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Expression of ryanodine receptor RyR3 produces Ca²⁺ sparks in dyspedic myotubes

Christopher W. Ward*, Martin F. Schneider*, Daniel Castillo†, Feliciano Protasi†, Yaming Wang†, S. R. Wayne Chen and Paul D. Allen†

1. Discrete, localized elevations of myoplasmic [Ca²⁺], Ca²⁺ 'sparks', were readily detected using the fluorescent Ca²⁺ indicator fluo-3 and laser scanning confocal microscopy in 'dyspedic' 1B5 myotubes, i.e. myotubes which do not express ryanodine receptors (RyRs), transduced with virions containing cDNA for RyR type 3 that were saponin permeabilized to allow dye entry. Ca²⁺ sparks were never observed in non-transduced RyR null myotubes.

2. The spatial locations of sparks observed in permeabilized myotubes roughly corresponded to regions of RyR protein expression in the same myotube as detected after subsequent fixation and antibody staining.

3. Permeabilized RyR3-transduced myotubes exhibited similar punctate peripheral RyR3 protein immunohistochemical patterns as myotubes fixed before permeabilization indicating that permeabilization did not affect the structural organization of the triad.

4. Ca²⁺ sparks, recorded in line scan mode, in permeabilized myotubes expressing RyR3 exhibited mean amplitudes (change in fluorescence/mean fluorescence, F/F: 1·20 ± 0·04) and temporal rise times (10-90 %; 6·31 ± 0·12 ms) similar to those of sparks recorded in permeabilized frog skeletal muscle fibres (0·98 ± 0·01; 6·11 ± 0·07, respectively) using the same confocal system. Spatial extent and temporal duration of the Ca²⁺ sparks were ~40 % larger in the RyR3-expressing myotube cultures than in frog fibres.

5. Ca²⁺ sparks recorded in line scan mode often occurred repetitively at the same spatial location in RyR3-expressing myotubes. Such repetitive events were highly reproducible in amplitude and spatio-temporal properties, as previously observed for repetitive mode sparks in frog skeletal muscle.

6. Ca²⁺ sparks recorded in xy mode were frequently compressed in the y (slower scan) direction compared to the x direction. This asymmetry was reproduced assuming spatially symmetric events having the time course of Ca²⁺ sparks recorded in line scan (xt) mode.

7. These expression studies demonstrate that the presence of RyR3 is sufficient for the production of Ca²⁺ sparks in a skeletal muscle system lacking the expression of any other RyR isoform.

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