Abstract
Genetic mutations in ryanodine receptors (RyRs), Ca2+-release channels in the sarcoplasmic reticulum essential for muscle contractions, cause various skeletal muscle and cardiac diseases. Because the main underlying mechanism of the pathogenesis is overactive Ca2+ release by gain-of-function of the RyR channel, inhibition of RyRs is expected to be a promising treatment for these diseases. Here, to identify inhibitors specific to skeletal muscle type 1 RyR (RyR1), we developed a novel high-throughput screening (HTS) platform using time-lapse fluorescence measurement of Ca2+ concentrations in the endoplasmic reticulum (ER) ([Ca2+]ER). Because expression of RyR1 carrying disease-associated mutation reduces [Ca2+]ER in HEK293 cells through Ca2+ leakage from RyR1 channels, specific drugs that inhibit RyR1 will increase [Ca2+]ER by preventing such Ca2+ leakage. RyR1 carrying R2163C mutation and R-CEPIA1er, a genetically-encoded ER Ca2+ indicator, were stably expressed in HEK293 cells and time-lapse fluorescence was measured using a FlexStation II fluorometer. False positives were effectively excluded by using cells expressing wild-type (WT) RyR1. By screening 1,535 compounds in a library of well-characterized drugs, we successfully identified four compounds that significantly increased [Ca2+]ER. They include dantrolene, a known RyR1 inhibitor, and three structurally-different compounds; oxolinic acid, 9-aminoacridine and alexidine. All the hit compounds, except for oxolinic acid, inhibited [3H]ryanodine binding of WT and mutant RyR1. Interestingly, they showed different dose-dependencies and isoform specificities. The highly quantitative nature and good correlation with the channel activity validated this HTS platform by [Ca2+]ER measurement to explore drugs for RyR-related diseases.
- Calcium
- Calcium imaging
- Drug discovery
- High throughput screening
- Ion channels
- Ryanodine receptors
- Skeletal muscle
- The American Society for Pharmacology and Experimental Therapeutics