Spark- and ember-like elementary Ca2+ release events in skinned fibres of adult mammalian skeletal muscle.

1. Using laser scanning confocal microscopy, we show for the first time elementary Ca2+ release events (ECRE) from the sarcoplasmic reticulum in chemically and mechanically skinned fibres from adult mammalian muscle and compare them with ECRE from amphibian skinned fibres. 2. Hundreds of spontaneously occurring events could be measured from individual single skinned mammalian fibres. In addition to spark-like events, we found ember-like events, i.e. long-lasting events of steady amplitude. These two different fundamental release types in mammalian muscle could occur in combination at the same location. 3. The two peaks of the frequency of occurrence for ECRE of mammalian skeletal muscle coincided with the expected locations of the transverse tubular system within the sarcomere, suggesting that ECRE mainly originate at triadic junctions. 4. ECRE in adult mammalian muscle could also be identified at the onset of the global Ca2+ release evoked by membrane depolarisation in mechanically skinned fibres. In addition, the frequency of ECRE was significantly increased by application of 0.5 mM caffeine and reduced by application of 2 mM tetracaine. 5. We conclude that the excitation-contraction coupling process in adult mammalian muscle involves the activation of both spark- and ember-like elementary Ca2+ release events.

A preferred amplitude of calcium sparks in skeletal muscle.

In skeletal and cardiac muscle, calcium release from the sarcoplasmic reticulum, leading to contraction, often results in calcium sparks. Because sparks are recorded by confocal microscopy in line-scanning mode, their measured amplitude depends on their true amplitude and the position of the spark relative to the scanned line. We present a method to derive from measured amplitude histograms the actual distribution of spark amplitudes. The method worked well when tested on simulated distributions of experimental sparks. Applied to massive numbers of sparks imaged in frog skeletal muscle under voltage clamp in reference conditions, the method yielded either a decaying amplitude distribution (6 cells) or one with a central mode (5 cells). Caffeine at 0.5 or 1 mM reversibly enhanced this mode (5 cells) or induced its appearance (4 cells). The occurrence of a mode in the amplitude distribution was highly correlated with the presence of a mode in the distribution of spark rise times or in the joint distribution of rise times and spatial widths. If sparks were produced by individual Markovian release channels evolving reversibly, they should not have a preferred rise time or amplitude. Channel groups, instead, could cooperate allosterically or through their calcium sensitivity, and give rise to a stereotyped amplitude in their collective spark.

Involvement of multiple intracellular release channels in calcium sparks of skeletal muscle.

In many types of muscle, intracellular Ca(2+) release for contraction consists of brief Ca(2+) sparks. Whether these result from the opening of one or many channels in the sarcoplasmic reticulum is not known. Examining massive numbers of sparks from frog skeletal muscle and evaluating their Ca(2+) release current, we provide evidence that they are generated by multiple channels. A mode is demonstrated in the distribution of spark rise times in the presence of the channel activator caffeine. This finding contradicts expectations for single channels evolving reversibly, but not for channels in a group, which collectively could give rise to a stereotyped spark. The release channel agonists imperatoxin A, ryanodine, and bastadin 10 elicit fluorescence events that start with a spark, then decay to steady levels roughly proportional to the unitary conductances of 35%, 50%, and 100% that the agonists, respectively, promote in bilayer experiments. This correspondence indicates that the steady phase is produced by one open channel. Calculated Ca(2+) release current decays 10- to 20-fold from spark to steady phase, which requires that six or more channels be open during the spark.

The spark and its ember: separately gated local components of Ca(2+) release in skeletal muscle.

Amplitude, spatial width, and rise time of Ca(2+) sparks were compared in frog fast-twitch muscle, in three conditions that alter activation of release channels by [Ca(2+)]. A total of approximately 17,000 sparks from 30 cells were evaluated. In cells under voltage clamp, caffeine (0.5 or 1 mM) increased average spark width by 28%, rise time by 18%, and amplitude by 7%. Increases in width were significant even among events of the same rise time. Spontaneous events recorded in permeabilized fibers with low internal [Mg(2+)] (0.4 mM), had width and rise times greater than in reference, and not significantly different than those in caffeine. The spark average in reference rides on a continuous fluorescence "ridge" and is continued by an "ember," a prolongation of width approximately 1 microm and amplitude <0.2, vanishing in approximately 100 ms. Ridge and ember were absent in caffeine and in permeabilized cells. Exposure of voltage-clamped cells to high internal [Mg(2+)] (7 mM) had effects opposite to caffeine, reducing spark width by 26% and amplitude by 27%. In high [Mg(2+)], the ember was visible in individual sparks as a prolongation of variable duration and amplitude up to 1.2. Based on simulations and calculation of Ca(2+) release flux from averaged sparks, the increase in spark width caused by caffeine was interpreted as evidence of an increase in radius of the release source-presumably by recruitment of additional channels. Conversely, spark narrowing suggests loss of contributing channels in high Mg(2+). Therefore, these changes in spark width at constant rise times are evidence of a multichannel origin of sparks. Because ridge and ember were reduced by promoters of Ca(2+)-dependent activation (caffeine, low [Mg(2+)]) and became more visible in the presence of its inhibitors, they are probably manifestations of Ca(2+) release directly operated by voltage sensors.

Spatially segregated control of Ca2+ release in developing skeletal muscle of mice.

1. Confocal laser scanning microscopy was used to monitor Ca2+ signals in primary-cultured myotubes, prepared from forelimbs of wild-type or ryanodine receptor type 3 (RyR3) knockout mice. Myotubes loaded with the acetoxymethyl ester (AM) form of fluo-3 were imaged at rest or under whole-cell patch clamp. 2. Discrete Ca2+ release events were detected in intact wild-type and RyR3-knockout myotubes. They showed almost no difference in amplitude and width, but were substantially different in duration. In wild-type myotubes (660 events, 57 cells) the amplitude was 1.27 (0.85, 1.97) (median (25 %, 75 %)) units of resting fluorescence, the full width at half-magnitude (FWHM) was 1.4 (0.9, 2.3) microm, and the full duration at half-magnitude (FDHM) was 25.3 (9.6, 51.7) ms. In RyR3-knockout myotubes (655 events, 83 cells) the amplitude was 1.30 (0.84, 2.08), FWHM was 1.63 (1.02, 2.66) microm, and FDHM was 43.6 (23.6, 76.9) ms. 3. Depolarization under voltage clamp of both wild-type and RyR3-knockout myotubes produced substantial Ca2+ release devoid of discrete Ca2+ events. Discrete events were still present but occurred without correlation with the applied pulse, largely at locations where the pulse did not elicit release. 4. The local correspondence between voltage control and absence of discrete events implies that the functional interaction with voltage sensors suppresses the mechanism that activates discrete events. Because it applies whether RyR3 is present or not, it is this exclusion by voltage of other control mechanisms, rather than isoform composition, that primarily determines the absence of discrete Ca2+ events in adult mammalian muscle.