PROPERTIES AND ROLES OF AN INTRAMEMBRANOUS CHARGE MOBILIZED AT HIGH VOLTAGES IN FROG SKELETAL-MUSCLE

1. Membrane Ca2+ currents (I-Ca), intramembranous charge movement curr ents and changes in intracellular Ca2+ concentrations were recorded in voltage clamped cut skeletal muscle fibres of the frog. Intra- and ex tracellular solutions, designed to prevent ionic current, and use of t he saponin-permeabilization procedure made possible the measurement of transfer of intramembranous charge up to high positive potentials. 2. Substantial charge moved at positive potentials. This charge was show n to be intramembranous in four tests of charge conservation, demonstr ating that the total displacement of charge depended only on the initi al and final voltages, and not on the history or pathway of intermedia te voltages. 3. On average, in twenty-three cells, the charge moved at 50 mV was 31 +/- 1.9 nC mu F-1 (mean +/- S.E.M.), and at 0 mV was 25 +/- 1.5 nC mu F-1. Approximately one-fifth of the total charge moved a bove 0 mV. 4. The charge that moved at high voltage could be fitted, i n most cases, with a Boltzmann distribution function. In twenty of twe nty-three cells, the total charge distribution could be fitted as the sum of two Boltzmann terms; the high voltage term was centred at 11 +/ - 3.9 mV, with a steepness factor of 12 +/- 1.6 mV and a magnitude of 8.6 +/- 1.1 nC mu F-1. The low voltage term was centred at -43 +/- 2.1 mV, with a steepness factor of 7.7 +/- 0.6 mV and a magnitude of 22 /- 1.8 nC mu F-1. Thus, the high voltage component comprised about one -quarter of the mobile charge. In four cells it was possible to fit th e sum of three Boltzmann terms to the distribution of mobile charge; t he parameters of the high voltage term then were similar to those foun d by fitting the sum of two Boltzmann terms to the same data. 5. The v oltage dependence of activation of I-Ca was determined in a buffered 2 mM Ca2+ external solution, from the tails of ionic current at -30 mV, after activating pulses to various voltages, the duration of which wa s sufficient to reach the peak of inward current. The voltage dependen ce was described by a Boltzmann function centred at 2.6 +/- 6.9 mV (n = 6), with a steepness factor of 20 +/- 1.4 mV. The voltages at which the high voltage charge moved were roughly the same as those at which I-Ca was activated. 6. Calcium release from the sarcoplasmic reticulum was determined from the Ca2+ transients. Calcium release continued to increase at potentials above 0 mV. On average, 15% of the maximum rel ease flux permeability activated above 0 mV.7. The results are consist ent with the hypothesis that high voltage charge moves in transitions required for activation of I-Ca. The conservation of charge, in pulses of different duration at high voltage, suggests that the slow transit ions which precede channel opening are not strongly voltage dependent. 8. The fact that calcium release continued to increase at high voltag es requires the activation of transitions in the voltage sensor. Such transitions should contribute to the charge measured at high voltages in the present experiments.