3h ryanodine

[³H]Ryanodine binding to rabbit skeletal SR was carried out as described previously (Schwartz et al., 2009 (link); Xiao et al., 2016 (link)). Briefly, Intrepicalcin was diluted directly into the incubation medium (10 μM CaCl₂, 0.2 M KCl, 10 mM Na-HEPES, pH 7.2) to obtain a final concentration of 1 pM to 20 μM. To evaluate the effect of Intrepicalcin on Ca²⁺ sensitivity of [³H]ryanodine binding activity, a fixed concentration of Intrepicalcin at 100 nM was added to the standard incubation medium containing 0.2 M KCl, 1 mM Na₂EGTA, 10 mM Na-PIPES (pH 7.2) and CaCl₂ to set free [Ca²⁺] in the range of 10 nM to 10 mM. Ca²⁺/EGTA ratio was determinated with the computer program MaxChelator (http://www.stanford.edu/~cpatton/maxc.html). [³H]Ryanodine (95 Ci/mmol, PerkinElmer, USA) was directly diluted in the incubation medium to a final concentration of 5 nM. Protein concentration of heavy SR was 0.3 mg/mL and was calculated by Bradford method. The incubation lasted for a period of 120 min at 36 °C. Samples (100 μL) were always run by duplicate, filtered on Whatman GF/C glass filters (Whatman, Clifton, NJ, USA) and washed twice with 5 mL of distilled water using a Brandel M24-R cell harvester (Gaithersburg, MD, USA). Non-specific binding was determined in the presence of 20 μM unlabelled ryanodine, and has been subtracted from each sample.

[³H]Ryanodine was obtained from Perkin Elmer Life Sciences, protease inhibitors from Sigma-Aldrich, and phospholipids from Avanti Polar Lipids.

[³H]Ryanodine was obtained from PerkinElmer (Waltham, MA), unlabeled ryanodine from Calbiochem (La Jolla, CA), protease inhibitors from Roche (Indianapolis, IN) and Sigma-Aldrich (St Louis, MO), and human embryonic kidney (HEK) 293 cells from ATCC. Full-length wild type RyR1 cDNA was provided by Dr. Gerhard Meissner (University of North Carolina, Chapel Hill). Full length wild type RyR2 and R1 chimera cDNAs were provided by Dr. Junichi Nakai (Saitama University, Japan).

To determine the maximum number of active RyRs, [³H]-ryanodine binding assays were performed using LV homogenates as previously reported [19 (link),22 (link)], with some modifications. Briefly, 50 μg of protein per tube were incubated with 7 nM [³H]-ryanodine (PerkinElmer, Waltham MA, USA) for 90 min at 37°C in an incubation medium containing (in mM): 200 KCl, 20 HEPES (pH 7.2 with KOH), and CaCl₂ necessary to set free Ca²⁺ at 10 μM, as calculated by MaxChelator (http://maxchelator.stanford.edu/index.html). The samples were filtered onto glass fiber filters (Whatman GF-B, GE Healthcare, Chicago IL, USA) and washed three times with distilled water in an automatic collector (Brandel M24-R). The filters were placed in scintillation vials with 5 mL of liquid scintillation mixture, and the retained radioactivity was measured in a Beckman LS-6500 counter. The specific [³H]-ryanodine binding was defined as the difference between the binding in the absence (total binding) and the presence (nonspecific binding) of 20 μM unlabeled ryanodine (MP Biomedicals LLC, Santa Ana CA, USA).