Mechanisms of stimulated 45Ca efflux in skinned skeletal muscle fibers.

Excitation-contraction (E-C) coupling in skeletal muscle can be studied in skinned fibers by direct assay of 45Ca efflux and simultaneous isometric force, under controlled conditions. Recent work provides evidence that such studies can address major current questions about the mechanisms of signal transmission between transverse tubules and sarcoplasmic reticulum and sarcoplasmic reticulum calcium release, as well as operation of the sarcoplasmic reticulum active Ca transport system in situ. Stimulation by imposed ion gradients at constant [K+][Cl-] product results in 45Ca release with two components: a large Ca2+-dependent efflux, responsible for contractile activation, and a small Ca2+-insensitive efflux. The Ca2+-insensitive stimulation is sustained, consistent with sustained depolarization, and appears to gradate the Ca2+-dependent stimulation; this component is likely to reflect intermediate steps in E-C coupling. Several lines of evidence suggest that the depolarizing stimulus acts on the transverse tubules. It is inhibited by the impermeant glycoside ouabain applied before skinning, which should specifically inhibit polarization of subsequently sealed transverse tubules. Sealed polarized transverse tubules also are the only plausible target for stimulation of 45Ca release by monensin and gramicidin D, which can rapidly dissipate Na+ and K+ gradients; a protonophore and the K+-specific ionophore valinomycin are ineffective. Ionophore stimulation is prevented by the permeant glycoside digitoxin; it is also highly Ca2+ dependent. Stimulation of 45Ca release by imposed ion gradients is potentiated by perchlorate, which potentiates charge movements and activation in intact fibers, and is inhibited selectively in highly stretched fibers, presumably by transverse tubule-sarcoplasmic reticulum uncoupling. These results relate the Ca2+-dependent sarcoplasmic reticulum efflux channel to the physiological transverse tubule-sarcoplasmic reticulum coupling pathway, which also could involve Ca2+.