Quantal Properties of Voltage-Dependent Ca2+ Release in Frog Skeletal Muscle Persist After Reduction of [Ca2+] in the Sarcoplasmic Reticulum.

In skeletal muscle, the Ca2+ release flux elicited by a voltage clamp pulse rises to an early peak that inactivates rapidly to a much lower steady level. Using a double pulse protocol the fast inactivation follows an arithmetic rule: if the conditioning depolarization is less than or equal to the test depolarization, then decay (peak minus steady level) in the conditioning release is approximately equal to suppression (unconditioned minus conditioned peak) of the test release. This is due to quantal activation by voltage, analogous to the quantal activation of IP3 receptor channels. Two mechanisms are possible. One is the existence of subsets of channels with different sensitivities to voltage. The other is that the clusters of Ca2+-gated Ryanodine Receptor (RyR) ß in the parajunctional terminal cisternae might constitute the quantal units. These Ca2+-gated channels are activated by the release of Ca2+ through the voltage-gated RyR α channels. If the RyR ß were at the basis of quantal release, it should be modified by strong inhibition of the primary voltage-gated release. This was attained in two ways, by sarcoplasmic reticulum (SR) Ca2+ depletion and by voltage-dependent inactivation. Both procedures reduced global Ca2+ release flux, but SR Ca2+ depletion reduced the single RyR current as well. The effect of both interventions on the quantal properties of Ca2+ release in frog skeletal muscle fibers were studied under voltage clamp. The quantal properties of release were preserved regardless of the inhibitory maneuver applied. These findings put a limit on the role of the Ca2+-activated component of release in generating quantal activation.

A study of store dependent Ca²âº influx in frog skeletal muscle.

Ca²âº influx across the plasma membrane upon drastic reduction of the sarcoplasmic reticulum Ca²âº content was studied in voltage clamped frog skeletal muscle fibers. Depletion was produced by the application of 30 µM cyclopiazonic acid (CPA) in Ca²âº-free, [Mg²âº] = 8 mM external salines and produced an increase in resting free myoplasmic [Ca²âº]. Once depletion was attained the external solution was changed to one containing the same concentration of the drug but with Ca²âº instead of Mg²âº. Of 27 fibers studied only nine showed a secondary increase in free myoplasmic [Ca²âº] upon readmitting Ca²âº in the external perfusate. In the presence of CPA the resting myoplasmic [Ca²âº] in Ca²âº-free external saline was 0.08 ± 0.01 µM (Mean ± SEM), and in Ca²âº-containing external saline 0.10 ± 0.02 µM when the intracellular solution contained [EGTA] = 5 mM (n = 18). In cells with lower (0.5 mM) intracellular [EGTA] resting [Ca²âº] went from 0.35 +/- 0.08 µM in Ca²âº-free external solution to 0.42 +/- 0.12 µM upon reapplication of Ca²âº(n = 9). In both cases the differences between means were not statistically significant (paired t test, p = 0.13 in high EGTA and p = 0.25 in low EGTA). In the nine fibers that showed a secondary increase of resting [Ca²âº] the holding current measured at -90 mV did not significantly change. These results suggest the Ca²âº entry secondary to store depletion is a labile mechanism in frog skeletal muscle and when present does not have an obvious electrical manifestation.