 1991;Schneider, 1994;Melzer et al. 1995). Analysis of the Ca 2+ signals recorded with optical indicator dyes revealed a characteristic time course of the global efflux of calcium from the SR during a step depolarization. An early large but transient flux component (peak), attributed to Ca 2+ -induced activation and inactivation (Melzer et al. 1987;Csernoch et al. 1993), could be distinguished from a rather persistent plateau component of Ca 2+ release flux. The latter showed a slow decline attributed to progressive store depletion .In recent years, cellular studies on muscle excitation-contraction (EC) coupling shifted their focus from amphibian preparations to the experimentally more challenging mammalian muscle cells. Of central importance are muscle cells of mice with genetic alterations of EC cou...

The purpose of this study was to quantify the Ca2+ fluxes underlying Ca2+ transients and their voltage dependence in myotubes by using the “removal model fit” approach. Myotubes obtained from the mouse C2C12 muscle cell line were voltage-clamped and loaded with a solution containing the fluorescent indicator dye fura-2 (200 μM) and a high concentration of EGTA (15 mM). Ca2+ inward currents and intracellular ratiometric fluorescence transients were recorded in parallel. The decaying phases of Ca2+-dependent fluorescence signals after repolarization were fitted by theoretical curves obtained from a model that included the indicator dye, a slow Ca2+ buffer (to represent EGTA), and a sequestration mechanism as Ca2+ removal components. For each cell, the rate constants of slow buffer and transport and the off rate constant of fura-2 were determined in the fit. The resulting characterization of the removal properties was used to extract the Ca2+ input fluxes from the measured Ca2+ transients during depolarizing pulses. In most experiments, intracellular Ca2+ release dominated the Ca2+ input flux. In these experiments, the Ca2+ flux was characterized by an initial peak followed by a lower tonic phase. The voltage dependence of peak and tonic phase could be described by sigmoidal curves that reached half-maximal activation at −16 and −20 mV, respectively, compared with −2 mV for the activation of Ca2+ conductance. The ratio of the peak to tonic phase (flux ratio) showed a gradual increase with voltage as in rat muscle fibers indicating the similarity to EC coupling in mature mammalian muscle. In a subgroup of myotubes exhibiting small fluorescence signals and in cells treated with 30 μM of the SERCA pump inhibitor cyclopiazonic acid (CPA) and 10 mM caffeine, the calculated Ca2+ input flux closely resembled the L-type Ca2+ current, consistent with the absence of SR Ca2+ release under these conditions and in support of a valid determination of the time course of myoplasmic Ca2+ input flux based on the optical indicator measurements.

Ca2+ transients elicited by action potentials were measured using MagFluo-4, at 20-22• C, in intact muscle fibres enzymatically dissociated from mice of different ages (7, 10, 15 and 42 days).

In the present study we describe the analysis of optically recorded whole cell Ca(2+) transients elicited by depolarization in cultured skeletal myotubes. Myotubes were obtained from the mouse muscle-derived cell line C2C12 and from mouse satellite cells. The cells were voltage-clamped and perfused with an artificial intracellular solution containing 15 mM EGTA to ensure that the bulk of the Ca(2+) mobilized by depolarization is bound to this extrinsic buffer. The apparent on- and off-rate constants of EGTA and the dissociation rate constant of fura-2 in the cell were estimated by investigating the Ca(2+)-dependence of kinetic components of the fluorescence decay after repolarization. These parameters were used to calculate the time course of the total voltage-controlled flux of Ca(2+) to the myoplasmic space (Ca(2+) input flux). The validity of the procedure was confirmed by model simulations using artificial Ca(2+) input fluxes. Both C2C12 and primary-cultured myotubes showed a very similar phasic-tonic time course of the Ca(2+) input flux. In most measurements, the input flux was considerably larger and showed a different time course than the estimated Ca(2+) flux carried by the L-type Ca(2+) channels, indicating that it consists mainly of voltage-controlled Ca(2+) release from the sarcoplasmic reticulum. In cells with extremely small fluorescence transients, the calculated input fluxes matched the kinetic characteristics of the Ca(2+) inward current, indicating that Ca(2+) release was absent. These measurements served as a control for the fidelity of the fluorimetric flux analysis. The procedures promise a deeper insight into alterations of Ca(2+) release gating in studies employing myotube expression systems for mutant or chimeric protein components of excitation-contraction coupling.

Functional impacts of the skeletal muscle-specific Ca2+ channel subunit γ1 have previously been studied using coexpression with the cardiac α1C polypeptide in nonmuscle cells and primary-cultured myotubes of γ1-deficient mice. Data from single adult muscle fibers of γ−/− mice are not yet available. In the present study, we performed voltage clamp experiments on enzymatically isolated mature muscle fibers of the m. interosseus obtained from γ+/+ and γ−/− mice. We measured L-type Ca2+ inward currents and intracellular Ca2+ transients during 100-ms step depolarizations from a holding potential of −80 mV. Ratiometric Ca2+ transients were analyzed with a removal model fit approach to calculate the flux of Ca2+ from the sarcoplasmic reticulum. Ca2+ current density, Ca2+ release flux, and the voltage dependence of activation of both Ca2+ current and Ca2+ release were not significantly different. By varying the holding potential and recording Ca2+ current and Ca2+ release flux induced by 100-ms test depolarizations to +20 mV, we studied quasi-steady-state properties of slow voltage–dependent inactivation. For the Ca2+ current, these experiments showed a right-shifted voltage dependence of inactivation. Importantly, we could demonstrate that a very similar shift occurred also in the inactivation curve of Ca2+ release. Voltages of half maximal inactivation were altered by 16 (current) and 14 mV (release), respectively. Muscle fiber bundles, activated by elevated potassium concentration (120 mM), developed about threefold larger contracture force in γ−/− compared with γ+/+. This difference was independent of the presence of extracellular Ca2+ and likely results from the lower sensitivity to voltage-dependent inactivation of Ca2+ release. These results demonstrate a specific alteration of voltage-dependent inactivation of both Ca2+ entry and Ca2+ release by the γ1 subunit of the dihydropyridine receptor in mature muscle fibers of the mouse.