Reconstitution of depolarization-induced Ca2+ release for validation of skeletal muscle disease mutations and drug discovery.

Abstract

In skeletal muscle, depolarization of the plasma membrane triggers Ca2+ release from the sarcoplasmic reticulum (SR),referred to as depolarization-induced Ca2+ release (DICR). DICR occurs via 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. It has recently become clear that mutations in these components cause various skeletal muscle diseases. However, functional validation is time-consuming because it must be performed in a skeletal muscle environment and no specific treatment has been developed yet. In this study, we established a platform of the reconstituted DICR in HEK293 cells. The essential components were effectively transduced using baculovirus vectors, and Ca2+ release was quantitatively measured with R-CEPIA1er, a fluorescent ER Ca2+indicator. High [K+] depolarization triggered rapid Ca2+ release, indicating successful reconstitution of DICR. 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. 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 validation of mutations and drug discovery for skeletal muscle diseases.