Ca2+‐induced Ca2+ release: physiological experiments on a new level

In his pioneering studies of Ca2+-induced Ca2+ release (CICR) and excitation-contraction coupling in heart, Fabiato (1985) triggered release of Ca2+ from sarcoplasmic reticulum (SR) with fast (millisecond) changes of Ca2+-buffered solutions. To expose the SR directly to the changes in [Ca2+], he used fragments of cardiac muscle cells, and to observe the Ca2+ released, he used the Ca2+-activated photoprotein aequorin. Today, the principle of controlling and observing intracellular [Ca2+] still applies, but the methodological goal for those studying CICR is to cause [Ca2+] to rise just in the local region of one or a few of the molecules of interest (viz. a single SR Ca2+ release channel or ryanodine receptor, RyR). Ideally this should involve no other confounding processes, such as changes in membrane voltage or the action of Ca2+-activated molecules that might be located elsewhere. For elevating [Ca2+] rapidly, photolysis of chemically ‘caged’ Ca2+ with conventional techniques now provides an improvement over the solution changes used by Fabiato, but ‘uncaging’ still occurs in a rather large volume of the cell (compared with molecular dimensions), even when using focused ultra-violet (UV) light from a laser. In this issue of The Journal of Physiology, Lipp & Niggli have used a relatively new technique, two-photon photolysis of caged Ca2+, to elevate [Ca2+] rapidly and controllably, in extremely small (10−15 l) volumes of mammalian cardiac cells. They used confocal microscopy of fluo-3 fluorescence to observe [Ca2+] simultaneously in the same volume in which uncaging occurred. The key to their achievement is multi-photon molecular excitation, first predicted by the theory of quantum mechanics in the 1930s. Two-photon photolysis (TPP) is a particular case of multi-photon excitation (see Denk et al. 1990) and involves the nearly simultaneous absorption of two red photons (λ, 705 nm) by a caging compound (e.g. DM-nitrophen) with resultant breaking of the molecular cage and release of Ca2+. On the molecular level, the result is the same as if a single UV photon (e.g. λ, 360 nm) had been absorbed. In two-photon photolysis, however, absorption is limited almost completely to the high-intensity region of the focused infra-red (IR) laser beam since the probability of two-photon absorption falls off with the square of the excitation intensity. Lipp & Niggli report that the size of this region, measured as the volume of two-photon excited fluorescence of indo-1, was approximately 0.6 × 10−15 l, certainly not yet confined to the region of single molecules, but a significant improvement over anything achieved previously in cardiac cells. Lipp & Niggli have used this technique to address an important instance of Ca2+ signalling, that of the putative elementary events of SR Ca2+ release in mammalian cardiac muscle, Ca2+‘sparks’ (Cheng et al. 1993). The voltage and time dependence of Ca2+ sparks elicited during voltage clamp (Lopez-Lopez et al. 1995) seems to provide the necessary support for the ‘local control’ theory of E-C coupling, in which the whole-cell Ca2+ transient comprises these elementary events of SR Ca2+ release, recruited (by CICR) independently of each other by the Ca2+ flowing through single L-type channels. Thus, Ca2+ sparks do seem to be elementary CICR events in cardiac E-C coupling, but are they elementary in the sense of being indivisible? If Ca2+ sparks arise from a single RyR, then we have the opportunity to study E-C coupling at its most fundamental level, the release of Ca2+ through a single Ca2+ release channel. In fact, however, smaller release events than sparks have been hypothesized to occur in skeletal muscle (Shirakova & Rios, 1997) and also in cardiac muscle (Lipp & Niggli, 1996), where they were termed Ca2+‘quarks’. Quarks were not observed directly, but were only inferred from the fact that low-level SR Ca2+ release occurred after photolysis of caged Ca2+ in the whole cell. Now, Lipp & Niggli report that, using TPP, they have actually triggered and observed very small Ca2+ transients that have a spatial width much less than that of the typical Ca2+ spark. Second, they observe these events only when the laser power is below the threshold for generating a normal Ca2+ spark. This implies that the physiological signals for spark generation by CICR (i.e. L-type Ca2+ channel current) are normally above this threshold and that sparks therefore represent release of Ca2+ from a larger number of RyRs than does the quark. The unequivocal identification of the novel small release events as ‘quarks’ (i.e. the event defined as the fundamental one) will require substantial further work. Nevertheless, the ability to control such small events in intact cells (with TPP), and to observe them reliably (with confocal microscopy) certainly constitutes a new level in the study of cell signalling and, in particular, the still puzzling phenomenon of Ca2+-induced Ca2+ release in mammalian heart.