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

Abstract

Sarcoplasmic reticulum (SR) Ca release was studied at 13-16~ in cut fibers (sarcomere length, 3.4-3.9 I~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 [Ca] 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 ApH signal measured with phenol red by -13/2. The value of 13, 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 I~M in the action-potential experiments and 2,544 IxM 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,'~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 was 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~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 was studied with small step depolarizations that open no more than one SR Ca channel in 7

A slow component of intramembranous charge movement during sarcoplasmic reticulum calcium release in frog cut muscle fibers.

“…1 [CaT]/dt trace then rapidly decreased to a local minimum of ~6 I~M/ms. This decrease, which occurred at about the same rate as that of the/t~st-I~o,t~ol trace, is similar to that observed in other experiments in fibers equilibrated with 20 mM EGTA and 1.76 mM Ca (Pape et al, 1995;Jong et al, 1995a) and attributed to Ca inactivation of Ca release (Baylor et al, 1983;Simon et al, 1985;Schneider and Simon, 1988;Simon et al, 1991;Jong et al, 1995a). After the early minimum, dA [CaT]/dt increased slightly and remained elevated for ~150 ms before eventually returning to zero.…”

“…This value is somewhat smaller than those reported by Pape et al (1995) in fibers equilibrated with the same internal and external solutions. The reason is that, in the experiments reported in Pape et al (1995), the duration of the recovery period between successive depleting depolarizations was 5 rain instead of 2 min, so that the SR was able to reaccumulate more Ca from the myoplasm.…”

“…The methods used for the determination of intramembranous charge movement are described by Chandler and Hui (1990) and Chandler (1990, 1991). The methods used for the estimation of SR Ca release with EGTA-phenol red are described in Pape et al (1995). Throughout this article, a 0.05-kHz digital Gaussian filter (Colquhoun and Sigworth, 1983) was used to filter the traces of SR Ca release.…”

“…1.76 mM Ca was added to the solutions that were used for the initial period of fiber equilibration; during the course of some experiments, as described in the text and figure legends, this solution was changed to one that contained 0 mM Ca. The pH was adjusted to 7.0 by the addition of CsOH, and the calculated concentration of free Mg was 1 mM (Pape et al, 1995). The composition of the central-pool solution was 110 mM TEA-gluconate, 10 mM MgSO 4, 1 IxM tetxodotoxin, and 10 mM MOPS, pH = 7.1; it was nominally Ca-free.…”

“…1 A shows A[CaT], the estimated amount of Ca that had been released from the SR (expressed as myoplasmic concentration). After depolarization, A [CAT] progressively increased during the pulse and reached a plateau value of 1,787 ~M after ~500 ms. After repolarization, A[CaT] remained elevated and decreased very slowly, as is typical of fibers equilibrated with 20 mM EGTA (Pape et al, 1995). The value of [CasR] R, the amount of readily releasable Ca that was inside the SR before stimulation (expressed as myoplasmic concentration), is assumed to be equal to the plateau value of A[CaT], 1,787 p~M.…”