Electrically stimulated intracellular Ca2+ ([Ca2+]i) transients in mammalian cardiac muscle are largely due to release of Ca2+ from the sarcoplasmic reticulum (Bers, 1991). One step in the mechanism that links membrane depolarization to release of intracellular Ca2+ stores is the influx of Ca2+ via L-type Ca2+ channels (Fabiato, 1985; Näbauer et al. 1989). This pathway for Ca2+ influx is routinely measured as the macroscopic Ca2+ current (ICa) and many investigations have demonstrated that the membrane potential (Vm) dependence of ICa can be similar to the Vm dependence of the [Ca2+]i transient (Cannell et al. 1987; Beuckelmann & Wier, 1988; Cleeman & Morad, 1991). Even so, more recent reports have shown that the relationship between ICa and [Ca2+]i transient amplitude is Vm dependent in a manner that suggests the efficacy of excitation-contraction (E-C) coupling is a function of single Ca2+ channel current amplitude rather than the total influx of Ca2+ during ICa (López-López et al. 1995; Santana et al. 1996) This apparent divergence between ICa amplitude and control of sarcoplasmic reticulum (SR) Ca2+ release is thought to be evidence in support of the ‘local control’ formalism for the Ca2+-induced Ca2+ release mechanism which postulates that localized changes of [Ca2+] in the dyad cleft, rather than global changes of [Ca2+]i, regulate SR Ca2+ release (Stern, 1992). If this is correct, measurements of ICa, by themselves, do not represent an accurate means to access the processes controlling localized increases in [Ca2+]i that trigger SR Ca2+ release. Thus, in the absence of single Ca2+ channel current measurements, a reasonable means must be found to extrapolate between ICa and events at the single channel level in order to understand how Ca2+ influx triggers SR Ca2+ release.
Localized increases of [Ca2+]i in resting myocytes, termed ‘Ca2+ sparks’ (Cheng et al. 1993) have been observed during stimulated increases of [Ca2+]i (López-López et al. 1994, 1995; Berlin, 1995; Cannell et al. 1995; Lipp & Niggli, 1996) and are thought to represent unitary SR Ca2+ release events, even if several functionally coupled ryanodine receptors actually contribute to Ca2+ release flux during each event (Lipp & Niggli, 1996). At the most basic level, the global increases in [Ca2+]i that occur during a [Ca2+]i transient can be thought of as the summation of these localized, unitary release events.
The ability to observe Ca2+ sparks offers a new avenue to study how opening of sarcolemmal Ca2+ channels and the subsequent Ca2+ influx regulate SR Ca2+ release. In this regard, several questions remain to be answered about the manner in which Ca2+ influx triggers unitary SR Ca2+ release events. For example, recent electron microscopic studies in developing chick heart have observed clusters of intramembrane particles localized in the surface membrane of the dyadic cleft (Protasi et al. 1996). By analogy to studies with skeletal muscle, these particles are presumed to be sarcolemmal Ca2+ channels, i.e. dihydropyridine receptors (Protasi et al. 1998). If correct, these results suggest that the dyad includes clusters of surface membrane Ca2+ channels in close apposition to clusters of ryanodine receptors on junctional SR membranes.
The presence of clusters of L-type Ca2+ channels in the dyadic junction raises the possibility that unitary SR Ca2+ release events are triggered by opening of multiple surface Ca2+ channels. In addressing this possibility, Cannell et al. (1995) and, more recently Santana et al. (1996), showed that an e-fold increase in the amplitude of Ca2+ current and incidence of ‘Ca2+ sparks’ occurred over approximately 7 mV in the range of Vm near the threshold for L-type Ca2+ channel opening. The similar voltage dependence for activation of these two processes suggests that the opening of a single Ca2+ channel on the surface membrane is sufficient to trigger a Ca2+ spark (Santana et al. 1996). Since E-C coupling normally occurs at more depolarized potentials, these authors also examined the relationship between ICa amplitude and the probability of Ca2+ spark occurrence over a wide range of Vm. By assuming that (1) single Ca2+ channel ion flux rates can be approximated using the Goldman-Hodgkin-Katz constant field equation and (2) instantaneous open probability of the SR release channel is a square function of dyadic [Ca2+], Santana et al. (1996) concluded that opening of a single L-type Ca2+ channel was sufficient to trigger a Ca2+ spark over a broad range of Vm values. However, given the difficulty in verifying these crucial assumptions, a more straightforward means to examine the relationship between ICa and Ca2+ spark occurrence is needed. Thus, in this study, the kinetics of ICa and Ca2+ spark occurrence were measured in voltage-clamped rat ventricular myocytes. Using Poisson statistics to describe ICa in terms of L-type Ca2+ channel openings, we were then able to analyse directly how changes in ICa related to the appearance of Ca2+ sparks during voltage-clamp depolarizations.