Voltage‐controlled Ca²⁺ release and entry flux in isolated adult muscle fibres of the mouse

Abstract

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...

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“…Using a two-electrode voltage clamp system, we recorded the L-type Ca 2ϩ inward current and derived the SR Ca 2ϩ release flux from simultaneous measurements of voltage-activated fura-2 fluorescence signals as described by Ursu et al (11). Fig.…”

“…Moreover, steady-state Ca 2ϩ entry caused by the L-type Ca 2ϩ channel window conductance, which is small compared with release (11), is even further reduced ( Fig. 6D and 6F, continuous red traces).…”

“…All animal procedures were approved by the local animal care committee. Interosseus muscle fiber dissociation was performed as previously described (11). Voltage Clamping and Data Analysis.…”

“…Voltage Clamping and Data Analysis. The experimental design was as described (11,27). Briefly, isolated fibers were voltage-clamped with a two-electrode system (Axoclamp 2B, Axon Instruments) and imaged using a fluorescence microscope (Axiovert 135 TV, 40x/0.75W objective, Zeiss).…”

“…To construct Ca 2ϩ current-voltage relationships, we averaged the last 10 milliseconds of each depolarization step. Ratiometric Ca 2ϩ transients were subjected to a removal model fit analysis as originally described by Melzer et al (28) to calculate the flux of Ca 2ϩ from the SR (11,29). The pipette concentrations of EGTA and fura-2 were used as the buffer concentrations for the removal analysis.…”