Single Sarcoplasmic Reticulum Ca2 Research Articles

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.

Cyclic ADP-ribose does not regulate sarcoplasmic reticulum Ca2+ release in intact cardiac myocytes.

Cyclic ADP-ribose (cADPR), an intracellular second messenger known to mobilize Ca2+ in sea urchin eggs, has been implicated in modulating Ca2+ release in a variety of mammalian tissues. On the basis of studies of isolated cardiac sarcoplasmic reticulum (SR) vesicles and single SR Ca2+ release channels, cADPR has also been proposed to be a modulator of SR Ca2+ release in heart. In the present study, we directly examined the ability of cADPR to trigger SR Ca2+ release and to modulate Ca(2+)-induced Ca2+ release (CICR) in intact rat ventricular myocytes. Voltage-clamped myocytes were dialyzed with up to 100 mumol/L caged cADPR and 0.6 mumol/L calmodulin along with the Ca(2+)-sensitive dye fluo 3. A step increase in the cADPR concentration induced by flash photolysis of caged cADPR neither directly triggered SR Ca2+ release nor modulated CICR in intact myocytes. In contrast, under similar conditions, extracellular application of caffeine (1 to 2.5 mmol/L) onto myocytes produced both effects. Under equivalent conditions, flash photolysis of caged cADPR-loaded sea urchin eggs resulted in large Ca2+ transients. Further, the sustained presence of high cytosolic concentrations of either cADPR or its antagonist, 8-amino-cADPR, was ineffective in altering normal CICR in myocytes. These findings indicate that cADPR does not regulate SR Ca2+ release in intact cardiac myocytes.

Submicroscopic calcium signals as fundamental events of excitation--contraction coupling in guinea-pig cardiac myocytes.

1. Subcellularly localized Ca2+ signals have been proposed to represent elementary events of cardiac Ca2+ signalling (Ca2+ sparks), whereby an individual sarcolemmal L-type Ca2+ channel locally controls opening of a single (or a few) Ca2+ release channels in the sarcoplasmic reticulum (SR). 2. To investigate directly the elementary nature of this Ca(2+)-induced Ca2+ release mechanism we used flash photolysis of caged Ca2+ while simultaneously measuring the intracellular Ca2+ concentration ([Ca2+]i) with a laser-scanning confocal microscope. 3. Power spectral analysis of the confocal images performed in the spatial domain revealed that only Ca2+ signalling events involving the L-type Ca2+ channel pathway gave rise to Ca2+ sparks. In contrast, SR Ca2+ release triggered by photolytic [Ca2+]i jumps resulted in Ca2+ transients that were always spatially homogeneous. 4. From these findings we conclude that the fundamental event of Ca2+ signalling in cardiac muscle may be smaller in size or amplitude than a Ca2+ spark. 5. We term this event a 'Ca2+ quark' possibly resulting from gating of a single SR Ca2+ release channel. It is proposed that concerted activation of several 'Ca2+ quarks' may be required for a Ca2+ spark. The 'Ca2+ quark' could also be the fundamental event in other cell types implementing a hierarchical Ca2+ signalling concept.

Activation of single cardiac and skeletal ryanodine receptor channels by flash photolysis of caged Ca2+

Single ryanodine-sensitive sarcoplasmic reticulum (SR) Ca2+ release channels isolated from rabbit skeletal and canine cardiac muscle were reconstituted in planar lipid bilayers. Single channel activity was measured in simple solutions (no ATP or Mg2+) with 250 mM symmetrical Cs+ as charge carrier. A laser flash was used to photolyze caged-Ca2+ (DM-nitrophen) in a small volume directly in front of the bilayer. The free [Ca2+] in this small volume and in the bulk solution was monitored with Ca2+ electrodes. This setup allowed fast, calibrated free [Ca2+] stimuli to be applied repetitively to single SR Ca2+ release channels. A standard photolytically induced free [Ca2+] step (pCa 7-->6) was applied to both the cardiac and skeletal release channels. The rate of channel activation was determined by fitting a single exponential to ensemble currents generated from at least 50 single channel sweeps. The time constants of activation were 1.43 +/- 0.65 ms (mean +/- SD; n = 5) and 1.28 +/- 0.61 ms (n = 5) for cardiac and skeletal channels, respectively. This study presents a method for defining the fast Ca2+ regulation kinetics of single SR Ca2+ release channels and shows that the activation rate of skeletal SR Ca2+ release channels is consistent with a role for CICR in skeletal muscle excitation-contraction coupling.

Abnormal human sarcoplasmic reticulum Ca2+ release channels in malignant hyperthermic skeletal muscle

Single sarcoplasmic reticulum (SR) Ca2+ release channels were reconstituted from normal and malignant hyperthermic (MH) human skeletal muscle biopsies (2-5 g samples). Conduction, gating properties, and myoplasmic Ca2+ dependence of human SR Ca2+ release channels were similar to those in other species (rabbit, pig). The MH diagnostic procedure distinguishes three phenotypes (normal, MH-equivocal, and MH-susceptible) on the basis of muscle contracture sensitivity to caffeine and/or halothane. Single channel studies reveal that human MH muscles (both MH phenotypes) contain SR Ca2+ release channels with abnormally greater caffeine sensitivity. Muscles from MH-equivocal and MH-susceptible patients appear to contain channels with the same abnormality. Further, our data (n = 115, 21 channels, 11 patients) reveals that human MH muscles (both phenotypes) may contain two populations of SR Ca2+ release channels, possibly corresponding to normal and abnormal isoforms. Thus, whole cell phenotypic variation (MH-equivocal vs. MH-susceptible) arises in muscles containing channels with similar caffeine sensitivity suggesting that human MH does not arise from a single defect. These results have important ramifications concerning (a) correlation of functional and genetic MH studies, (b) identification of other, yet to be determined, factors which may influence MH expression, and (c) characterization of normal SR Ca2+ release channel function by exploring genetic channel defects.