Spatially segregated control of Ca super(2+) release in developing skeletal muscle of mice

Confocal laser scanning microscopy was used to monitor Ca super(2+) 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. Discrete Ca super(2+) 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 times 27 (0 times 85, 1 times 97) (median (25 %, 75 %)) units of resting fluorescence, the full width at half-magnitude (FWHM) was 1 times 4 (0 times 9, 2 times 3) mu m, and the full duration at half-magnitude (FDHM) was 25 times 3 (9 times 6, 51 times 7) ms. In RyR3-knockout myotubes (655 events, 83 cells) the amplitude was 1 times 30 (0 times 84, 2 times 08), FWHM was 1 times 63 (1 times 02, 2 times 66) mu m, and FDHM was 43 times 6 (23 times 6, 76 times 9) ms. Depolarization under voltage clamp of both wild-type and RyR3-knockout myotubes produced substantial Ca super(2+) release devoid of discrete Ca super(2+) events. Discrete events were still present but occurred without correlation with the applied pulse, largely at locations where the pulse did not elicit release. 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 Ca super(2+) events in adult mammalian muscle.