The effects of co-expressing TRIC-A or TRIC-B with RyR2 on the relationship between Po and cytosolic [Ca ²⁺ ]. (A) Relationship between [Ca²⁺] and Po for RyR2 derived from HEK293 cells expressing RyR2-only (black), RyR2+TRIC-A (blue), and RyR2+TRIC-B (red) at −30 mV. Lines display non-linear bell-shaped fitted curves. EC₅₀ values are 3.6 µM for RyR2-only, 5.8 µM for RyR2+TRIC-A, and 11.7 µM for RyR2+TRIC-B, and IC₅₀ values are 87 µM for RyR2-only, 1.08 mM for RyR2+TRIC-A, and 0.84 mM for RyR2+TRIC-B. (B) The effect of holding potential (−30 mV on the left; +30 mV on the right) on Po for RyR2 derived from HEK293 cells expressing RyR2-only (black), RyR2+TRIC-A (blue), and RyR2+TRIC-B (red). For the statistical analysis, we used a linear mixed model with two independent variables: (1) “cytosolic [Ca²⁺]” and (2) “type of HEK293 cells.” To preserve correlations within recordings, a Random Effects factor was included in the model that determined the identity of the single-channel recording for each measurement (as discussed in Materials and methods). For the −30 mV on the left: interaction effect for cytosolic [Ca²⁺] by type of HEK293 cells P = 0.000008; main effect of cytosolic [Ca²⁺], P = 4.794 × 10⁻¹¹; main effect of type of HEK293 cells, P = 0.000025 (n_RyR2 = 6, n_RyR2+TRIC-A = 3–8, n_RyR2+TRIC-B = 13). Adjusted P values (Sidak correction) for simple effects are listed in Tables S1 and S2. For the +30 mV on the right: interaction effect for cytosolic [Ca²⁺] by type of HEK293 cells, P = 0.0219; main effect of cytosolic [Ca²⁺], P = 0.0024; main effect of type of HEK293 cells; P = 0.0423 (n_RyR2 = 6, n_RyR2+TRIC-A = 4–5, n_RyR2+TRIC-B = 6). Adjusted P values (Sidak correction) for simple effects are listed in Tables S3 and S4. Simple effects tests for type of HEK293 cells comparing (1) RyR2-only, (2) RyR2+TRIC-A, and (3) RyR2+TRIC-B are indicated by asterisks (*: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001). Values are of the mean ± SEM.