45Ca efflux from skinned muscle fibers is stimulated transiently, by a highly Ca2+-dependent mechanism, by KCl replacement of K propionate. In the present studies, Cl replaced the much less permeant anion methanesulfonate (Mes) either (a) at constant [K], in which increased [K][Cl] permits net KCl and water flux across internal membranes, or (b) at constant [K][Cl] (choline substitution), in which the imposed gradients and diffusion potentials should dissipate slowly. 45Ca efflux and isometric force were measured simultaneously on segments of frog semitendinosus fibers skinned by microdissection. EGTA was applied to chelate released 45Ca either (a) shortly after high [Cl] (interrupted response), to minimize reaccumulation, (b) before high [Cl] (pretreated response), to evaluate Ca2+ dependence, or (c) under control conditions in KMes. KCl replacement of KMes stimulated release of 65% fiber 45Ca within 1 min in interrupted responses; EGTA pretreatment was only moderately inhibitory with substantial residual stimulation. In contrast, choline Cl replacement of KMes induced release of 26-35% fiber 45Ca in interrupted responses; EGTA pretreatment was strongly inhibitory, but release significantly exceeded control with a small, sustained increase in Ca2+-insensitive efflux. These differences in 45Ca release and EGTA inhibition suggest that Cl replacement of Mes at constant [K] stimulates efflux by osmotic effects as well as imposed diffusion potentials; at least half the stimulated 45Ca loss (above control) in interrupted KCl responses is attributable to an osmotic component with low Ca2+ sensitivity. In the highly Ca2+-sensitive stimulation at constant [K][Cl], 45Ca release (above control) in interrupted responses correlated well with that in the pretreated responses of segments from the same fiber, with a slope of 8.4. This relationship suggests that imposed diffusion potentials stimulate a small Ca2+-insensitive component that gradates a much larger Ca2+-dependent efflux. The Ca2+-insensitive component apparently reflects intermediate steps in the excitation-contraction coupling that require positive feedback to result in sufficient Ca release for contraction.
Thin Filament-Reconstituted Skinned Muscle Fibers for the Study of Muscle Physiology