Effect of MH mutations in Cav1.1 on DICR activity. (A) Schematic drawing of the location of MH mutations (R174W, R1086H, and T1354S) in Cav1.1. Note that WT and mutant Cav1.1s also carry N617D Ca²⁺ non-conducting mutation. (B) Resting ER [Ca²⁺] in cells expressing WT and mutant Cav1.1s. Data are means ± SD (n = 12; N = 3) and were analyzed by one-way ANOVA with Dunnett’s multiple comparisons test. (C) Typical results of R-CEPIA1er fluorescence in cells infected with 5 BV carrying WT (left), R174W (center), and R1086H (right) Cav1.1. High [K⁺] solution ranging from 5 to 61 mM (symbols shown in left) was applied at 30 s after starting (blue arrowheads). (D) [K⁺] dependence of R-CEPIA1er fluorescence corrected by ER [Ca²⁺] in WT (black), R174W (red), R1086H (blue), and T1354S (green) Cav1.1 cells. Data are means ± SD (n = 9, N = 3). (E and F) Fluorescence change by 61 mM [K⁺] corrected by ER Ca²⁺ in WT (E) and EC₅₀ values for [K⁺] (F). Data are means ± SD (n = 9, N = 3) and were analyzed by one-way ANOVA with Dunnett’s multiple comparisons test. (G) Caffeine dependence of R-CEPIA1er fluorescence corrected by ER Ca²⁺ in WT (black), R174W (red), R1086H (blue), and T1354S (green) Cav1.1 cells. Data are means ± SD (n = 9, N = 3). (H and I) Fluorescence change by 20 mM caffeine corrected by ER Ca²⁺ in WT (H) and EC₅₀ values for caffeine (I). Data are means ± SD (n = 9, N = 3) and were analyzed by one-way ANOVA with Dunnett’s multiple comparisons test. n is the number of wells and N is the number of independent experiments.