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

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 Ca2+ inward current exhibiting half-maximal activation at - 7.60 +/- 1.29 and 3.0 +/- 1.44 mV, respectively. The rate of Ca2+ release from the SR was calculated after fitting the relaxation phases of fluorescence ratio signals with a kinetic model to quantify overall Ca2+ removal. Results obtained with the two indicators were similar. Ca2+ release was 2-3 orders of magnitude larger than the flux carried by the L-type Ca2+ 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 Ca2+ 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.