Determination of resting [Ca²⁺]_ER in cells expressing WT or mutant RyR1s.

<p>HEK293 cells expressing WT or mutant RyR1 channels were transfected with G-GECO1.1 and R-CEPIA1er to determine [Ca²⁺]_i and [Ca²⁺]_ER, respectively. Measurements were carried out at RT. <b>A.</b> Typical traces for [Ca²⁺]_i (upper) and [Ca²⁺]_ER (lower) signals. Caffeine was initially applied to deplete Ca²⁺ in the ER (white bars) and then Ca²⁺ in the ER was determined at the plateau after the removal of caffeine. Finally, <i>F</i>_max for indicators was determined by the application of 20 μM ionomycin and 20 mM Ca²⁺ (black bars). <b>B.</b> [Ca²⁺]_ER of WT (filled column), MH mutations (open columns) and MH/CCD mutations (hatched columns). Data are means ± SE (<i>n</i> = 35–99). *<i>p</i> < 0.05 compared with WT. <b>C</b> and <b>D.</b> Peak caffeine-induced Ca²⁺ transients <b>(C)</b> and resting [Ca²⁺]_i<b>(D)</b> of WT (filled circle), MH mutations (open circles) and MH/CCD mutations (crosses) were plotted against [Ca²⁺]_ER. Note that the peak caffeine-induced Ca²⁺ transients (<i>R</i>² = 0.79) and resting [Ca²⁺]_i (<i>R</i>² = 0.82) correlated strongly with [Ca²⁺]_ER (dashed lines).