Skeletal Muscle

The modulation by internal free [Mg 2 1 ] of spontaneous calcium release events (Ca 2 1 “sparks”) from the sarcoplasmic reticulum (SR) was studied in depolarized notched frog skeletal muscle fibers using a laser scanning confocal microscope in line-scan mode ( x vs. t ). Over the range of [Mg 2 1 ] from 0.13 to 1.86 mM, decreasing the [Mg 2 1 ] induced an increase in the frequency of calcium release events in proportion to [Mg 2 1 ] 2 1.6 . The change of event frequency was not due to changes in [Mg-ATP] or [ATP]. Analysis of individual SR calcium release event properties showed that the variation in event frequency induced by the change of [Mg 2 1 ] was not accompanied by any changes in the spatiotemporal spread (i.e., spatial half width or temporal half duration) of Ca 2 1 sparks. The increase in event frequency also had no effect on the distribution of event amplitudes. Finally, the rise time of calcium sparks was independent of the [Mg 2 1 ], indicating that the open time of the SR channel or channels underlying spontaneous calcium release events was not altered by [Mg 2 1 ] over the range tested. These results suggest that in resting skeletal fibers, [Mg 2 1 ] modulates the SR calcium release channel opening frequency by modifying the average closed time of the channel without altering the open time. A kinetic reaction scheme consistent with our results and those of bilayer and SR vesicle experiments indicates that physiological levels of resting Mg 2 1 may inhibit channel opening by occupying the site for calcium activation of the SR calcium release channel. key words: excitation–contraction coupling • Ca sparks • ryanodine receptor • magnesium • confocal microscopy i n t r o d u c t i o n During excitation–contraction coupling of skeletal muscle, depolarization of the transverse tubule membrane results in the activation of the dihydropyridine receptor voltage sensors, which cause the opening of the ryanodine receptor (RyR) 1 calcium channels, located in the sarcoplasmic reticulum (SR) (see Schneider, 1994, for references). The subsequent elevation of intracellular calcium arises from the summation of localized discrete SR calcium release events, or calcium sparks (Tsugorka et al., 1995; Klein et al., 1996; Schneider and Klein, 1996). In a recent study, we showed that the average properties of individual SR calcium release events are independent of membrane potential (Lacampagne et al., 1996), suggesting that the global calcium transient and the rate of release is modulated by a change in the frequency of SR calcium release event occurrence (Klein et al., 1997). As in cardiac cells (Cheng et al., 1993), “spontaneous” SR calcium release events also have been described to occur in skeletal muscle at a very low frequency independently of activation of the voltage sensor (Klein et al., 1996). From that study, it was proposed that spontaneous events presumably result from the activation of SR calcium release channels by calcium-induced calcium release (Klein et al., 1996). Mg 2 1 ions have been shown to suppress SR calcium release in a variety of skeletal muscle preparations. In mechanically skinned frog or toad skeletal fibers, spontaneous contractions produced by reducing [Mg 2 1 ] in the bathing solution have been described (HerrmannFrank, 1989; Lamb and Stephenson, 1991). In these preparations, much of the release of calcium from the SR may have been due to activation of voltage sensors due to the polarization of sealed transverse tubules (TT). From their experiments, Lamb and Stephenson (1991) suggested that magnesium, in the physiological range, inhibits the SR calcium channel and that activation of the voltage sensor by TT membrane depolarization can reduce the affinity of the channel for Mg 2 1 . Lamb and Stephenson (1994) found a comparable modulatory effect of magnesium ions on mammalian and frog skeletal fibers. In frog skeletal muscle fibers, Portions of this work were previously published in abstract form (Lacampagne, A., M.G. Klein, K. Bagley, and M.F. Schneider. 1997. Biophys. J. 72: A 43). Address correspondence to M.F. Schneider, Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Greene St., Baltimore, MD 21201. Fax: 410-7068297; E-mail: [email protected] 1 Abbreviations used in this paper: CICR, calcium-induced calcium release; RyR, ryanodine receptor; SR, sarcoplasmic reticulum; TT, transverse tubule. on A ril 8, 2017 D ow nladed fom Published February 1, 1998