Figure 1. The effects of coexpressing TRIC-A or TRIC-B with RyR2 on the function of RyR2 channels incorporated into bilayers at 2 μM free Ca ²⁺ . (A) Representative current fluctuations of RyR2 channels from the HEK293 cells expressing RyR2-only (left), RyR2+TRIC-A (middle), and RyR2+TRIC-B (right) at the holding potentials of ±30 mV in symmetrical 210 mM KPIPES, 2 μM free Ca²⁺. The Po (measured over 2 min) is shown above each trace. The zero current level is indicated by C, a single full open channel level by O1, and double full open channel level by O2. The red asterisks highlight subconductance events. (B) Single-channel K⁺ conductance of RyR2 channels from HEK293 cells expressing RyR2-only (black circles), RyR2+TRIC-A (blue squares), and RyR2+TRIC-B (red triangles) in the same solutions as in A. The amplitudes in I–V curve of conductance were measured from multiple traces at ±30 and 0 mV. One-way ANOVA for the independent variable, “type of HEK293 cells,” is not significant with P = 0.72 (n_RyR2 = 10, n_RyR2+TRIC-A = 7, n_RyR2+TRIC-B = 10). Symbols indicate values from individual experiments and bars indicate mean ± SEM. More about this image found in The effects of coexpressing TRIC-A or TRIC-B with RyR2 on the function of R...

Figure 2. The effects of coexpressing TRIC-A or TRIC-B with RyR2 on the response of RyR2 channels to increasing cytosolic [Ca ²⁺ ]. Typical gating behavior of RyR2 is shown from the HEK293 cells expressing RyR2-only (left), RyR2+TRIC-A (middle), and RyR2+TRIC-B (right) at −30 mV in symmetrical 210 mM KPIPES, at the indicated free [Ca²⁺]. In symmetrical 210 mM KPIPES and 10 µM cytosolic Ca²⁺, RyR single-channel conductance from the RyR2-only, RyR2+TRIC-A, and RyR2+TRIC-B cells was 735.7 ± 12.7 pS, 735.0 ± 19.2 pS, and 726.3 ± 9.0 pS, respectively. When cytosolic Ca²⁺ was raised to 1 mM, RyR conductance decreased to 544 ± 22 pS, 549 ± 34 pS, and 557 ± 10 pS for RyR2-only, RyR2+TRIC-A, and RyR2+TRIC-B cells, respectively (n_RyR2 = 10, n_RyR2+TRIC-A = 7, n_RyR2+TRIC-B = 10). The Po (measured over 2 min) is shown above each trace. The zero current level is indicated by C and the fully open channel levels by O1 and O2. The red asterisk highlights subconductance events. More about this image found in The effects of coexpressing TRIC-A or TRIC-B with RyR2 on the response of R...

Figure 3. 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. More about this image found in The effects of co-expressing TRIC-A or TRIC-B with RyR2 on the relationship...

Figure 4. Voltage-dependent inactivation of RyR2. (A) Example of voltage-dependent inactivation of RyR2 channels that results from switching the holding potential from −30 to +30 mV in a bilayer where multiple RyR2 channels were gated prior to the voltage change. The blue arrow indicates the time of the voltage change and the orange arrows indicate the times of individual channel inactivation. The dashed line indicates 0 pA. (B) Graph comparing the times to channel inactivation after changing the holding potential from −30 to +30 mV in the RyR2 derived from HEK293 cells expressing RyR2-only (black), RyR2+TRIC-A (blue), and RyR2+TRIC-B (red). The curves were fitted according to the equation: y = n ( 1 − e − k t ) , where t = time in seconds, n is the number of channels (n_RyR2 = 15, n_RyR2+TRIC-A = 15, n_RyR2+TRIC-B = 18), and k is the rate constant (k_RyR2 = 0.264, k_RyR2+TRIC-A = 0.422, k_RyR2+TRIC-B = 4.89). The time for half the channels to become inactivated (half decay time, t_1/2) was 2.62, 1.64, and 0.14 s for RyR2-only, RyR2+TRIC-A, and RyR2+TRIC-B, respectively. Kruskal–Wallis test: P = 0.000003. Post-hoc multiple comparison with Dunn’s correction: significant difference between the t_1/2 of RyR2 derived from HEK293 cells expressing RyR2+TRIC-B and the t_1/2 of cells expressing RyR2-only (indicated by **** for P = 0.000007) or RyR2+TRIC-A (indicated by *** for P = 0.000483). More about this image found in Voltage-dependent inactivation of RyR2. (A) Example of voltage-dependent i...

