Table 1.
Disease-associated mutations used in this study.
Fig 1.
Caffeine-induced Ca2+ transients in cells expressing WT and mutant RyR1s.
A–D. HEK293 cells expressing WT or mutant RyR1 channels were loaded with fluo-4 AM and stimulated by different concentrations (0.1–10 mM) of caffeine. Measurements were carried out at room temperature (RT). A. Representative traces of fluo-4 signals for WT and three mutants (R164L, Y523S and R615C). Caffeine was applied at the time points indicated by the short horizontal bars. Fluo-4 signals were normalized by Fmax (see Materials and Methods). B. The magnitude of the Ca2+ transients were plotted against caffeine concentrations and fitted to the dose-response curve. C and D. The maximum Ca2+ transients (C) and EC50 for caffeine (D) of WT (filled column), MH mutations (open columns) and MH/CCD mutations (hatched columns). Data are means ± SE (n = 78–150). E. HEK293 cells of WT (filled column), MH mutations (open columns) and MH/CCD mutations (hatched columns) were loaded with fura-2 AM and resting [Ca2+]i was determined. Data are means ± SE (n = 207–494). F. The maximum caffeine-induced Ca2+ transients correlate well with resting [Ca2+]i. (R2 = 0.76, dashed line).
Fig 2.
Determination of resting [Ca2+]ER in cells expressing WT or mutant RyR1s.
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. A. 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, Fmax for indicators was determined by the application of 20 μM ionomycin and 20 mM Ca2+ (black bars). B. [Ca2+]ER of WT (filled column), MH mutations (open columns) and MH/CCD mutations (hatched columns). Data are means ± SE (n = 35–99). *p < 0.05 compared with WT. C and D. Peak caffeine-induced Ca2+ transients (C) and resting [Ca2+]i (D) 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 (R2 = 0.79) and resting [Ca2+]i (R2 = 0.82) correlated strongly with [Ca2+]ER (dashed lines).
Fig 3.
Caffeine-induced Ca2+ oscillations by monitoring ER Ca2+.
ER Ca2+ of HEK293 cells expressing WT or mutant RyR1 channels (C36R, G249R and R615C) was monitored with R-CEPIA1er as described in Fig 2. A. Typical traces for ER Ca2+ signals. Cells were incubated for 2 min with normal Krebs solution containing 2 mM Ca2+ and 2 mM caffeine (grey bar). Ca2+ in Krebs solution was then increased to 5 mM (black bars). After depletion of Ca2+ by 30 mM caffeine, Fmax for indicators was determined by the application of 20 μM ionomycin and 20 mM Ca2+. B. Ca2+ oscillation frequencies determined with normal (2 mM Ca2+) and 5 mM Ca2+ Krebs in the presence of 2 mM caffeine. C. Caffeine-induced Ca2+ oscillations by monitoring ER Ca2+. ER [Ca2+]ER levels in normal and 5 mM Ca2+ Krebs solution with 2 mM caffeine. For WT and R615C, upper levels during Ca2+ oscillations (threshold levels for Ca2+ release) were measured.
Fig 4.
Ca2+-dependent [3H]ryanodine binding of WT and mutant RyR1s.
A–D. Ca2+-dependent [3H]ryanodine binding was determined at 25°C in 0.17 M NaCl, 20 mM MOPSO, pH 6.8, 2 mM dithiothreitol, 1 mM AMP and various concentrations of Ca2+ buffered with 10 mM EGTA. Curves without data points in B–D indicate WT. Data are means ± SE (n = 3–5). Note that the mutants show greater [3H]ryanodine binding than WT. E–H. Activity profiles of the mutant channels. The three parameters, Amax, KA and KI, were obtained by fitting analysis (see Materials and Methods) and plotted on the radar charts relative to WT. 1/KA was used as the parameter for activating Ca2+ dissociation constants, in which a larger value represents higher sensitivity. Note that the size of the triangle represents the magnitude of the channel activity.
Fig 5.
Estimation of the activity of mutant channels at resting [Ca2+]i.
A. Ryanodine binding of mutant channels under resting conditions (pCa 7) was estimated by substituting the three parameters (KA and KI and Amax) into eqs (1)–(3) and plotted relative to WT in ascending order. B. [Ca2+]ER for WT (filled circle), MH mutations (open circles) and MH/CCD mutations (crosses) were plotted against their estimated ryanodine binding at pCa 7.
Fig 6.
Ca2+-dependent [3H]ryanodine binding of WT and mutant RyR1s at 37°C.
A–D. Ca2+-dependent [3H]ryanodine binding was determined as in Fig 4 at 37°C instead of RT. Curves without data points in B–D indicate WT. Data are means ± SE (n = 3–5). E–H. Activity profiles of the mutant channels.
Fig 7.
Comparison of the activity profiles between 25 and 37°C.
The three parameters, Amax (A), KA (B) and KI, (C) for WT (filled circles), MH (open circles), MH/CCD (crosses) and C36R (triangles) at 25 and 37°C are plotted. Note that high linear correlations (R2 > 0.8) were found between mutants for all parameters. D. Estimated ryanodine binding at pCa 7.
Fig 8.
Cellular Ca2+ homeostasis of cells expressing WT or mutant RyR1s at 36°C.
A. [Ca2+]ER of WT (filled column), MH mutations (open columns) and MH/CCD mutations (hatched columns). Data are means ± SE (n = 30–67). *p < 0.05 compared with WT. B. [Ca2+]ER for WT (filled circle), MH (open circles) and MH/CCD (crosses) were plotted against their estimated ryanodine binding at pCa 7 at 37°C. C. ER Ca2+ for MH (open circles) and MH/CCD (crosses) relative to WT (filled circle) at RT and 36°C was plotted. The dotted line represents y = x equation. Therefore, mutants under the line indicate that their ER Ca2+ relative to WT is lower at 36°C than at RT. D. Caffeine-induced Ca2+ transients of WT (open circles), C36R (open triangles) and R615C (filled circles) at 36°C. Note that caffeine sensitivity for C36R was not increased, whereas R615C exhibited a marked enhancement in caffeine sensitivity.