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

1. Discrete, localized elevations of myoplasmic [Ca2+], Ca2+ 'sparks', were readily detected using the fluorescent Ca2+ indicator fluo-3 and laser sca nning confocal microscopy in 'dyspedic' 1B5 myotubes, i.e. myotubes which d o not express ryanodine receptors (RyRs), transduced with virions containin g cDNA for RyR type 3 that were saponin permeabilized to allow dye entry Ca 2+ sparks were never observed in non-transduced RyR null myotubes. 2. The spatial locations of sparks observed in permeabilized myotubes rough ly 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 periph eral RyR3 protein immunohistochemical patterns as myotubes fixed before per meabilization indicating that permeabilization did not affect the structura l organization of the triad. 4. Ca2+ sparks, recorded in line scan mode, in permeabilized myotubes expre ssing RyR3 exhibited mean amplitudes (change in fluorescence/mean fluoresce nce, Delta 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 m uscle fibres (0.98 +/- 0.01; 6.11 +/- 0.07, respectively) using the same co nfocal system. Spatial extent and temporal duration of the Ca2+ sparks were similar to 40% larger in the RyR3-expressing myotube cultures than in frog fibres. 5. Ca2+ sparks recorded in line scan mode often occurred repetitively at th e same spatial location in RyR3-expressing myotubes. Such repetitive events were highly re-producible 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 (slo wer scan) direction compared tu the x direction. This asymmetry was reprodu ced assuming spatially symmetric events having the time course of Ca2+ spar ks recorded in line scan (xt) mode. 7. These expression studies demonstrate that the presence of RyR3 is suffic ient for the product;ion of Ca2+ sparks in a skeletal muscle system lacking the expression of any other RyR isoform.