A reconstituted depolarization-induced Ca²⁺ release platform for validation of skeletal muscle disease mutations and drug discovery

In skeletal muscle excitation–contraction (E–C) coupling, depolarization of the plasma membrane triggers Ca²⁺ release from the sarcoplasmic reticulum (SR), referred to as depolarization-induced Ca²⁺ release (DICR). DICR occurs through the type 1 ryanodine receptor (RyR1), which physically interacts with the dihydropyridine receptor Cav1.1 subunit in specific machinery formed with additional essential components including β1a, Stac3 adaptor protein, and junctophilins. Exome sequencing has accelerated the discovery of many novel mutations in genes encoding DICR machinery in various skeletal muscle diseases. However, functional validation is time-consuming because it must be performed in a skeletal muscle environment. In this study, we established a platform of the reconstituted DICR in HEK293 cells. The essential components were effectively transduced into HEK293 cells expressing RyR1 using baculovirus vectors, and Ca²⁺ release was quantitatively measured with R-CEPIA1er, a fluorescent ER Ca²⁺ indicator, without contaminant of extracellular Ca²⁺ influx. In these cells, [K⁺]-dependent Ca²⁺ release was triggered by chemical depolarization with the aid of inward rectifying potassium channel, indicating a successful reconstitution of DICR. Using the platform, we evaluated several Cav1.1 mutations that are implicated in malignant hyperthermia and myopathy. We also tested several RyR1 inhibitors; whereas dantrolene and Cpd1 inhibited DICR, procaine had no effect. Furthermore, twitch potentiators such as perchlorate and thiocyanate shifted the voltage dependence of DICR to more negative potentials without affecting Ca²⁺-induced Ca²⁺ release. These results well reproduced the findings with the muscle fibers and the cultured myotubes. Since the procedure is simple and reproducible, the reconstituted DICR platform will be highly useful for the validation of mutations and drug discovery for skeletal muscle diseases.