Fig. 4.
Determining the Ca2+ dependency of HBDorg effect on RyR1. For this, SR vesicles (0.1 mg/ml) were incubated in binding buffer consisting of 500 mM KCl, 20 mM Tris·HCl, 0.1 mM EGTA, pH 7.4, 6.7 nM [3H]ryanodine, with 2.0 μg/ml HBDorg and increasing amounts of Ca2+ (2–3,900 μM) 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. *Significantly different from [3H]ryanodine bound in absence of HBDorg (P < 0.05).