Molecular basis for gating of cardiac ryanodine receptor explains the mechanisms for gain- and loss-of function mutations

Figures

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  Fig. 1. Conformational changes upon Ca2+ binding. a Overlay of RyR2 in the closed (light blue) and open (yellow) states viewed from the direction parallel to the lipid bilayer is shown as a ribbon model. Two facing protomers in the RyR2 tetramer are shown. b Magnified view of the dotted box in (a). In the left protomer, each domain is colored (N-Central, light pink; C-Central, purple; U-motif, magenta; S1–S5, wheat; S2–S3 linker domain, light green; S4–S5 linker, warm pink; S6, blue; CTD, orange). S4–S5 linker and S6 moved outside upon Ca2+ binding as indicated by the red arrows. Three regions parallel to the membrane are defined as CTD, U-motif, and S4–S5 layers. Ca2+, shown as a cyan ball; Zn2+, shown as a gray ball. c–e Cross-section views of CTD, U-motif, and S4–S5 layers. The closed state is colored in light blue and the open state is colored according to (b) or yellow. In (e), Cα representation overlaid with cylindrical TM helices is used. Ca2+ binding causes clockwise rotation of CTD (green arrow in (c)), U-motif (green arrow in (d)), and S1-S4 TM helices and outward movement of S4-S5 linker and S6 (red arrows in (e)). f C-Central/U-motif/S6cyto/CTD complex. Closed (light blue)…

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  Fig. 2. Key interactions between U-motif and S2-S3 linker domain upon channel opening. a–c Interface of U-motif and S2-S3 linker domain in the closed state (a), open state (b), and an overlay of both states (c) viewed parallel to the membrane is shown as a Cα model. Amino acid residues involved in key interactions are shown as stick models. The color of carbon atoms is the same as that of Cα; oxygen, red; nitrogen, blue. The TM region forming α-helices is overlaid with the cylinder model. Hydrogen bonds or salt bridges are shown as orange dotted lines. Density maps around side chains shown in (a) and (b) are superimposed and contoured at 0.025. d–g Functional analysis of mutants involved in U-motif/S2–S3 linker domain interaction. d Ca2+-dependent [3H]ryanodine binding of WT and representative mutants. Data are shown as means ± SD (n = 4). e Summary of [3H]ryanodine binding of WT and mutants at pCa 4.5. Data are shown as means ± SD (n = 6 and 4 for WT and mutants, respectively) and were analyzed by one-way ANOVA with Dunnett’s multiple comparisons test. ***p < 0.001 from WT. f Representative traces of cytoplasmic ([Ca2+]cyt) and ER ([Ca2+]ER) Ca2+ signals of HEK293 cells expressing WT or…

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  Fig. 3. Key interactions in the transmembrane region upon channel opening. a–c The S1–S4 bundle. Closed state (a), open state (b), and overlay of the structures in both states (c) are shown as a Cα model and overlaid with cylinder models. Hydrogen bonds/salt bridges are shown as orange dotted lines. Density maps around side chains shown in (a) and (b) are superimposed and contoured at 0.03. d–g Functional analysis of mutants involved in the S1/S2, S2/S3, S1/S4, or S3/S4 interaction. d Ca2+-dependent [3H]ryanodine binding of WT and representative mutants. Data are shown as means ± SD (n = 4). e Summary of [3H]ryanodine binding of WT and mutants at pCa 4.5. Data are shown as means ± SD (n = 6 and 4 for WT and mutants, respectively) and were analyzed by one-way ANOVA with Dunnett’s test. ***p < 0.001 from WT. f Representative traces of [Ca2+]cyt and [Ca2+]ER signals of HEK293 cells expressing Y4589A or D4744A. g Summary of the upper level of [Ca2+]ER signals in WT and mutants. Data are shown as box-and-whisker plots, with the median for all subjects shown as the center line, the box representing the 25–75 percentile, and the lines showing the range of the data (n = 97, 69, 65, 66, 35, 56,…

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  Fig. 4. Key interactions between the U-motif and S6cyto/CTD. a Structure around the U-motif in the open state (U-motif, magenta; S6cyto, blue; CTD, orange). Ca2+ and Zn2+ are shown as cyan and gray spheres, respectively. b, c Details of the U-motif/S6cyto interaction. Structures in the closed (b) and open (c) states fitted to the N-terminal region of the U-motif are shown as a full atomic model. Density maps around the interaction are superimposed and contoured at 0.025. d Details of the U-motif/CTD interaction around F4888. Overlay of the structures in the closed (light blue) and open (colored) states. e–g Compaction in U-motif. Closed state (e), open state (f), and overlay of the structures in both states (g) are shown as a Cα model. Inset of (g) shows the magnified view of the F4171/F4191 stacking looking from aromatic ring of F4191. F4171 is stacked in parallel with F4191 in both the closed and the open states, but it slightly moves away from F4191 in the open state. The structures are fitted in the C-terminal side of U-motif (4183–4205). The closed state and the open state is colored with light blue and yellow, respectively. The N-terminus side of the U-motif of the WT in the open…

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  Fig. 5. Structural basis of loss-of-function mutation and gating mechanism upon Ca2+ binding. a Overlay of K4593A mutant in the presence of EGTA (cyan) and K4593A mutant in the presence of Ca2+ (salmon) viewed from the direction parallel to the lipid bilayer is shown as a ribbon model. Two facing protomers in the RyR2 tetramer are shown. b Magnified view of the dotted box in (a). In the left protomer, each domain is colored (N-Central, light pink; C-Central, purple; U-motif, magenta; S1–S5, wheat; S2–S3 linker domain, light green; S4–S5 linker, warm pink; S6, blue; CTD, orange). S6 moved outside upon Ca2+ binding as indicated by the red arrow. Three regions parallel to the membrane are defined as CTD, U-motif, and S4–S5 layers. Ca2+, shown as cyan ball; Zn2+, shown as gray ball. c–e Cross-section views of CTD, U-motif, and S4-S5 layers. K4593A mutant in the presence of EGTA is colored with cyan and in the presence of Ca2+ is colored according to (b) or salmon. In (e), Cα representation overlaid with cylindrical TM helices are used. Ca2+ binding causes clockwise rotation of CTD (green arrow in (c)), but U-motif and S1-S4 TM helices show no considerable movement as shown in Figs. 1d, e. f…

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  Fig. 6. Details of interactions and schematic diagram of the channel gating of WT and mutant RyR2 upon Ca2+ binding. a Details of interactions identified in this study. Amino-acid residues shown in red letter and blue letter indicate gain-of-function and loss-of-function by alanine-substituted or pathogenic mutations, respectively. Arrows indicate interactions. Purple and green arrows indicate interactions only found in the closed and open states, respectively. b–d Schematic diagram of the channel gating of WT and mutant RyR2 upon Ca2+ binding. The left and right diagrams show the states in the absence and presence of Ca2+, respectively. The black lines, the domain interactions; The dotted line, CTD/U-motif interaction via F4888; The blue T-shaped lines, U-motif/S6cyto interaction that acts as a suppressor; The black lines with arrowhead, signals for channel opening. The domains shown in gray-scale and colored indicate the domains in the inactive and active states, respectively. b In WT., the channel pore is closed, since the movement of S4-S5 linker is locked. Ca2+ binding unlocks the S4-S5 linker and induces the outward leaning of S6, resulting in the pore opening. c Loss-of-function…