Figure 5. The effects of co-expressing TRIC-A or TRIC-B with RyR2 on the frequency of subconductance events. Bar chart showing the frequency of subconductance open events >2 ms in duration at 10 µM cytosolic free [Ca²⁺] at −30 mV, in the same solutions as in Fig. 2 , for RyR2 derived from HEK293 cells expressing RyR2-only (black), RyR2+TRIC-A (blue), and RyR2+TRIC-B (red). The Kruskal–Wallis test for one independent variable, type of cell, P = 0.0271. Post-hoc multiple comparison with Dunn’s correction: significant difference between the event frequency of RyR2 derived from HEK293 cells expressing RyR2-only or cells expressing RyR2+TRIC-A is indicated by * for P = 0.0287 (n_RyR2 = 6, n_RyR2+TRIC-A = 6, n_RyR2+TRIC-B = 8). Symbols indicate values from individual experiments and bars show mean ± SEM. More about this image found in The effects of co-expressing TRIC-A or TRIC-B with RyR2 on the frequency of...

Figure 6. Comparison of mouse skeletal SR K ⁺ single-channel events with those derived from HEK293 cell vesicles coexpressing TRIC-A or TRIC-B with RyR2 and with detergent-purified TRIC-B. (A) Typical SR K⁺ channel openings were observed after incorporating mouse skeletal muscle SR vesicles into bilayers in symmetrical 210 mM KPIPES, 2 µM free Ca²⁺ at the holding potential of +30 mV. The zero current level is indicated by “closed” and the fully open channel level by “open.” The orange and blue arrows indicate the characteristic rapidly gating subconductance states and slower gating full conductance openings. So that direct comparison can be made with the TRIC channel currents under identical recording conditions, B–D indicate the amplitude of a full SR K⁺ channel opening with a black arrow and the zero current level with “closed.” (B) Example of the currents observed after incorporating vesicles from HEK293 cells coexpressing RyR2+TRIC-A under the same experimental conditions as in A. (C) Example of the currents observed after incorporating vesicles from HEK293 cells coexpressing RyR2+TRIC-B under the same experimental conditions as in A. (D) Example of current fluctuations obtained after bilayer incorporation of CHAPS-purified TRIC-B channels expressed in yeast under the same experimental conditions as in A. More about this image found in Comparison of mouse skeletal SR K ⁺ single-channel events with...

Figure 7. Effect of co-expressing TRIC-A or TRIC-B on background currents. (A) The bar chart illustrates the percentage of the bilayer in which no background current was detected after the incorporation of vesicles from HEK293 cells expressing RyR2-only, RyR2+TRIC-A, and RyR2+TRIC-B. Significant differences were detected across the three groups using a χ-square test with a P value of 0.000065. The percentage of bilayers with no background current is indicated in each bar (total number of bilayers: n_RyR2 = 21, n_RyR2+TRIC-A = 24, n_RyR2+TRIC-B = 26). (B) Mean current amplitudes in KCl gradient conditions after vesicles incorporation are compared for HEK293 cells expressing RyR2-only, RyR2+TRIC-A, and RyR2+TRIC-B. One-way ANOVA: P = 0.000065. Post-hoc multiple comparison with Tukey’s correction: significant differences between RyR2-only and RyR2+TRIC-A vesicle preparations are indicated by *** for P = 0.0003 and between the RyR2+TRIC-A and RyR2+TRIC-B indicated by *** with P = 0.0005 (n_RyR2 = 21, n_RyR2+TRIC-A = 24, n_RyR2+TRIC-B = 26). Symbols indicate values from individual experiments and bars indicate mean ± SEM. More about this image found in Effect of co-expressing TRIC-A or TRIC-B on background currents. (A) The b...

