A REPETITIVE MODE OF ACTIVATION OF DISCRETE CA2+ RELEASE EVENTS (CA2+ SPARKS) IN FROG SKELETAL MUSCLE FIBRES

Ca2+ release events (Ca2+‘sparks’), which are believed to arise from the opening of a sarcoplasmic reticulum (SR) Ca2+ release channel or a small cluster of such channels that act as a release unit, have been measured in single, frog (Rana pipiens) skeletal muscle fibres. Under conditions of extremely low rates of occurrence of Ca2+ sparks we observed, within individual identified triads, repetitive Ca2+ release events which occurred at a frequency more than 100-fold greater than the prevailing average event rate. Repetitive sparks were recorded during voltage-clamp test depolarizations after a brief (0.3–2 s) repriming interval in fibres held at 0 mV and in chronically depolarized, ‘notched’ fibres. These repetitive events are likely to arise from the re-opening of the same SR Ca2+ release channel or release unit operating in a repetitive gating mode (‘rep-mode’), rather than from the random activation of multiple, independent channels or release units within a triad. A train of rep-mode events thus represents a series of Ca2+ sparks arising from a single location within the fibre. Rep-mode events are activated among different triads in a random manner after brief repriming. The frequency of repetitive events among all identified events during voltage-clamp depolarization to 0 mV after brief repriming was 3.9 ± 1.3%. The occurrence of repetitive events was not related to exposure of the fibre to laser illumination. The events observed within a rep-mode train exhibited a relatively uniform amplitude. Analysis of intervals between identified events in triads exhibiting rep-mode trains indicated similar variations of fluorescence as in neighbouring, quiescent triads, suggesting there was not a significant number of small, unidentified events at the triads exhibiting rep-mode activity. The distribution of rep-mode interspark intervals exhibited a paucity of events at short intervals, consistent with the need for recovery from inactivation before activation of the next event in a repetitive train. The mean interspark interval of repetitive sparks during voltage-clamp depolarizations was 88 ± 5 ms, and was independent of membrane potential. The individual Ca2+ sparks within a rep-mode train were similar in average amplitude and spatiotemporal extent to singly occurring sparks, suggesting a common mechanism for termination of the channel opening(s) underlying both types of events. The average properties of the sparks did not vary during a train. The relative amplitude of a spark within a rep-mode was not correlated with its rise time. Repetitive Ca2+ release events represent a mode of gating of SR Ca2+ release channels which may be significant during long depolarizations and which may be influenced by the biochemical state of the SR ryanodine receptor Ca2+ release channels. Depolarization of a skeletal muscle fibre results in the release of calcium ions (Ca2+) from the sarcoplasmic reticulum (SR). The activation of transverse (t-) tubular membrane voltage sensors (dihydropyridine receptors, DhpR) causes the opening of Ca2+ release channels (ryanodine receptors, RyR) in the membrane of the adjacent, junctional SR, resulting in release of Ca2+ into the myoplasm and consequent contractile activation. Recently, measurements of discrete Ca2+ release events, or Ca2+‘sparks’, have been described in skeletal muscle. These events are thought to arise from the opening of single RyR Ca2+ channels, or small clusters of such channels. Ca2+ sparks have been measured in voltage-clamped skeletal fibres during small depolarizations in fully polarized fibres (Tsugorka et al. 1995; Klein et al. 1996; Schneider & Klein, 1996), and during small and large depolarizations following brief (0.5-2 s) repriming intervals at -90 mV in fibres held at 0 mV (Lacampagne et al. 1996; Klein et al. 1997). Finally, spontaneous Ca2+ sparks having similar rise time, amplitude and spatial spread have also been recorded in polarized or depolarized fibres (Klein et al. 1996), albeit at a much lower frequency. The activation of these spontaneous events is modulated not by depolarization, but by the binding of myoplasmic ligands such as Ca2+ and Mg2+ to the RyRs (Klein et al. 1996; Lacampagne et al. 1998). Calcium release during depolarization of skeletal muscle is steeply dependent on membrane voltage. However, the average properties (rise time, amplitude, spatiotemporal spread) of discrete Ca2+ sparks are voltage independent (Lacampagne et al. 1996), so depolarization does not appear to modulate the properties of individual events. Rather, depolarization modulates the pattern and frequency of occurrence of Ca2+ sparks over the voltage range of activation of calcium release, accounting for both the increase in release with increasing depolarization, and the time course of release during a maintained depolarization (Klein et al. 1997). Thus, much of the voltage dependence of calcium release can be accounted for by the pattern and frequency of occurrence of individual Ca2+ sparks with similar characteristics at all voltages. The present article describes the properties of a repetitive mode of activation of Ca2+ sparks in skeletal muscle fibres, which is manifest as a brief train of several sparks activated by membrane depolarization or by ligand binding, even when the overall event rate in the fibre is extremely low. These repetitive events appear to arise from the repeated activation of a single SR Ca2+ release unit within a triad, and thus provide the opportunity for studying the gating properties of an individual Ca2+ release unit. This heretofore uncharacterized mode of gating of Ca2+ sparks in skeletal muscle may arise from direct modulation of SR RyR Ca2+ release channels, and may contribute to the slowly inactivating component of Ca2+ release during a prolonged depolarization.