Fig. 3.
Determining the affinity of HBDorg for RyR1. Displacement [3H]ryanodine binding assays were used to determine the affinity of HBDorg for RyR1. For this, SR vesicles (0.1 mg/ml) were incubated in binding buffer consisting of 500 mM KCl, 20 mM Tris·HCl, 0.3 mM CaCl2, 0.1 mM EGTA, pH 7.4, and 6.7 nM [3H]ryanodine with 12 concentrations of HBDorg (0.007–15.75 μg/ml) for 2 h at 37°C. At the end of the incubation, samples were rapidly filtered through GF/C filters using a cell harvester (Brandel) and washed three times with 3 ml of ice-cold binding buffer, and the amount of [3H]ryanodine bound to the filters was determined by liquid scintillation counting. Data for each compound represent means ± SE from four experiments. For comparison, ryanodine was also used in this assay.