Calcium Release in Frog Cut Twitch Fibers Exposed to Different Ionic Environments under Voltage Clamp

Abstract

Calcium release was measured in highly stretched frog cut twitch fibers mounted in a double Vaseline-gap voltage clamp chamber, with the internal solution containing 20 mM EGTA plus 0.4 or 1.8 mM added calcium. Rise in myoplasmic [Ca 2+] was monitored with antipyrylazo III as the indicator at a temperature of 13 to 14°C. The waveform of calcium release rate (Rel) computed from the absorbance change showed an early peak (Rel p) followed by a maintained phase (Rel m). Each Rel p-versus-V plot was fitted with a Boltzmann distribution function. The maximum value of Rel p (Rel p,max) was compared in various calcium-containing external solutions. The average value in a Cl − solution was about one-third larger than those in a CH 3SO 3 − or gluconate solution, whereas the values in the CH 3SO 3 − and gluconate solutions had no statistically significant difference. In external solutions containing CH 3SO 3 − or gluconate, a replacement of the Ca 2+ with Mg 2+ reduced Rel p,max by 30 to 50%, on average. The values of Rel p,max also had no statistically significant difference among calcium-free external solutions containing different impermeant anions. An increase of the nominal free [Ca 2+] in the end-pool solution from a reduced to the normal physiological level increased the value of Rel p,max, and also slowed the decay of the maintained phase of the Rel waveform. The Rel waveforms in the Cl − and CH 3SO 3 − solutions were compared in the same fiber at a fixed potential. CH 3SO 3 − increased the time to peak, reduced Rel p, and increased Rel m, and the effects were partially reversible. Under the hypothesis that the decay of the peak was due to calcium inactivation of calcium release, the inactivation was larger in Cl − than in CH 3SO 3 −, in qualitative agreement with the ratio of Rel p in the two solutions. Under the alternative hypothesis that the peak and the maintained phase were separately gated by calcium and depolarization, respectively, then CH 3SO 3 − appeared to decrease the calcium-gated component and increase the voltage-gated component.