Four manifestations of excitation-contraction (E-C) coupling were derived from measurements in cut skeletal muscle fibers of the frog, voltage clamped in a Vaseline-gap chamber: intramembranous charge movement currents, myoplasmic [Ca2+] transients, flux of calcium release from the sarcoplasmic reticulum (SR), and the intrinsic optical transparency change that accompanies calcium release. In attempts to suppress Ca release by direct effects on the SR, three interventions were applied: (a) a conditioning pulse that causes calcium release and inhibits release in subsequent pulses by Ca-dependent inactivation; (b) a series of brief, large pulses, separated by long intervals (greater than 700 ms), which deplete Ca2+ in the SR; and (c) intracellular application of the release channel blocker ruthenium red. All these reduced calcium release flux. None was expected to affect directly the voltage sensor of the T-tubule; however, all of them reduced or eliminated a component of charge movement current with the following characteristics: (a) delayed onset, peaking 10-20 ms into the pulse; (b) current reversal during the pulse, with an inward phase after the outward peak; and (c) OFF transient of smaller magnitude than the ON, of variable polarity, and sometimes biphasic. When the total charge movement current had a visible hump, the positive phase of the current eliminated by the interventions agreed with the hump in timing and size. The component of charge movement current blocked by the interventions was greater and had a greater inward phase in slack fibers with high [EGTA] inside than in stretched fibers with no EGTA. Its amplitude at -40 mV was on average 0.26 A/F (SEM 0.03) in slack fibers. The waveform of release flux determined from the Ca transients measured simultaneously with the membrane currents had, as described previously (Melzer, W., E. Ríos, and M. F. Schneider. 1984. Biophysical Journal. 45:637-641), an early peak followed by a descent to a steady level during the pulse. The time at which this peak occurred was highly correlated with the time to peak of the current suppressed, occurring on average 6.9 ms later (SEM 0.73 ms). The current suppressed by the above interventions in all cases had a time course similar to the time derivative of the release flux; specifically, the peak of the time derivative of release flux preceded the peak of the current suppressed by 0.7 ms (SEM 0.6 ms). The magnitude of the current blocked was highly correlated with the inhibitory effect of the interventions on Ca2+ release flux.(ABSTRACT TRUNCATED AT 400 WORDS)
The effects of reducing the extracellular calcium concentration on the twitch in isolated frog's skeletal muscle fibres.
