Calcium inactivation of calcium release in frog cut muscle fibers that contain millimolar EGTA or Fura-2.

Abstract

Cut muscle fibers from Rana teraporaria (sarcomere length, 3.4-4.2 p.m) were mounted in a double and equilibrated with end-pool solutions that contained 20 mM EGTA and 1.76 mM Ca. Sarcoplasmic reticulum (SR) Ca release was estimated from changes in pH (Pape, P. C., D.-S.Jong, and W. I~ Chandler. 1995.Journal of GeneralPhysiology. 106:000-000). Although the amplitude and duration of the [Ca] transient, as well as its spatial spread from the release sites, are reduced by EGTA, SR Ca release elicited by either depolarizing voltage-clamp pulses or action potentials behaved in a manner consistent with Ca inactivation of Ca release. After a step depolarization to -20 or 10 mV, the rate of SR Ca release, corrected for SR Ca depletion, reached a peak value within 5-15 ms and then rapidly decreased 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. Immediately after an action potential or a 10-15 ms prepulse to -20 mV, the peak rate of SR Ca release elicited by a second stimulation, as well as the fractional amount of release, were substantially decreased. The rising phase of the rate of release was also reduced, suggesting that at least 0.9 of the ability of the SR to release Ca had been inactivated by the first stimulation. There was little change in intramembranous charge movement, suggesting that the changes in SR Ca release were not caused by changes in its voltage activation. These effects of a first stimulation on the rate of SR Ca release elicited by a second stimulation recovered during repolarization to -90 mV; the time constant of recovery was ~25 ms in the action-potential experiments and ~50 ms in the voltage-clamp experiments. Fura-2, which is able 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 greater amount, did not prevent Ca inactivation of Ca release, even at con-

Calcium release and its voltage dependence in frog cut muscle fibers equilibrated with 20 mM EGTA.

“…This explanation is consistent with the rapid speed with which Ca inactivation of Ca release is able to develop, with a time constant of 2-4 ms ( Fig. 13 and Jong et al, 1995).…”

“…Such inactivation cannot account for the smaller rates of release in our voltageclamp experiments because similar small rates were observed with 1.8 mM Ca, instead of 10 mM Mg, in the external solution (Jong et al, 1995).…”

“…For example, the quasi-steady to peak ratio of dA [CAT]/dt during a step depolarization was larger in our EGTA experiments than in experiments in which the [Ca] transient was not modified. Additional information about Ca inactivation of Ca release and its reduction by EGTA is presented in the following article (Jong et al, 1995).…”

“…8 B). Additional information about this inactivation and its reduction by EGTA is given in the following article (Jong et al, 1995).…”

“…As far as we can tell, the presence of EGTA appears to have had relatively litde effect on the function of SR Ca channels and their gating by the voltage sensors in the membranes of the transverse tubular system. The principle modulatory effect of EGTA appears to be a reduction in Ca inactivation of Ca release, as described in the following article (Jong, Pape, Baylor, and Chandler, 1995). Many of the theoretical properties of the EGTAphenol red method are described in Appendix A.…”