CALCIUM INACTIVATION OF CALCIUM-RELEASE IN FROG CUT MUSCLE-FIBERS THAT CONTAIN MILLIMOLAR EGTA OR FURA-2

Cut muscle fibers from Rana temporaria (sarcomere length, 3.4-4.2 mu m ) were mounted in a double Vaseline-gap chamber (14-15 degrees C) and equilibrated with end-pool solutions that contained PO mM EGTA and 1.7 6 mM Ca. Sarcoplasmic reticulum (SR) Ca release was estimated from cha nges in pH (Pape, P. C., D.-S. Jong, and W. K. Chandler. 1995. Journal of General Physiology. 106:000-000). Although the amplitude and durat ion of the [Ca] transient, as weH as its spatial spread from the relea se sites, are reduced by EGTA, SR Ca release elicited by either depola rizing voltage-clamp pulses or action potentials behaved in a manner c onsistent with Ca inactivation of Ca release. After a step depolarizat ion to -20 or 10 mV, the rate of SR Ca release, corrected for SR Ca de pletion, reached a peak value within 5-15 ms and then rapidly decrease d to a quasi-steady level that was about half the peak value; the time constant of the last half of the decrease was usually 2-4 ms. Immedia tely after an action potential or a 10-15 ms prepulse to -20 mV, the p eak rate of SR Ca release elicited by a second stimulation, as well as the fractional amount of release, were substantially decreased. The r ising phase of the rate of release was also reduced, suggesting that a t least 0.9 of the ability of the SR to release Ca had been inactivate d by the first stimulation. There was little change in intramembranous charge movement, suggesting that the changes in SR Ca release were no t caused by changes in its voltage activation. These effects of a firs t stimulation on the rate of SR Ca release elicited by a second stimul ation recovered during repolarization to -90 mV; the time constant of recovery was similar to 25 ms in the action-potential experiments and similar to 50 ms in the voltage-clamp experiments. Fura-2, which is ab le to bind Ca more rapidly than EGTA and hence reduce the amplitude of the [Ca] transient and its spatial spread from release sites by a gre ater amount, did not prevent Ca inactivation of Ca release, even at co ncentrations as large as 6-8 mM. These effects of Ca inactivation of C a release can be simulated by the three-state, two-step model proposed by Schneider, M. F., and B. J. Simon (1988. Journal of Physiology. 40 5:727-745), in which SR Ca channels function as a single uniform popul ation of channels. They are not readily reconciled with the idea that SR Ca channels exist in two functionally distinct populations, voltage -gated and Ca-gated, and that only the Ca-gated channels are susceptib le to Ca inactivation