Fig. 4.
Normalized response against 4-chloro-m-cresol (4-CmC). (A) Fluorescence ratio 340/380 was monitored in real time and recorded at 0.5-s intervals. A typical example of fluorescence change in a response to 500 µm 4-CmC addition is shown. Data are shown for one region analyzed for wild type (WT) and each mutant. 4-CmC was added at zero in this time scale. Kinetic measurements were not possible for comparison of response time to 4-CmC between WT and mutants using our experimental conditions. (B) The response at 1,000 µm of 4-CmC was set as 100%. Normalized data were plotted as mean ± SD, and sigmoidal fitted curves were drawn using OriginPro 8.5 software (OriginLab Corporation, Northampton, MA) using the DoseResp function with four parameters and EC50 values were calculated using those fitted curves. All malignant hyperthermia-linked mutants (R44C; number of regions analyzed n = 21, EC50 = 80.5 µm, R163C; n = 12, EC50 = 170.4 µm, R401C; n = 20, EC50 = 56.5 µm, R533C; n = 20, EC50 = 121.0 µm, R533H; n = 22, EC50 = 21.2 µm, H4833Y; n = 23, EC50 = 71.3 µm) showed lower EC50 for 4-CmC activation compared with WT (n = 18, EC50 = 453.2 µm). Differences between mutants and WT were statistically significant (* P < 0.001) in two-way factorial ANOVA. (n) = number of regions analyzed.