CALCIUM-RELEASE AND ITS VOLTAGE-DEPENDENCE IN FROG CUT MUSCLE-FIBERS EQUILIBRATED WITH 20 MM EGTA

Sarcoplasmic reticulum (SR) Ca release was studied at 13-16 degrees C in cut fibers (sarcomere length, 3.4-3.9 mu m) mounted in a double Vaseline-gap chamber. The amplitude and duration of the action-potential stimulated free [Ca] transient were reduced by equilibration with end-pool solutions that contained 20 mM EGTA with 1.76 mM Ca and 0.63 mM phenol red, a maneuver that appeared to markedly reduce the amount of Ca complexed by troponin. A theoretical analysis shows that, under these conditions, the increase in myoplasmic free [Gal is expected to be restricted to within a few hundred nanometers of the SR Ca release sites and to have a time course that essentially matches that of release. Furthermore, almost all of the Ca that is released from the SR is expected to be rapidly bound by EGTA and exchanged for protons with a 1:2 stoichiometry. Consequently, the time course of SR Ca release can be estimated by scaling the Delta pH signal measured with phenol red by - beta/2. The value of beta, the buffering power of myoplasm, was determined in fibers equilibrated with a combination of EGTA, phenol red, and fura-2; its mean value was 22 mM/pH unit. The Ca content of the SR (expressed as myoplasmic concentration) was estimated from the total amount of Ca released by either a train of action potentials or a depleting Voltage step; its mean value was 2,685 mu M in the action-potential experiments and 2,544 mu M in the voltage-clamp experiments. An action potential released, on average, 0.14 of the SR Ca content with a peak rate of release of similar to 5 %/ms. A second action potential, elicited 20 ms later, released only 0.6 times as much Ca (expressed as a fraction of the SR content), probably because Ca inactivation of Ca release nas produced by the first action potential, During a depolarizing voltage step to 60 mV, the rate of Ca release rapidly increased to a peak value of similar to 3 %/ms and then decreased to a quasi-steady level that was only 0.6 times as large; this decrease was also probably due to Ca inactivation of Ca release. SR Ca release tvas studied with small step depolarizations that open no more than one SR Ca channel in 7,000 and increase the value of spatially averaged myoplasmic free [Ca] by only 0.2 nM. With such small depolarizations, SR Ca release is steeply voltage dependent, with a mean e-fold factor of 3.5 mV. Under these conditions, if the spatial distribution of open release sites is random, EGTA is expected to shield each open site from any significant increase in free [Ca] produced by Ca flux through a neighboring open site. Consequently, the open state of any particular site would not be expected to have been caused by Ca from another open site. For this reason, it seems unlikely that SR Ca release itself - by Ca-induced Ca release, by the positive feedback model proposed by Pizarro, Csernoch, Uribe, Rodriguez, and Rios (1991. Journal of General Physiology. 97:913-947), or by some other mechanism - was able to contribute to the steep voltage dependence of Ca release. Rather, it seems likely that the steep voltage dependence of Ca release directly reflects the activity of the voltage sensor in the membranes of the transverse tubular system that regulates a single release site. Such a site might consist of a single SR Ca channel or a collection of channels that function as a singly gated unit (for example, a single voltage-gated channel and neighboring channel[s] that are slaved to it).