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. 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. 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. 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. 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.

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. (4, 5), and smooth muscle (6). Sparks are fundamental in health and disease (7), but their mechanism, especially whether one or many channels are involved in the spark generator or ''release unit,'' remains unclear (8-10). Answering this question, which pervades the field since its inception (2), will help understand how the channels are coaxed by their agonists (Ca, membrane voltage; reviewed in refs. 11 and 12) and restrained by their antagonists (Ca, Mg) to shape these events.Here we improve a technique for detection of massive numbers of sparks (13) Materials and MethodsExperiments were carried out at 17°C in cut skeletal muscle fibers from Rana pipiens semitendinosus muscle, stretched at 3-3.5 m͞sarcomere, either voltage-clamped in a two-Vaseline gap chamber or permeabilized and immersed in internal solution, on an inverted microscope. Adult frogs anaesthetized in 15% ethanol were killed by pithing. The external solution contained 10 mM Ca(CH 3 SO 3 ) 2 , 130 mM tetraethylammonium-CH 3 SO 3 , 5 mM Tris maleate, 1 mM 3,4 diaminopyridine, and 1 M tetrodotoxin. The internal solution contained 110 mM Cs-glutamate, 1 mM EGTA, 5 mM glucose, 5 mM Mg-ATP, 5 mM phosphocreatine, 10 mM Hepes, 0.2 mM fluo-3, with 100 nM free [Ca 2ϩ ] and 1.8 mM [Mg 2ϩ ]. For permeabilized cells the internal solution contained 0.05 mM fluo-3 and 0.34 mM [Mg 2ϩ ] and included 4% 10-kDa dextran. Solutions were adjusted to pH 7 and 270 mosmol͞kg. The scanning microscope (MRC 1000, Bio-Rad) was in fluo-3 configuration (14) using a 40ϫ, 1.2 numerical aperture water immersion objective (Zeiss). Images shown are of fluorescence determined at 2-ms intervals (4.3 ms in toxin experiments) along a parallel to the fiber axis. Fluorescence F(x,t) is presented normalized to its average F 0 (x) before the voltage pulse. Sparks are located on a spatially filtered version of...