[3H]Ryanodine binding assays.

Binding assays were carried out following a modified version of a protocol previously described (53). Binding mixtures were prepared containing 50–100 μg of cell lysate, 0.2 M KCl, 20 mM Na-HEPES (pH 7.4), 6.5 nM [³H]ryanodine (NET950, PerkinElmer), and enough CaCl₂ to set free [Ca²⁺] between 100 nM–100 μM. EGTA (1 mM) was used to buffer Ca²⁺. The Ca²⁺/EGTA ratio for these solutions was determined using MaxChelator (WEBMAXCLITE v1.15, https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/webmaxc/webmaxclite115.htm). The binding reactions were incubated for 2 hours at 37°C and were then filtered through Whatman GF/B filters presoaked in 5% polyethyleneimine to maximize protein retention and washed 3 times with 5 ml of distilled water in a Brandel M24-R Harvester. Nonspecific binding was determined in the presence of 20 μM unlabeled ryanodine (2153770, MP Biomedicals). [³H]ryanodine binding was measured by liquid scintillation using Bio-Safe II counting cocktail (RPI Research). Corrections for RyR2 expression were determined by dividing the total [³H]ryanodine binding by the intensity from Western blots, expressed as a percentage of the WT. Hill’s equation was used to determine the maximum [³H]ryanodine binding and the EC₅₀ in Origin 2018b (Origin Lab).