[3H]Ryanodine ([3H]Ry) binding analysis

Specific [³H]Ry binding to mouse skeletal muscle RyR1 microsomes in suboptimal (1 µM), optimal (50 µM), and inhibitory (1 mM) Ca²⁺ concentrations ([Ca²⁺]) was measured as established previously (Pessah and Zimanyi 1991; Pessah et al., 1985). Each experiment was performed in triplicate (n = 3) and replicated twice. Skeletal microsomes (100 µg/ml) were incubated with ice-cold binding buffer (pH 7.4) composed of (in mM) 250 KCl, 15 NaCl, and 20 HEPES, with Ca²⁺ buffered to a concentration of 1 µM, 50 µM, or 1 mM for binding of 5 nM [³H]Ry under equilibrium assay conditions (3 h; 37°C). Assays were performed in the absence or presence of increasing concentration of CP or FD (0.01–100 µM) with constant shaking. Bound and Determined (BAD) program was used to calculate the concentration of ethylene glycol-bis(2-aminoethylether)-N, N, N′, N′-tetraacetic acid (EGTA) necessary to achieve the desired free [Ca²⁺] (Brooks and Storey, 1992). Non-specific binding was determined by incubating with 1000-fold (5 µM) excess of unlabeled ryanodine in the absence or presence of specified compound. Bound from free ligand was separated by filtration through Whatman GF/B glass fiber filters (Whatman, Clifton, New Jersey) using a cell harvester (Brandel, Gaithersburg, Maryland) and washed thrice with 5 mL of ice-cold harvesting buffer (pH 7.4) consisting of (in mM) 140 KCl, 0.1 CaCl₂, and 10 HEPES. Filters were completely submerged in 4 mL of scintillation fluid (ScintiVerse BD Cocktail, Fisher Scientific, Waltham, Massachusetts) and [³H]Ry trapped to the filter quantified using a Beckman Coulter LS6500 spectrometer (Beckman Coulter, Indianapolis, Indiana). Each experiment was performed in triplicates (n = 3) and replicated using different membrane preparations with final DMSO concentration ≤1% (v/v).