Assays for Modulators of Ryanodine Receptor (RyR)/Ca2+ Release Channel Activity for Drug Discovery for Skeletal Muscle and Heart Diseases.

The ryanodine receptor (RyR) is a Ca²⁺ release channel that is present in the sarcoplasmic reticulum and endoplasmic reticulum (ER) and that plays a central role in excitation-contraction coupling in skeletal and cardiac muscle. Hyperactivation of RyR by genetic mutations or posttranslational modification can cause various skeletal muscle and arrhythmogenic heart diseases. Inhibitors of RyR are therefore expected to be potential drugs for treatment of such diseases. This article describes assays to evaluate RyR channel activity, including an ER Ca²⁺ measurement assay that is compatible with high-throughput screening and a [³ H]-ryanodine binding assay that provides a quantitative measure of RyR channel activity as a second screen for compound hits. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: [Ca²⁺ ]_ER assay for ryanodine receptor (RyR) channel activity Support Protocol 1: Determination of dose dependence and isoform selectivity of RyR inhibitors using [Ca²⁺ ]_ER assay Basic Protocol 2: [³ H]-Ryanodine binding assay for RyR channel activity Support Protocol 2: Isolation of microsomes from RyR-expressing HEK293 cells.