Regulation of Ca 2 Sparks by Ca 2 and Mg 2 in Mammalian and Amphibian Muscle . An RyR Isoform-specific Role in Excitation – Contraction Coupling ?

Ca 2 and Mg 2 are important mediators and regulators of intracellular Ca 2 signaling in muscle. The effects of changes of cytosolic [Ca 2 ] or [Mg 2 ] on elementary Ca 2 release events were determined, as functions of concentration and time, in single fast-twitch permeabilized fibers of rat and frog. Ca 2 sparks were identified and their parameters measured in confocal images of fluo-4 fluorescence. Solutions with different [Ca 2 ] or [Mg 2 ] were rapidly exchanged while imaging. Faster and spatially homogeneous changes of [Ca 2 ] (reaching peaks 100 M) were achieved by photolysing Ca NP-EGTA with laser flashes. In both species, incrementing cytosolic [Ca 2 ] caused a steady, nearly proportional increase in spark frequency, reversible upon [Ca 2 ] reduction. A greater change in spark frequency, usually transient, followed sudden increases in [Ca 2 ] after a lag of 100 ms or more. The nonlinearity, lag, and other features of this delayed effect suggest that it requires increase of [Ca 2 ] inside the SR. In the frog only, increases in cytosolic [Ca 2 ] often resulted, after a lag, in sparks that propagated transversally. An increase in [Mg 2 ] caused a fall of spark frequency, but with striking species differences. In the rat, but not the frog, sparks were observed at 4–40 mM [Mg 2 ]. Reducing [Mg 2 ] below 2 mM, which should enable the RyR channel’s activation (CICR) site to bind Ca 2 , caused progressive increase in spark frequency in the frog, but had no effect in the rat. Spark propagation and enhancement by sub-mM Mg 2 are hallmarks of CICR. Their absence in the rat suggests that CICR requires RyR3 para-junctional clusters, present only in the frog. The observed frequency of sparks corresponds to a channel open probability of 10 7 in the frog or 10 8 in the rat. Together with the failure of photorelease to induce activation directly, this indicates a basal inhibition of channels in situ. It is proposed that relief of this inhibition could be the mechanism by which increased SR load increases spark frequency. key words: sarcoplasmic reticulum • excitation–contraction coupling • Ca channels • ryanodine receptors • calcium photorelease I N T R O D U C T I O N After the action potential depolarizes the transverse (T) tubular membrane in muscle, a sequence of events takes place that result in rapid activation of Ca 2 release from the SR, followed by fast and synchronous closing of the release channels and removal of released Ca 2 . The speed of the resulting Ca 2 transient’s onset and termination is crucial for a rapid contraction–relaxation cycle (Rome et al., 1996). Although a reasonably complete picture of the major aspects of excitation– contraction coupling has emerged, many aspects, especially those controlling the speed of activation and termination of Ca 2 release, remain to be understood. The presence of different RyR isoforms in variable proportions in mammals and amphibians suggests that mechanisms may be different in these two taxonomic classes. In both, channels of the “skeletal” isoform (RyR1 in mammals, in amphibians) are located in two parallel rows facing the T tubular membrane. In this membrane, tetrads of dihydropyridine (DHP) receptors face and perhaps make contact with the large cytoplasmic domain of every other RyR, in a precise skipping pattern (Block et al., 1988). In the frog, in addition to junctional RyRs, there are isoforms, homologous to RyR3 of mammals (for review see Sutko and Airey, 1996; Ogawa et al. 1999). receptors are located in a parajunctional region close to the triad. Although they are ordered, the geometric arrangement of their clusters is different from that of their junctional counterparts (Felder and Franzini-Armstrong, 2002). Most importantly, they seem to be too far from DHP receptors for any physical interaction with them. Studies of cell-averaged Ca 2 transients have found differences in the waveform of Ca 2 release in response Address correspondence to Eduardo Ríos, Department of Molecular Biophysics and Physiology, Rush University School of Medicine, 1750 W. Harrison St., Suite 1279JS, Chicago, IL 60612. Fax: (312) 9428711. email: erios@rush.edu Abbreviations used in this paper: DHP, dihydropyridine; EDL, extensor digitorum longus; T, transverse. D ow naded rom http://rupress.org/jgp/24/4/409/1220229/jgp1244409.pdf by gest on 23 Feruary 2022