The concentration of free Ca(2+) in the sarcoplasmic reticulum of frog cut twitch skeletal muscle fibers estimated with tetramethylmurexide

Abstract

One aim of this article was to determine the resting concentration of free Ca(2+) in the sarcoplasmic reticulum (SR) of frog cut skeletal muscle fibers ([Ca(2+)](SR,R)) using the calcium absorbance indicator dye tetramethylmurexide (TMX). Another was to determine the ratio of [Ca(2+)](SR,R) to TMX's apparent dissociation constant for Ca(2+) (K(app)) in order to establish the capability of monitoring [Ca(2+)](SR)(t) during SR Ca(2+) release - a signal needed to determine the Ca(2+) permeability of the SR. To reveal the properties of TMX in the SR, the surface membrane was rapidly permeabilized with saponin to rapidly dissipate myoplasmic TMX. Results indicated that the concentration of Ca-free TMX in the SR was 2.8-fold greater than that in the myoplasm apparently due to binding of TMX to sites in the SR. Taking into account that such binding might influence K(app) as well as a dependence of K(app) on TMX concentration, the results indicate an average [Ca(2+)](SR,R) ranging from 0.43 to 1.70mM. The ratio [Ca(2+)](SR,R)/K(app) averaged 0.256, a relatively low value which should not depend on factors influencing K(app). As a result, the time course of [Ca(2+)](SR)(t) in response to electrical stimulation is well determined by, and approximately linearly related to, the active TMX absorbance signal.