Ca²⁺ homeostasis in cells expressing WT or mutant RYR1s in the N-terminal region. (A) Caffeine-induced changes in [Ca²⁺]_cyt determined by fura-2 in WT and mutants (Q156K, R402C, Y523C, and Y523S). Caffeine was applied at the time points indicated by the black horizontal bar. (B and C) Comparison of the peaks of [Ca²⁺]_cyt transiently induced by 0.3 mM (B; n = 31–322) and 10 mM (C; n = 68–318) caffeine in the WT (black) or the mutants (gray). (D) Comparison of resting [Ca²⁺]_cyt in the WT (black) and mutants (gray; n = 148–651). (E) Half-maximal effective concentration (EC₅₀) for caffeine in the WT (black) and the mutants (gray). Y523S was excluded because caffeine’s maximum and minimum response magnitudes are too small for accurate calculations. (F) The relationship between the maximum caffeine-induced Ca²⁺ transient and resting [Ca²⁺]_cyt in cells expressing WT (black) or mutant (white) RYR1 (R² = 0.68). Values are shown as mean ± SEM (n = 63–651). *P < 0.0001 by t test compared with the WT.