Analysis of Calcium (Ca) Transients during Excitation-Contraction Coupling in Frog Twitch Muscle Fibers

Abstract

Singly-dissected fibers were micro-injected with furaptra, a rapidly-responding Ca indicator. Resting fluorescence (FR) and action-potential evoked changes (ΔF) were measured at 16 oC; ΔF/FR was scaled to units of ΔfCaD, the change in fraction of furaptra in the Ca-bound form. Measured ΔfCaD was compared with ΔfCaD simulated with a kinetic model of the underlying myoplasmic Ca movements. During the period 30-200 ms after an action potential, simulated ΔfCaD decayed toward baseline more slowly than measured ΔfCaD. If ΔfCaD was simulated with a modified model that incorporated competition between Mg and Ca for occupancy of the regulatory sites on troponin (assumed dissociation constant of Mg's reaction with the regulatory sites, 2.2 mM; assumed myoplasmic free [Mg], 1 mM), good agreement with measured ΔfCaD was observed. The results support the conclusion that Mg, at physiological levels, competes with Ca for occupancy of the regulatory sites, as indicated in some experiments from fragmented preparations, including tension-pCa measurements in skinned fibers and biochemical studies of isolated troponin molecules. ΔfCaD in frog was also compared with our previous measurements and simulations of ΔfCaD in mouse fibers (reviewed in J. Gen. Physiol., 2012). In frog, the SR Ca release flux elicited by an action potential appears to be the sum of two components. The time course of the first component is similar to that of the entire flux waveform in both fast-and slow-twitch mouse fibers, while that of the second is several-fold slower; the fractional release amounts are ∼0.85 (first component) and ∼0.15 (second component). An anatomical basis for two release components in frog is the presence of both junctional and para-junctional Ca release channels, whereas the mouse fibers have only junctional channels (Felder and Franzini-Armstrong, 2002). Supported by NIH (GM 086167)