Effects of extracellular calcium on calcium movements of excitation-contraction coupling in frog skeletal muscle fibres

Abstract

1. The effect of low extracellular free calcium ion concentration ([Ca2+]o) on the transient changes in cytoplasmic [Ca2+] associated with membrane depolarization (Ca2+ transients) was studied on single cut skeletal muscle fibres of the frog, voltage clamped in a double-Vaseline-gap chamber. The Ca2+ transients were monitored with the dye Antipyrylazo III diffused intracellularly. 2. The Ca2+ transients were substantially reduced in external salines with low [Ca2+] (10(-5) M or less and Mg2+ substituted for Ca2+). This decrease was more noticeable at late times during 100 ms or longer depolarizing pulses. 3. The rates of the processes that remove Ca2+ from the myoplasmic solution were not altered by the low [Ca2+]o. This implies that the input flux of Ca2+ into the myoplasm was reduced. 4. The Ca2+ input flux, equal to release flux from the sarcoplasmic reticulum (SR) plus Ca2+ influx via the T-tubule membrane Ca2+ channel, was derived from the Ca2+ transient. In low [Ca2+]o the peak input flux was reduced by 45% (n = 16 fibres) and decayed more rapidly during a depolarizing pulse. 5. The reduction in Ca2+ influx via the T-tubule membrane Ca2+ channel due to the reduced [Ca2+]o could not account for more than 5% of the reduction in Ca2- input flux, which was thus interpreted as an actual reduction of release from the SR. 6. The inward (T-tubular) Ca2+ current was not associated with this effect of extracellular Ca2+ as the effect was voltage independent at high intracellular voltages at which the Ca2+ inward current was strongly voltage dependent. 7. Low [Ca2+]o made Ca2+ release more readily inactivatable; the effect of low [Ca2-]o is best described as a left shift by 29 mV of the 'inactivation curve' of Ca2+ release, relating peak release flux to membrane holding potential. 8. The reduction of Ca2+ release by low [Ca2+]o was not accompanied by changes in the voltage dependence of Ca2+ release or in the threshold voltage for just-detectable release. 9. The results are consistent with a primary effect of Ca2+ on the T-tubular-membrane voltage sensor of excitation-contraction coupling.