Figure 8. Effects of coexpressing TRIC-A or TRIC-B with RyR2 on the currents elicited by voltage ramps. (A) Representative current fluctuations from individual experiments after incorporating vesicles from HEK293 cells expressing only RyR2 in which a single RyR2 channel was incorporated (black) or when a background current was detected (green). The black (RyR2-only) and green (background current) arrows indicate the E_rev for the individual experiment. (B) Representative currents from individual experiments after incorporating vesicles from HEK293 cells coexpressing RyR2+TRIC-A (blue) or coexpressing RyR2+TRIC-B (red) into the bilayer. The blue (RyR2+TRIC-A) and red (RyR2+TRIC-B) arrows indicate the E_rev for the corresponding experiment. Vesicles were incubated with the bilayer for 5–10 min in 740 mM cytosolic:210 mM luminal KCl gradient to allow multiple fusion events (evidenced by step changes in conductance) to occur. Vesicle fusion was halted by perfusing the cytosolic chamber with 210 mM KCl. The 740 mM cytosolic:210 mM luminal KCl gradient was then reapplied. The bilayers were held at 0 mV and then switched to −50 mV for 1 s before applying the voltage ramp (red) from −50 to +50 mV. More about this image found in Effects of coexpressing TRIC-A or TRIC-B with RyR2 on the currents elicited...

Figure 9. Effect of coexpressing TRIC-A or TRIC-B with RyR2 on the reversal potential. The mean E_rev values obtained after the application of a voltage ramp are compared for RyR2+TRIC-A (blue squares; n =11) and RyR2+TRIC-B (red triangles; n = 13) vesicle preparations. Ramp currents were recorded in triplicates for each bilayer experiment and the three E_revs measured were averaged and plotted as a single data point. An unpaired t test gives P = 0.0055, indicated by **. Symbols indicate values from individual experiments and bars indicate mean ± SEM. More about this image found in Effect of coexpressing TRIC-A or TRIC-B with RyR2 on the reversal potential...

Figure 10. Example of single-channel recordings of TRIC-B expressed in wheat germ. (A) Liposomes containing TRIC-B expressed in the wheat germ system were fused into bilayers under KCl gradient conditions (740 mM KCl cis; 210 mM KCl trans), 2 µM free Ca²⁺. The recording illustrates the typical multiple-channel fluctuations observed in the KCl gradient and at holding potentials of +40 and −40 mV. The dashed line represents the zero current level. The large current observed at −40 mV indicates that TRIC-B is predominantly permeable to Cl⁻ ions. (B) Example of current fluctuations of TRIC-B from wheat germ during the application of a voltage ramp under gradient (pink trace) conditions. E_rev is indicated by the arrow. (C) The mean E_rev values for each bilayer are shown for RyR2+TRIC-B (red triangles; n = 13; data from Fig. 9 ), RyR2+TRIC-B without outliers (orange triangles; n = 11) and TRIC-B expressed in wheat germ (pink squares; n = 8). One-way ANOVA shows no significant difference with P value = 0.3678. More about this image found in Example of single-channel recordings of TRIC-B expressed in wheat germ. (A)...

Figure 1. DHPs in complexes with calcium channels. (A) Structure of the DHP antagonist R-Bay k 8644. The dihydropyridine ring has a flattened-boat conformation with the polar group NO₂ at the starboard, the hydrophobic group COOCH₃ at the portside, and phenyl-trifluoromethyl group at the bowsprit. (B) Localization of the DHP binding site in the interface between repeats III and IV. The selectivity filter (SF) and the activation gate regions are marked. (C and D) Intracellular (C) and membrane (D) views of 3-D-aligned structures of DHP-bound calcium channels (PDB IDs: 6jp5 , 6jp8 , 7jpl , and 7jpw ). The backbone helices are shown only for the Cav1.1 channel with R-Bay k 8644 ( 7jpw ). Repeats I, II, III, and IV are colored green, yellow, cyan, and magenta, respectively. A calcium ion in the SF region is shown as a blue sphere. (E) Structure of the Cav1.1 channel with the DHP antagonist nifedipine ( 6jp5 ) bound in the fenestration between repeats III and IV. Surrounding residues from helices P1_III, S6_III, and S6_IV are shown as thin sticks. More about this image found in DHPs in complexes with calcium channels. (A) Structure of the DHP antagoni...

Figure 2. Interactions of DHP ligands with helix S6 _IV in MC-minimized structures . (A) The portside methoxy group of antagonist R-Bay k 8644 fits in the hydrophobic pocket between Ala⁴ⁱ¹⁵ and Ile⁴ⁱ¹⁹ (7jpw-based model). (B) A small NO₂ group of agonist S-Bay k 8644 does not fill up the pocket, leaving a void space (7jpl-based model). (C) The void space in the 7jpl-based model is filled up by a water molecule that donates two H-bonds to the NO₂ group of the agonist. More about this image found in Interactions of DHP ligands with helix S6 _IV in MC-minimized s...