Regulation of Ca²⁺ Sparks by Ca²⁺ and Mg²⁺ in Mammalian and Amphibian Muscle. An RyR Isoform-specific Role in Excitation–Contraction Coupling?

Ca²⁺ and Mg²⁺ are important mediators and regulators of intracellular Ca²⁺ signaling in muscle. The effects of changes of cytosolic [Ca²⁺] or [Mg²⁺] on elementary Ca²⁺ release events were determined, as functions of concentration and time, in single fast-twitch permeabilized fibers of rat and frog. Ca²⁺ sparks were identified and their parameters measured in confocal images of fluo-4 fluorescence. Solutions with different [Ca²⁺] or [Mg²⁺] were rapidly exchanged while imaging. Faster and spatially homogeneous changes of [Ca²⁺] (reaching peaks >100 μM) were achieved by photolysing Ca NP-EGTA with laser flashes. In both species, incrementing cytosolic [Ca²⁺] caused a steady, nearly proportional increase in spark frequency, reversible upon [Ca²⁺] reduction. A greater change in spark frequency, usually transient, followed sudden increases in [Ca²⁺] after a lag of 100 ms or more. The nonlinearity, lag, and other features of this delayed effect suggest that it requires increase of [Ca²⁺] inside the SR. In the frog only, increases in cytosolic [Ca²⁺] often resulted, after a lag, in sparks that propagated transversally. An increase in [Mg²⁺] 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 [Mg²⁺]. Reducing [Mg²⁺] below 2 mM, which should enable the RyR channel's activation (CICR) site to bind Ca²⁺, caused progressive increase in spark frequency in the frog, but had no effect in the rat. Spark propagation and enhancement by sub-mM Mg²⁺ 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.