Host: The Japanese Pharmacological Society
Name : The 95th Annual Meeting of the Japanese Pharmacological Society
Number : 95
Location : Fukuoka
Date : March 07, 2022 - March 09, 2022
In skeletal muscle excitation-contraction coupling, depolarization of transverse tubule membrane causes conformational change in dihydropyridine receptor (DHPR), which in turn opens type 1 ryanodine receptor (RyR1) to release Ca2+ from sarcoplasmic reticulum. This 'depolarization-induced Ca2+ release' (DICR) occurs through a supramolecular complex composed of several proteins including RyR1, Cav1.1, β1a, Stac3, and junctophilin. However, it remains so far unclear about molecular mechanism of DICR, especially conformational change in RyR1. Recently, Perni et al. reported successful reconstitution of DICR by co-expressing these essential components in tsA201 cells (PNAS, 114: 13822-13827, 2017). In this study, we developed a high-throughput platform of reconstituted DICR in HEK293 cells. Baculovirus infection of essential components into HEK293 cells expressing RyR1 greatly increased their transduction efficiency, and fluorescent ER Ca2+ indicator (R-CEPIA1er) quantitatively measured Ca2+ release without contaminant of Ca2+ influx. We demonstrated [K+]o-dependent Ca2+ release by chemical depolarization in the baculovirus-infected cells, indicating a successful reconstitution of DICR. Our high-throughput platform will accelerate elucidation of molecular mechanism of DICR.
