Figure 5.

[³H]Ryanodine binding assays to determine the dose-dependent effect of hits on the RyR1 function. (A) In control experiments, HSR vesicles were incubated with 0 or 800 nM CaM in the presence of [³H]ryanodine, at 30 nM and 30 μM free Ca²⁺. Results are shown normalized relative to the values for (−)CaM controls. (B–F) Dose dependent (0–100 μM) effect of screen Hit compounds on [³H]ryanodine binding to skeletal SR membranes when incubated in the absence of CaM (open symbol) or in the presence of 800 nM CaM (solid symbol), at 30 nM (blue) and 30 μM (red) free Ca²⁺. Results are shown normalized relative to the values for no-drug control (dashed line), mean ±SE, n = 6–10. (G) Effect of 10 μM non-reproducible hit compounds from screen on [³H]ryanodine binding to skeletal SR membranes when incubated in absence or presence of 800 nM CaM at 30 nM Ca²⁺. *Significantly different from control (DMSO only) using unpaired Student’s t-test, p> 0.05.