Figure 5.
Calcium transients from ryanodine-pretreated E4032A-expressing 1B5 cells are not affected by ryanodine. 1B5 myotubes infected with viruses containing E4032A or wtRyR1 cDNA (3 × 105 IU/ml) were imaged for calcium as described in Materials and Methods. (A) Pretreatment of E4032A with 500 μM ryanodine for 24 h restored responsiveness to 40 mM caffeine (clear bar). Addition of 40 mM caffeine supplemented with 500 μM ryanodine produced a calcium transient whose activation and deactivation kinetics were similar to transients produced by 40 mM caffeine alone. Responses to subsequent applications of 40 mM caffeine were unaffected. (B) In an RyR1-expressing 1B5 myotube, addition of 40 mM caffeine/500 μM ryanodine resulted in a long-lived rise in calcium that persisted after washout of these RyR agonists. Response to a subsequent application of caffeine was inhibited. (Bar = 0.05 340/380 ratio units vs. 30 (A) or 100 (B) sec.)