Figure 5.

Dissociation of [³H]ryanodine from the high-affinity binding site of cardiac muscle RyR2. Microsomes were incubated with 10 nM [³H]ryanodine in a binding buffer containing 100 μM free Ca²⁺ for 90 min at 37°C to reach equilibrium. Incubation was then continued after the addition of different agents: □, no addition (control); ●, 1 mM EGTA; ■, 2 mM EGTA; ⧫, 2 mM EGTA plus 100 μM ruthenium red; ▴, 2 mM EGTA plus 100 μM ryanodine; ▾, 100 μM ruthenium red. At different time points, duplicate 0.2-ml aliquots were filtered. The arrow indicates the addition of 1 mM CaCl₂ at a time point of 120 min for the 1 mM EGTA curve. The data are normalized to the values obtained after the first 90-min incubation, which is shown as time 0.