CaM Regulation of Ryanodine Receptor Binding

The effect of CaM (calmodulin) on [³H]ryanodine binding was assessed as described.^{37 (link),38 (link)} SR-enriched vesicles were obtained from IZ and NIZ, using Tris-buffered solution (0.9% NaCl, 10 mmol/L Tris-HCl, pH 6.8) plus protease inhibitors (PIs) and collected by 3 steps of centrifugation. The 40 000-g pellets were resuspended in Tris-buffered solution with PI, plus 10% sucrose. After protein quantification, the [3H]ryanodine-bind-ing assay was performed. Briefly, 100 μl of [3H]ryanodine-binding buffer containing 200 mmol/L KCl, 25 mmol/L Tris, 50 mmol/L Hepes (pH 7.4), 1 mmol/L EGTA, 5 nmol/L [3H]ryano-dine (68.4 Ci-mmol-1; Dupont NEN), and CaCl₂ was added to set free [Ca²⁺] to pCa 5. Ca²⁺/EGTA ratio was calculated with Max-Chelator (www.stanford.edu/~cpatton/maxc.html), and 50 μg of SR-enriched solution was incubated for 2 hours at 37°C, filtered on GF/B glass filters (Whatman) presoaked with water, and washed twice with 5 mL of distilled water with an M24-R cell harvester (Brandel). Nonspecific binding was determined in the presence of 20 μM unlabeled ryanodine and subtracted from each sample.